The human cytomegalovirus

www.elsevier.com/locate/pharmthera

Pharmacology & Therapeutics 98 (2003) 269–297

Associate editor: S. Pestka

The human cytomegalovirus

Santo Landolfoa,*, Marisa Garigliob, Giorgio Gribaudoa, David Lemboa

aDepartment of Public Health and Microbiology, University of Turin, Via Santena 9, 10126 Turin, ItalybDepartment of Medical Sciences, University of Eastern Piedmont, Novara, Italy

Abstract

Human cytomegalovirus (HCMV), a betaherpesvirus, represents the major infectious cause of birth defects, as well as an important

pathogen for immunocompromised individuals. The viral nucleocapsid containing a linear double-stranded DNA of 230 kb is surrounded by

a proteinaceous tegument, which is itself enclosed by a loosely applied lipid bilayer. Expression of the HCMV genome is controlled by a

cascade of transcriptional events that leads to the synthesis of three categories of viral proteins designated as immediate-early, early, and late.

Clinical manifestations can be seen following primary infection, reinfection, or reactivation. About 10% of infants are infected by the age of 6

months following transmission from their mothers via the placenta, during delivery, or by breastfeeding. HCMV is a significant post-allograft

pathogen and contributes to graft loss independently from graft rejection. Histopathologic examination of necropsy tissues demonstrates that

the virus enters via the epithelium of the upper alimentary, respiratory, or genitourinary tracts. Hematogenous spreading is typically followed

by infection of ductal epithelial cells. Infections are kept under control by the immune system. However, total HCMV clearance is rarely

achieved, and the viral genome remains at selected sites in a latent state. Virological and molecular detection of HCMV, as well as serological

demonstration of a specific immune response, are used for diagnosis. Treatment of HCMV infections is difficult because there are few

options. The presently available drugs produced a significant clinical improvement, but suffer from poor oral bioavailability, low potency,

development of resistance in clinical practice, and dose-limiting toxicities.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: HCMV; Gene expression; Epidemiology; Transplant; Diagnosis; Antiviral therapy

Abbreviations: ab, antibody; AIDS, acquired immunodeficiency syndrome; AP, assembly protein; AP-1, activator protein-1; ATF, activating transcription

factor; BMT, bone marrow transplant; CDV, cidofovir; CMV, cytomegalovirus; CPE, cytopathic effect; CREB, cyclic AMP-responsive element binding

protein; crs, cis-repression signals; CTL, cytotoxic T lymphocyte; DB, dense bodies; E, early; ER, endoplasmic reticulum; GCV, ganciclovir; GPCR, G-

protein-coupled receptor; HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; IE, immediate-early; Ig, immunoglobulin; L, late; mCP,

minor capsid protein; MCP, major capsid protein; MHC, major histocompatibility complex; MIE, major immediate-early; NF, nuclear factor; NK, natural killer;

ORF, open reading frame; PBL, peripheral blood leukocytes; PCR, polymerase chain reaction; PFA, foscarnet; p.i., post-infection; Rb, Retinoblastoma; TAP,

transporter associated with antigen processing; UL, unique long; US, unique short.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

2. The human cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

2.1. Virus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

2.2. Virus genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

2.3. Virus growth cycle and viral gene expression . . . . . . . . . . . . . . . . . . . . . . . 273

2.3.1. Range of cell permissivity and in vitro growth . . . . . . . . . . . . . . . . . . 273

2.3.2. Virus binding and penetration . . . . . . . . . . . . . . . . . . . . . . . . . . 273

2.3.3. Regulation of viral gene expression . . . . . . . . . . . . . . . . . . . . . . . 274

2.3.4. Characteristics and functions of the immediate-early proteins . . . . . . . . . . 274

2.3.5. Characteristics and functions of the early proteins . . . . . . . . . . . . . . . . 276

2.3.6. Viral DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

0163-7258/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0163-7258(03)00034-2

* Corresponding author. Tel.: +39-11-6706604; fax: +39-11-6636436.

E-mail address: [email protected] (S. Landolfo).

S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297270

2.3.7. Characteristics and functions of the late proteins . . . . . . . . . . . . . . . . . 278

2.3.8. Virion assembly, maturation, and egress . . . . . . . . . . . . . . . . . . . . . 278

3. Epidemiology of human cytomegalovirus infection . . . . . . . . . . . . . . . . . . . . . . . . 278

4. Infection routes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

5. Pathogenesis and pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

6. Host defences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

6.1. Cell-mediated immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

6.2. Humoral immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

6.3. Immune evasion by human cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . 282

6.4. Persistence and release from the host . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

7. Clinical features associated with human cytomegalovirus infection. . . . . . . . . . . . . . . . 284

7.1. Infection in normal hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

7.2. Congenital infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

7.3. Cytomegalovirus infection in the immunocompromised host . . . . . . . . . . . . . . . 284

8. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

8.1. Virus detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

8.1.1. Viremia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

8.1.2. Antigenemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

8.1.3. DNAemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

8.1.4. RNAemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

8.2. Detection of the immune response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

9. Prevention of human cytomegalovirus infection and disease . . . . . . . . . . . . . . . . . . . 286

9.1. Human cytomegalovirus vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

10. Antiviral treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

10.1. Currently available drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

10.2. Therapeutic approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

10.2.1. Prophylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

10.2.2. Pre-emptive treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

10.2.3. Treatment of an established disease . . . . . . . . . . . . . . . . . . . . . . . 288

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

1. Introduction

The human cytomegalovirus (HCMV) is a widespread

pathogen responsible for generally asymptomatic and per-

sistent infections in healthy people. It may, however, cause

severe disease in the absence of an effective immune

response, as in immunologically immature and immunocom-

promised individuals. Its impact, therefore, has increased in

recent decades due to the rise in organ allografting, immu-

nosuppressive treatment, and human immunodeficiency

virus (HIV)-infected patients. Furthermore, it is the leading

infectious agent causing birth defects (Griffiths, 2000; Pass,

2001).

HCMV is a member of the betaherpesvirinae subfamily,

whose virion structure, kinetics of viral gene expression,

and persistence for the lifetime of their host are typical of

other herpesviruses. However, its strict species specificity,

salivary gland tropism, and slow growth in cell cultures

differentiate it as the prototype betaherpesvirus (Mocarski &

Courcelle, 2001).

This paper provides an overview of the general character-

istics of HCMV structure, growth, and replication, as well as

the pathogenesis, epidemiology, diagnosis, and management

of the infections it causes.

2. The human cytomegalovirus

2.1. Virus structure

The virion of HCMV consists of a 100-nm diameter

icosahedral nucleocapsid containing a 230-kbp, double-

stranded linear DNA genome surrounded by a proteinaceous

layer defined as the tegument or matrix, which, in turn, is

enclosed by a lipid bilayer containing a large number of

viral glycoproteins. The mature virion particle is 150–200

nm in diameter.

HCMV-infected cell cultures produce the infectious viri-

ons and another two types of morphological particles: non-

infectious enveloped particles and dense bodies (DB).

Noninfectious enveloped particles are defective viral par-

ticles composed of enveloped immature capsids (type B)

that lack DNA, but contain the viral scaffolding/assembly

protein (AP) normally absent from fully mature nucleocap-

sids (C-capsids). DB are enveloped particles that lack an

assembled nucleocapsid and viral DNA, but contain several

tegument proteins of which ppUL83 (pp65 or lower matrix

protein) is the most abundant. The relative amounts of the

three viral forms depends on the number of passages in cell

culture and the viral strain (Mocarski & Courcelle, 2001).

Table 1

HCMV proteins discussed in this review1

Gene Kinetic

class

Protein

name(s)

Function(s)

UL36 IE pUL36 Inhibition of apoptosis

UL37 IE vMIA Inhibition of apoptosis

UL122 IE IE1491aa,

IE1–72

Regulation of viral and

host gene expression

UL123 IE IE2579aa,

IE2–86



UL123 IE IE2425aa,

IE2–55



IRS1 IE pIRS1 Transactivator of viral

gene expression

US3 IE gpUS3 Down-modulation of MHC

Class I expression

TRS1 IE pTRS1 Transactivator of viral

gene expression

UL4 E gpUL4, gp48 Minor envelope glycoprotein

UL40 E-L gpUL40 Immune evasion

UL44 E-L ppUL44, p52 DNA processivity factor

UL46 E-L mC-BP Minor capsid-binding protein,

capsid structure

UL54 E DNA pol,

pUL54

DNA polymerase

UL55 E gB Major envelope

glycoprotein,

constituent of gCI

UL57 E pUL57,

ssDNA BP

Single-stranded

DNA-binding protein

UL69 E-L ppUL69 Transactivator,

dysregulation of

host cell cycle

UL70 E-L Helicase-

primase

Subunit of the helicase-

primase complex

UL78 E pUL78 Similar to glucocorticoid

receptors

UL84 E-L ppUL84 Initiation of oriLyt-specific

DNA replication

UL85 E-L mCP mCP, capsid structure

UL97 E-L pUL97 phosphotransferase,

GCV-activating enzyme

UL102 E Helicase-

primase


primase complex

UL105 E Helicase-

primase


primase complex

UL112 E Early pp

family

Organization of viral

DNA replication centers

UL113 E Early pp

family

Organization of viral

DNA replication centers

UL114 E pUL114 Uracil DNA glycosylase

US2 E gpUS2 Down modulation of MHC

Class I expression

US6 E-L gpUS6 Inhibition of TAP-mediated

peptide translocation

US11 E gpUS11 Down modulation of MHC

Class I expression

US27 E pUS27 Similar to glucocorticoid

receptors

US28 E pUS28 C-C chemokine receptor,

immune evasion

UL32 L pp150,

ppUL32

Major tegument component,

basic phosphoprotein

UL33 L gpUL33 Similar to glucocorticoid

receptors, minor envelope gp

Gene Kinetic

class

Protein

name(s)

Function(s)

UL48.5 L SCP Smallest capsid protein,

capsid structure

UL73 L gN Envelope glycoprotein,

constituent of gCII

UL74 L gO Envelope glycoprotein,

constituent of gCIII

UL75 L gH Envelope glycoprotein,


UL80 L Assemblin

precursor

Assemblin protease (pUL80a),

capsid assembly

UL80.5 L AP AP scaffolding protein,

capsid assembly

UL82 L pp71,

ppUL82

Transactivator,

dysregulation of

host cell cycle

UL83 L pp65,

ppUL83

Major tegument component,

lower matrix protein

UL86 L MCP MCP, capsid structure

UL94 L pUL94 Virion protein?

UL99 L pp28 Tegument protein

UL100 L gM Envelope glycoprotein,

constituent of gCII

UL115 L gL Envelope glycoprotein,


UL123 L gIE2338aa Regulation of viral and


1 See Mocarski and Courcelle (2001) for a complete list of all HCMV

genes, their products, and their functions.

Table 1 (continued)

S. Landolfo et al. / Pharmacology & Therapeutics 98 (2003) 269–297 271

Of the more than 30 viral proteins found in the complete

infectious virion, four constitute the capsid; namely, pUL46,

pUL48.5, the minor capsid protein (mCP), and the major

capsid protein (MCP) encoded by UL85 and UL86, respect-

ively (Table 1). Three APs encoded by UL80 associate with

capsid and play roles in maturation. The 162 capsomer shell

of HCMV is composed of hexameric and pentameric units

of the MCP located at the vertices of a T = 16 icosahedral

lattice, where adjacent capsomers are joined by surface

structures produced by the association of pUL48.5 and

pUL46.

The phospholipid envelope contains 6 virus encoded

glycoproteins, including gpUL55 (gB), gpUL73 (gN), gp

UL74 (gO), gpUL75 (gH), UL100 (gM), and gpUL115

(gL). These glycoproteins play essential roles in virus entry

into host cells, cell-to-cell spread, and virion maturation

(Britt & Mach, 1996). Mutational analysis has revealed that

disruption of gB, gH, gL, and gM open reading frames

(ORFs) results in the failure to produce infectious progeny,

and underscores the essential role of these proteins for

productive replication. Only gO is dispensable for viral

growth in cultured fibroblasts (Hobom et al., 2000). Two

other glycoproteins, gpUL4 (gp48) and the seven-spanning

transmembrane gpUL33, are found in the virion and are also

probably located in the envelope. The protein products of

the six major glycoprotein-encoding genes associate to form

three complexes that are highly conserved within herpesvi-

Fig. 1. Structure of the four HCMV genome isomers.


ruses and previously have been designated as gCI, gCII, and

gCIII (Gretch et al., 1988). At least two are required for viral

entry: gCI, composed of homodimeric gB molecules, and

gCIII, a heteroligomeric complex composed of gH, gL, and

gO (Theiler & Compton, 2001). gB is an essential gly-

coprotein that plays a crucial role in virus binding, since it is

the major cell surface, heparan sulfate proteoglycan-binding

glycoprotein. It also participates in viral entry, cell-to-cell

spread, and cell fusion. By virtue of its abundance and

ability to elicit neutralizing antibodies (abs), it has been

proposed as a molecule of choice for a recombinant subunit

vaccine (Gonczol & Plotkin, 2001). gCIII is necessary for

the final stage of virus entry via pH-independent fusion

between the viral envelope and the cell membrane (Huber &

Compton, 1998). gCII results from the association of gM

and gN (Kari et al., 1994). All three complexes induce

neutralizing abs. Because of their interaction with the

immune system, the genes encoding gB, gH, and gN have

been detected as highly polymorphic loci that can be

clustered in different genotypes in clinical isolates. The

occurrence of ab-escaping mutant viruses selected by the

pressure of the humoral immune response may be related to

cell tropism and virulence, and is thought to contribute to

the pathogenicity of HCMV.

The remaining 20–25 structural virion proteins probably

are located in the still poorly characterized amorphous layer

between the nucleocapsid and the envelope (Baldick &

Shenk, 1996). The tegument proteins may be involved in

the maturation of progeny virions, or may influence viral

and cellular events in the early stages of infection, such as

release of viral DNA from disassembling virus particles or

the regulation of viral and cellular promoters. Most tegu-

ment proteins are phosphorylated and are highly immuno-

genic. The most abundant are ppUL32 (pp150 or basic

phosphoprotein) and ppUL83. Due to its large amounts,

pp65 is the target antigen in antigenemia assays for rapid

diagnosis of HCMV-substained clinical infections. Tegu-

ment proteins such as ppUL69 and ppUL82 (pp71) may

play important regulatory roles in both viral and cellular

gene expression. They are, in fact, transactivators of viral

gene expression, and deeply impact on host cell physiology

by dysregulating cycle progression. UL69 stimulates

expression from the ie1/ie2 enhancer-promoter. It is required

for efficient viral replication, and arrests cells in G1 by an

unknown mechanism (Lu & Shenk, 1999; Hayashi et al.,

2000). UL82 is a transcriptional activator of activating

transcription factor (ATF)/cyclic AMP-response element

binding protein (CREB) or the activator protein-1 (AP-1)-

containing promoter and, thus, stimulates the activity of the

ie1/ie2 enhancer-promoter. It also increases the infectivity of

transfected viral DNA, is required for viral replication at low

multiplicities of infection, and accelerates the G1 to S

transition of quiescent cells (Liu & Stinski, 1992; Baldick

et al., 1997; Bresnahan & Shenk, 2000b). Two more trans-

activator proteins (pIRS1 and pTRS1) are associated with

virions and DB (Romanowski et al., 1997).

Five viral RNAs (designated as virion RNAs) have been

detected in preparations of highly purified, infectious

HCMV particles. These HCMV transcripts probably are

located in the tegument and are delivered to the host cell on

infection. However, the functional significance of their

presence in the virions and that of their protein products

remains to be defined (Bresnahan & Shenk, 2000a).

2.2. Virus genome

The HCMV genome is the largest of all herpesviruses and

has a high G+C content. Like that of herpes simplex virus-1,

it contains an arrangement of unique long (UL), unique short

(US), and repeat regions. Since each long and short region

can be oriented in either direction, four genome isomers are

produced in viral progeny (Class E structure) (Fig. 1). In

contrast, the genomes of animal CMV, as well as those of

other betaherpesviruses, are linear without repeat regions

(Class F genomes). Inversion of UL and US regions is

mediated by direct repeat sequences (a, b, c) at the genome

termini and by inverted repeat elements at the UL-US

junction (a0, b0, c0). The repeated a sequence that occurs as a

direct element at the termini and in the inverted orientation at

the UL/US junction promotes genome isomerization, since it

contains the cis-acting pac (packaging) elements needed for

DNA cleavage (Mocarski & Courcelle, 2001).

The AD169 laboratory strain is the only completely

sequenced HCMV. Analysis of its 230-kbp genome has

revealed that it encodes 225 ORFs of � 100 or more amino

acids (Chee et al., 1990a; Novotny et al., 2001). These

ORFs are designated sequentially according to their location

within the unique and repeated regions. Additional ORFs

have been identified in the Towne and Toledo laboratory

strains. In the latter, as well as in clinical isolates, the

inverted b0 repeat is deleted and replaced by an additional

UL region of � 15 kbp, containing 19 additional ORFs that

are absent in the AD169 genome (Cha et al., 1996). The

unique ORF UL1–154 and US1–36 blocks are separated

by duplicated IRL1–14 and J1I genes and the partially

repeated IRS1 gene. The UL region is flanked at the 50 end

by the duplicated TRL1–14 and J1L (identical to IRL1–14

Fig. 2. HCMV cytopathic effect on a human fibroblasts monolayer.


and J1I), whereas the US gene block is flanked at its 30 end

by the TRS1 gene and by the third copy of a J1 gene (J1S).

Comparison of the AD169 amino acid sequences with

those of other herpesvirus genomes has revealed that the

protein products of more than 40 ORFs share high similarity

to proteins encoded by alpha- and gammaherpesviruses

(Chee et al., 1990a; Karlin et al., 1994), and provided

further evidence of a common origin of the three subfam-

ilies. Of the herpesvirus-conserved ORFs, � 25% appear to

encode functions related to viral DNA metabolism and

replication, whereas the remaining 75% are thought to be

involved in the maturation and structural organization of

virions. The UL ORF-encoding functions involved in DNA

replication and repair, nucleotide metabolism, or virion

structure are grouped in seven conserved gene blocks (A–

G) also found in other herpesviruses, such as herpes simplex

virus-1 and Epstein-Barr virus, although in a different order

in the alpha- and gammaherpesviruses. The occurrence of

these conserved blocks in a characteristic order specific to

each subfamily suggests that the functions encoded are

probably conserved in all herpesviruses. The US ORF and

those located within the repeated regions of the HCMV

genome are less well conserved than in the other herpesvi-

ruses (Chee et al., 1990a).

Sequence homology searches and experimental biochem-

ical and/or genetic studies have assigned functional roles to

only some of the more than 200 HCMV ORFs (Novotny et

al., 2001). However, analysis of the phenotypes of spontan-

eous deletion mutants of the AD169 strain, as well as those

of virus-bearing deletions or inactivation at specific loci, has

indicated that the products of more than 50 HCMV ORFs

are dispensable for productive replication in fibroblast

cultures. These findings, along with observations that the

proteins responsible for functions common to all herpesvi-

ruses, such as basic DNA replication, virion organization,

and maturation, do not account for all the ORFs, indicate

that many ORFs still await functional characterization. It is

likely, therefore, that much of the coding capability has

evolved to optimize infection by influencing dissemination,

growth in target tissues and pathogenesis, and in counter-

acting host immune reactivity (Mocarski & Courcelle,

2001).

2.3. Virus growth cycle and viral gene expression

2.3.1. Range of cell permissivity and in vitro growth

Autopsy specimens show that HCMV infects a wide

range of epithelial tissues. The ductal epithelial cell is most

commonly infected, and develops a typical cytopathology

(Pass, 2001; Bissinger et al., 2002). However, during natural

infection, it is thought that HCMV replicates productively in

epithelial cells, endothelial cells, smooth muscle cells,

mesenchymal cells, hepatocytes, granulocytes, and mono-

cyte-derived macrophages (Plachter et al., 1996; Sinzger &

Jahn, 1996; Sinzger et al., 1996; Kahl et al., 2000; Bissinger

et al., 2002). Latent viral DNA, thus, can be detected in

macrophage-granulocyte progenitors in the bone marrow

and in peripheral monocytes (Kondo et al., 1994; Soder-

berg-Naucler et al., 1997). In contrast, in vitro, the only cells

fully permissive for replication of laboratory strains are

human skin or lung fibroblasts, whereas clinical isolates

replicate preferentially on endothelial cell cultures. Some

transformed cell lines derived from glioblastomas, such as

U373MG, as well as primary arterial smooth muscle cells,

support productive infection, although at lower levels than

in fibroblasts (Sinzger et al., 1995; Plachter et al., 1996). In

fibroblasts, the replication cycle is slow, with a typical

cytopathic effect (CPE) characterized by cell rounding and

enlargement with both intra- and perinuclear inclusions (Fig.

2). Only laboratory strains, such as AD169 or Towne,

produce a generalized CPE, with lysis of the entire cell

monolayer, and release high titers of virions starting from 3

to 4 days post-infection (p.i.). Recently, isolated strains

mainly spread on fibroblast monolayers by cell-to-cell

contact and produce a limited CPE, with dispersed foci

and poor virion yields (Pass, 2001).

2.3.2. Virus binding and penetration

Virus attachment and penetration are rapid and efficient

in both permissive and nonpermissive cell types. However,

since productive replication is observed in a very restricted

range of human cells, a post-penetration block to viral gene

expression is thought to restrict replication in nonpermissive

cells (Sinzger et al., 2000). The poorly characterized recep-

tor(s) for HCMV is widely distributed among host cell

types, and contributes to the broad viral tropism observed

during natural infections. Viral entry is the result of a

cascade of interactions between viral and cellular proteins

that culminate in fusion of the virion envelope with the

cellular plasma membrane by a pH-independent mechanism.

During the initial virus-cell interactions, as observed with

other herpesviruses, HCMV attaches to the cell surface by

low-affinity binding of gB to heparan sulfate proteoglycans

(Compton et al., 1993). The subsequent interaction of gB

with its nonheparin receptor then turns the weak adhesion of

the viral particle into a more stable binding or docking state.

S. Landolfo et al. / Pharmacology & T274

However, final fusion of the viral envelope with the cell

membrane to allow viral penetration is thought to require a

further priming event mediated by the heteroligomeric gH-

gL-gO complex with as yet unidentified receptors (Theiler

& Compton, 2001). Fusion of the virus and cell membranes

is followed by entry into the host cytoplasm of the nucle-

ocapsid and tegument proteins, and their rapid translocation

into the nucleus, where pp65 is detected < 1 hr p.i. Inter-

action of HCMV glycoproteins with their receptors is

enough to generate an intracellular signal transduction

pathway, leading to the alteration of cellular gene expres-

sion. Most changes in the profiles of host gene activity

resemble those induced by binding of interferons to their

receptors (Zhu et al., 1997, 1998; Browne et al., 2001). The

specific viral ligand triggering this response is gB, and its

interaction with its as yet unidentified receptor is thought to

be the main mechanism by which HCMV modifies host cell

gene expression in the very early phases of infection (Boyle

et al., 1999; Simmen et al., 2001). In addition, engagement

of gB and gH with their receptors is sufficient to induce the

activity of the cellular transcription factors nuclear factor-kB(NF-kB) and Sp1 (Yurochko et al., 1997).

2.3.3. Regulation of viral gene expression

During productive infection, the HCMV genome is

expressed in a temporally coordinated and regulated cascade

of transcriptional events that lead to the synthesis of three

categories of viral proteins described as immediate-early (IE

or a), early (E or b), and late (L or g) (Fig. 3). Failure in the

expression of early gene and subsequent viral DNA replica-

tion rather than attachment and/or penetration may be the

restricting event in nonpermissive cells. HCMV genes are

transcribed in the infected cell nucleus by RNA polymerase

II and the associated basal transcription machinery, with the

intervention of host-encoded transcription factors whose

activity may be stimulated by viral transactivators (Fortu-

nato & Spector, 1999; Mocarski & Courcelle, 2001).

Fig. 3. HCMV gene expression and viral gene pro

2.3.4. Characteristics and

functions of the immediate-early proteins

HCMV gene expression initiates from a few IE proteins

within l hr p.i. without de novo protein synthesis. The IE

genes include the major IE (MIE) UL122/123 genes (IE1

and IE2) and auxiliary genes, such as UL36–UL38,

UL115–UL119, IRS1/TRS1, and US3 (Table 1). The MIE

proteins, alone or in synergism, are required for subsequent

expression by acting as transactivators and autostimulators

of viral genes. In addition, these proteins have a deep impact

on host cell physiology since they regulate the expression of

a large number of host cell genes (Fortunato & Spector,

1999).

MIE proteins are encoded by the ie1/ie2 genes (UL122/

UL123), whose expression is regulated by a complex

enhancer-modulator element that functions in a tissue- and

cell-type-specific manner (Nelson et al., 1990; Meier &

Stinski, 1996), and exerts its strong transcriptional activity

through interactions with several host transcription factors

whose binding sites are closely distributed within the

regulatory element. An � 500-bp segment upstream from

the TATA box of the ie1/ie2 genes contains several repeat

elements with binding sites for NF-kB, AP-1, Sp1, and

CREB/ATF (Boshart et al., 1985). Since their cognate

DNA-binding activities are rapidly activated by HCMV

infection, their binding to the corresponding sites is thought

to contribute to the very strong activity of the MIE enhancer.

The CREB/ATF and AP-1 sites are also responsive to the

tegument transactivator ppUL82 (Liu & Stinski, 1992). In

addition, NF-1, serum response factor, Elk-1, CCAAT/

enhancer-binding protein, and YY1 sites have been iden-

tified within the enhancer segment. The cell type- and

differentiation state-specific enhancer activity is related to

the availability of appropriate transcription factors in a

specific cell type, and is thought to depend on the modulator

region. This element spans � 500 bp upstream from the

core of the MIE enhancer. In transient transfection assays,

herapeutics 98 (2003) 269–297

duct functions during productive infection.


the modulator region represses the transcriptional activity of

the enhancer in undifferentiated cells lines, although these

findings could not be reproduced with mutagenized viruses

in which the modulator element was removed (Nelson et al.,

1990; Meier & Stinski, 1997).

The MIE region produces a set of transcripts synthesized

from different start sites and differentially spliced and

polyadenylated to give rise to four protein products. The

most abundant in IE times of infection are IE1491aa and

IE2579aa. These share the same amino terminal 85 amino

acid long segment, but their differentially spliced exons are

4 (UL123) and 5 (UL122), respectively. In addition, a less

abundant IE2 isoform (IE2425aa) is encoded by a spliced

transcript bearing an in-frame deletion within UL122. The

fourth IE protein expressed from the MIE region is a true

late 40-kDa polypeptide (gIE2338aa) encoded by a polyade-

nylated transcript that originates late in infection from a start

site within UL122. Both IE1491aa and IE2579aa are expressed

throughout replication, albeit with slightly different kinetic

profiles. Their ability to regulate transcription of several

viral and cellular genes has been studied extensively (For-

tunato & Spector, 1999; Mocarski & Courcelle, 2001).

IE1479aa is a nuclear phosphoprotein of � 72 kDa (IE1-

72), whose functions are not needed for viral growth in

fibroblast cultures infected at high multiplicity of infection,

but are required after a low multiplicity of infection. The

presence of IE1-72, thus, is crucial when infection starts

from a single virion (Mocarski et al., 1996). Generation of

mutants lacking UL123 and, hence, IE1-72 expression has

confirmed in infected cells its functions, as predicted by

transfection assays. The first role of IE1-72 is consistent

with its ability to positively autoregulate expression of the

ie1/ie2 and US3 genes by activating the corresponding

enhancer elements through NF-kB-binding sites (Mocarski

et al., 1996). In addition, IE1-72 cooperates with IE2579aa to

regulate the expression of viral genes belonging to the

subsequent categories, as predicted by transient transfection

assays in which co-expression of the two IE proteins were

found to be required for maximal activity of the promoters

of E (UL44 and UL54), as well as L (UL83), genes

(Fortunato & Spector, 1999). IE1-72 is a weaker and less

promiscuous transactivator than IE2579aa. In these assays,

however, it stimulates the activity of several cellular pro-

moters, including those of dihydrofolate reductase, DNA

polymerase a, c-fos, c-myc, NF-kB p65 subunit, and thy-

midylate synthase genes (Hagemeier et al., 1992a; Wade et

al., 1992; Hayhurst et al., 1995; Yurochko et al., 1995;

Gribaudo et al., 2002). In addition, IE1 may affect the host

cell cycle regulatory pathway by targeting the Retinoblas-

toma (Rb) protein family and E2F-mediated transcriptional

mechanisms since it interacts with cellular p107, a member

of the Rb family, and activates an E2F-responsive promoter,

such as that of the DHFR gene, by alleviating p107-

mediated repression (Poma et al., 1996; Johnson et al.,

1999). Moreover, it interacts physically with E2F1 and

transactivates the DHFR promoter only if its E2F sites are

retained (Margolis et al., 1995). Lastly, IE1-72 is endowed

with a kinase activity that autophosphorylates and phos-

phorylates the p107 and p130, as well as E2F1, -2, and -3.

The putative catalytic domain of IE1-72 is also required for

the activation of E2F-dependent transcription (Pajovic et al.,

1997).

IE2579aa is an 86-kDa (IE2-86) nuclear phosphorylated

polypeptide whose activities are critical for viral replication.

A bacterial artificial chromosome clone of HCMV with a

deletion of exon 5 unique to IE2-86 (the UL122 ORF), in

fact, did not activate E gene expression nor yield progeny

virus upon transfection in permissive cells (Marchini et al.,

2001). Since it is difficult to generate mutant viruses lacking

IE2-86 and complementing long-term cell lines expressing

fully functional wild-type IE2-86, most studies have relied

on transient transfection experiments, many of which have

indeed established that IE2-86 behaves as a strong tran-

scriptional regulator by either stimulating or repressing both

HCMV and cellular genes (Fortunato & Spector, 1999;

Mocarski & Courcelle, 2001; Song & Stinski, 2002). During

productive infection, IE2-86 is the key protein regulating the

transition from the IE to the E, and very likely to the L

phases of HCMV gene expression. Characterization of its

function in transient transfection assays, in fact, has dem-

onstrated that alone or in combination with IE1, it trans-

activates several E and L promoters (Fortunato & Spector,

1999), and IE2-86-binding sites have been identified just

upstream from the TATA box of three E promoters. These

transactivating properties probably are attributable to the

interaction of IE2-86 with factors of the basal-transcription

machinery and a variety of promoter-specific transcription

factors. Recombinant IE2-86 produced in bacteria, in fact,

interacts with the TATA-binding protein, TFIIB, TAFII-130,

and TFIID (Hagemeier et al., 1992b; Caswell et al., 1993;

Jupp et al., 1993; Lukac et al., 1997). It also interacts in

vitro with other transcription factors, such as histone acetyl-

transferase, CREB, Sp1, c-Jun, and ATF-2, as well as with

cell-cycle regulatory factors, including p53, pRb, and p21

(Speir et al., 1993; Hagemeier et al., 1994; Sommer et al.,

1994; Fortunato et al., 1997). These interactions indicate

that IE2-86 may act as an adapter-like protein by stabilizing

basal and specific transcription factors on the promoter

region (Mocarski & Courcelle, 2001). Consistent with this

model, the occurrence of cis-acting regulatory elements;

namely, AP-1, CREB/ATF, and Sp1, upstream from some E

promoters contributes to the overall promoter responsive-

ness to IE2-86-mediated transactivation (Fortunato & Spec-

tor, 1999).

In addition to their regulation of the expression of E and

L genes, IE2-86 and gIE2338aa repress expression of ie1/ie2

and US3 (Fortunato & Spector, 1999; Mocarski & Cour-

celle, 2001). This down-regulation is mediated by IE2

binding to cis-repression signals (crs) sequences located

between the TATA box and the transcription initiation sites

of these genes (Pizzorno & Hayward, 1990; Cherrington et

al., 1991). By down-regulating transcription from its own


promoter, therefore, IE2-86 mediates autoregulation of its

own expression and contributes to the reduction of IE gene

expression in the late stages of infection. In vitro trans-

fection assays have established that binding of IE2-86 to crs

confers the IE2-dependent repression of homologous and

heterologous promoters, provided that the sequence element

is located near the transcription start site. Moreover, purified

bacterial IE2-86 binds to crs through its C-terminus and

alters RNA polymerase pre-initiation complex formation in

the in vitro repression of transcription assays.

IE2-86 also modulates several cell processes, including

cycle control and apoptosis (Zhu et al., 1995; Kalejta &

Shenk, 2002). In fact, expression of IE2-86 by either

transient transfection or replicative-defective adenovirus

arrests cells in an early S phase by mechanisms that are

not known yet (Wiebush & Hagemeier, 1999, 2001; Murphy

et al., 2000). Their limited DNA synthesis and high levels of

cyclin E-dependent kinase activity show that these cells are

blocked one step further than the G1 restriction point of the

cycle. IE2-86 stimulates quiescent, serum-starved cells into

the early S phase, but prevents them from progressing

through this phase, and cell division does not occur. Its

ability to arrest the cycle thus greatly contributes to the late

G1/early S phase synchronization and host DNA synthesis

inhibition observed in HCMV-infected cells (Jault et al.,

1995; Lu & Shenk, 1996; Bresnahan et al., 1996; Dittmer &

Mocarski, 1997; Salvant et al., 1998). These modifications

lead to a more favorable environment for viral replication,

since the precursors for DNA synthesis produced by the host

are available, but are not utilized by the DNA replication

machinery of the cell (Fortunato et al., 2000; Kalejta &

Shenk, 2002).

The functions of the other IE genes are heterogeneous.

TRS1 and IRS cooperate with IE1-72 and IE2-86 in the

transactivation of E promoters. The glycoproteins encoded

by the US3 gene are involved in the establishment of

immune evasion in infected cells, since these endoplasmic

reticulum (ER)-located proteins prevent the transport of the

assembled major histocompatibility complex (MHC) Class I

from ER to the Golgi and, thus, down-modulate MHC Class

I antigen presentation (Stasiak & Mocarski, 1992; Jones et

al., 1996). In contrast, the UL36–38 region encodes pro-

teins such as UL36 and UL37 that are endowed with anti-

apoptotic properties and that act at different steps in the

caspase cascade (Skaletskaya et al., 2001; Goldmacher,

2002). UL36, UL37, US3, and IRS1 are all dispensable

for replication in cell culture.

2.3.5. Characteristics and functions of the early proteins

Expression of E or b genes depends on the presence of

functional IE proteins and is unaffected by inhibitors of viral

DNA replication (Fortunato & Spector, 1999). They are

divided into two subclasses: b1 (E) and b2 (E-L) according

to their time of expression. b1 genes are transcribed within

4–8 hr p.i., b2 transcription 8–24 hr p.i.. The functional

data indicate that E genes encode mostly non-structural

proteins, including viral DNA replication factors, repair

enzymes, and proteins involved in immune evasion (Mocar-

ski & Courcelle, 2001).

The expression profiles of microarrays of viral DNA

recently have provided a temporal map of IE, E, and L

genes in the entire viral genome (Chambers et al., 1999).

Hybridization of such microarrays to cDNAs prepared from

HCMV-infected cells treated with ganciclovir (GCV) to

block viral DNA replication has revealed that 36% of the

more than 150 ORFs scored positive for expression were

unaffected by GCV and, therefore, classified as E. These E

genes are dispersed throughout the HCMV genome. Unlike

the genes in the UL region, most US genes show E class

expression properties. However, several E genes, such as

UL4, UL44, UL54, and UL112/113, are also transcribed late

in infection through several mechanisms, including activa-

tion of a promoter different and independent from that

transcriptionally active in the E times, initiation of tran-

scription from a new start site, and alteration of the splicing

pattern as infection proceeds (Fortunato & Spector, 1999;

Mocarski & Courcelle, 2001).

It is believed that transcription of E genes is stimulated by

IE2-86 alone or in cooperation with IE1-72 through trans-

activation of the corresponding promoters in a TATA box-

dependent manner that requires both the host basal transcrip-

tion initiation complex and sequence-specific transcription

factors, such as CREB/ATF and Sp1 (Fortunato & Spector,

1999). Both E and L transcripts may have a polycistro-

nic structure due to the relatively few polyadenylation sig-

nals in the genome that generate families of 30 co-terminal

transcripts. In addition, expression of several E genes

studied in some detail is regulated by both transcriptional

and post-transcriptional mechanisms (Mocarski & Cour-

celle, 2001).

Functional roles in viral DNA replication have been

identified for the UL112/113 family of DNA-binding pro-

teins that contribute to organization of the so-called replica-

tion centers within the nucleus of infected cells for the viral

DNA polymerase encoded by the UL54 ORF and the UL44

gene product that acts as a polymerase processivity factor

(Mocarski & Courcelle, 2001). Several other E proteins,

however, are involved in establishment of immune evasion

in the productively infected cell, such as the glycoproteins

encoded by US2 and US11, which bind the MHC Class I

heavy chains and transport them in a retrograde fashion

from the ER into the cytosol, where they are degraded by

the proteasome (Shamu et al., 1999; Story et al., 1999). The

E-expressed US27 and US28 ORFs have homology to the

CC chemokine receptors (Chee et al., 1990b). However,

US28 alone is a receptor for the CC chemokines RANTES

and monocyte chemoattractant peptide-1. It sequesters them

from the extracellular environment by internalization and,

thus, prevents elimination of HCMV-infected cells by che-

mokine-activated immune cells (Gao & Murphy, 1994;

Bodaghi et al., 1998). In addition, the E gene UL4 encodes

an early structural glycoprotein (gp48). This is a non-


essential component of the viral envelope, since a mutant

virus with disruption of the UL4 gene produces virus

progeny without impaired replication kinetics (Hobom et

al., 2000) (Table 1).

2.3.6. Viral DNA replication

HCMV genome replication, inversion, and packaging

occur in the nucleus of the infected cells. Viral DNA synthesis

begins later than 16 hr p.i. It requires the activities of essential

and specific viral proteins and the active contribution of

several cellular proteins (Mocarski & Courcelle, 2001).

Examination of CMV genome sequences has shown that,

unlike other herpesviruses, they do not encode deoxyribo-

nucleotide biosynthetic enzymes, such as thymidine kinase,

dihydrofolate reductase, thymidylate synthase, and an active

form of ribonucleotide reductase (Chee et al., 1990a; Raw-

linson et al., 1996). Thus, the virus must depend on the host

cell metabolism to ensure a sufficient supply of dNTPs for

its DNA replication. As a result, it does not shut off host

macromolecular synthesis, but stimulates cellular transcrip-

tion and translation. CMV evidently has developed strat-

egies to stimulate the biochemical pathways involved in the

biosynthesis of DNA precursors. Several reports have dem-

onstrated that the early consequences of CMV infection are

similar to those observed in serum-deprived cells exposed to

growth factors (Fortunato et al., 2000). These include cell

cycle functions, such as nuclear translocation of Cdk2;

induction of cyclin E and B; pRb hyperphosphorylation;

activation of E2F-dependent transcription; and activation of

c-myc, c-jun, and c-fos proto-oncogenes (Jault et al., 1995;

Margolis et al., 1995; Bresnahan et al., 1996, 1997; Salvant

et al., 1998). Moreover, there is a substantial increase in the

expression of cellular enzymes involved in nucleotide

metabolism, including thymidine kinase (Estes & Huang,

1977), ornithine decarboxylase (Isom, 1979) and topoiso-

merase II (Benson & Huang, 1990), dihydrofolate reductase

(Wade et al., 1992; Lembo et al., 1999; Song & Stinski,

2002), folylpolyglutamate synthetase (Cavallo et al., 2001),

thymidylate synthase (Gribaudo et al., 2000, 2002; Song &

Stinski, 2002), deoxycytidilate deaminase (Gribaudo et al.,

manuscript in preparation), and ribonucleotide reductase

(Lembo et al., 2000; Song & Stinski, 2002). Despite the

induction of an S phase-like state, CMV-infected cells, as

discussed in Section 2.3.4, fail to undergo cellular DNA

replication and division as a result of blocks in cell

cycle progression that prevent the host DNA replication

machinery from competing with the virus for access to

DNA precursors (Fortunato et al., 2000; Kalejta & Shenk,

2002). The ability of HCMV to stimulate the expression of

cellular enzymes for DNA precursor synthesis is crucial for

its productive replication in the quiescent or terminally

differentiated non-dividing cells it encounters during natural

infection, since their expression is stringently repressed in

these cells.

Six herpesvirus-conserved ORFs in the HCMV genome

provide the core replication proteins for viral DNA replica-

tion. Among them, the single-stranded DNA-binding pro-

tein ppUL57 prevents the reannealing of DNA strands

following unwinding by the helicase-primase complex.

This, in turn, is composed of three subunits encoded by

UL70, UL102, and UL105, the DNA polymerase encoded

by UL54 and the DNA polymerase processivity factor UL44

that prevents dissociation of UL54 from the template

(Griffiths, 2000; Mocarski & Courcelle, 2001). Replication

also requires other viral proteins to maximize DNA replica-

tion, such as UL84, UL112/113, and UL114. UL84 encodes

a 75-kDa phosphoprotein, which stably interacts with IE2-

86, functions as an origin-specific initiator factor, and

stimulates the viral origin (oriLyt)-dependent DNA syn-

thesis (Sarisky & Hayward, 1996). The phosphoproteins

encoded by the UL112/113 region regulate the establishment

of the so-called replication centers corresponding to sub-

nuclear sites of HCMV DNA synthesis. UL112/113 localize

to small intranuclear globular sites representing the early

precursors of the replication centers and recruit the core

replication proteins and enzymes (Penfold & Mocarski,

1997; Ahn et al., 1999). Finally, the protein encoded by

UL114 expresses a functional uracil DNA glycosylase

activity that appears to be required for efficient viral DNA

replication in post-mitotic cells, since a mutant virus with a

substitution mutation in UL114 showed a defect in transition

to high-level, late-phase DNA replication (Courcelle et al.,

2001). IE proteins, such as the transactivators encoded by

the ie1/ie2 and TRS1/IRS1 genes and those expressed by the

UL36-38 region, are also required for transient complemen-

tation of oriLyt-dependent DNA synthesis, although their

roles in this process are not known. HCMV DNA replica-

tion proceeds through initial circularization of the input

genome within 4 hr p.i., followed by DNA synthesis via a

bidirectional q mechanism from a single origin (oriLyt) of

replication that undergoes a switch to a late-phase rolling

circle form of DNA replication (Mocarski & Courcelle,

2001). The latter is responsible for most of the viral DNA

produced during the late stages of infection in the form of

large concatemeric replicating units lacking terminal frag-

ments that are subsequently cleaved into lengths that can be

encapsulated. The oriLyt of HCMV maps within the UL

region close to UL57 (Masse et al., 1992). It spans � 2000

bp, containing repeated nucleotide sequences, as well as

transcription factor-binding sites, and encodes a number of

short transcripts. The integrity of this region is needed for

efficient replication, since mutations targeting different

repeated sequence elements dramatically reduce the ini-

tiation of viral DNA synthesis (Mocarski & Courcelle,

2001).

During the late stages of viral DNA replication, newly

synthesized genomes mature through their inversion, cleav-

age, and packaging (McVoy & Adler, 1994). Inversion

occurs in concatemeric units and leads to the generation

of progeny genomes as a pool of four isomers that only

differ in the orientation of their L and S components (Fig.

1). The mechanism leading to inversion is thought to require


a recombination that needs the repeated a sequences at the

termini and the UL/US junction. Packaging of the genome

into preformed B capsids then follows its cleavage at the

essential cleavage/packaging signals pac1 and pac2. These

sequences are highly conserved among the herpesvirus

genomes and are contained within a 220-bp element located

in the S component of the HCMV DNA near the repeated a

sequence (Mocarski & Courcelle, 2001).

2.3.7. Characteristics and functions of the late proteins

The L proteins are the last class of gene products

expressed during HCMV replication (Table 1). Their tran-

scription begins more than 24 hr p.i. and requires prior viral

DNA replication (Fortunato & Spector, 1999). Late or

g gene expression leads to the synthesis of two subclasses

of L proteins (g1 and g2) in accordance with their time of

expression and sensitivity to viral DNA replication inhib-

itors. g1 (leaky late) transcription occurs 24–36 hr p.i., and

is reduced by such inhibitors. g2 (true late) transcription

occurs 24–48 hr p.i., and is strictly dependent on DNA

replication. The L proteins have mainly structural roles and

primarily contribute to the assembly and morphogenesis of

the virion (Mocarski & Courcelle, 2001).

Most HCMV genes belong to the L class. Examination of

the expression profiles of DNA microarrays from the whole

HCMV genome has shown that at 72 hr p.i., in the absence

of a viral DNA synthesis inhibitor, 26% and 32% of the

transcriptionally active genes are g1 and g2, respectively

(Chambers et al., 1999).

Little is known about the transcriptional regulation of L

genes and the requirement of viral and/or cellular factors for

their expression. Of the L genes studied in detail, the g1

UL83 gene promoter is transactivated by the combination of

IE2-86 and IE1-72, whereas deletion analysis of the g2

UL94 and UL99 promoters revealed that they require a

minimal promoter element containing little more then the

TATA box for full gene activation in late infection (For-

tunato & Spector, 1999; Mocarski & Courcelle, 2001).

2.3.8. Virion assembly, maturation, and egress

Formation of HCMV capsids and packaging of viral

DNA occur in the nucleus. Subsequently, nucleocapsids

acquire a primary envelopment by budding at the nuclear

membrane, and further mature through a de-envelopment/re-

envelopment process in the cytoplasm before leaving the

cell via an exocytotic-like pathway (Mocarski & Courcelle,

2001; Mettenleiter, 2002).

Nucleocapsid particles accumulate in inclusions that

confer the typical ‘‘owl’s eye’’ appearance of the infected

cell nucleus. The initial step is the interaction of pUL86 with

the scaffolding AP pUL80.5 (the AP precursor) in the

cytoplasm and subsequent translocation into the nucleus

(Gibson, 1996), where oligomerization of these complexes

catalyzed by AP leads to the formation of hexons and

pentons that interact with pUL85-pUL46 complexes to form

the B capsid precursor shell. The subsequent association of

capsid intermediates with pUL48.5 completes the formation

of B capsids that are now ready to package viral DNA and,

after insertion of viral genomes, remove AP (Butcher et al.,

1998). During capsid formation, a series of proteolytic

cleavages catalyzed by the assemblin protein (pUL80a)

leads to maturation of the UL80 precursor to assemblin/

APs and the dissociation of UL86 from AP (Gibson, 1996).

Capsids are initially enwrapped through budding at the

nuclear membrane, where they acquire a primary envelope

derived from its inner leaflet (Gibson, 1996). They then

cross the lumen, fuse with the outer leaflet of the nuclear

membrane or the ER membrane with which it is contiguous,

lose their primary envelope, and move into the cytoplasm.

Here, HCMV virion particles further mature by acquiring

their tegument. The tegumented capsids then receive their

definitive envelope by budding into vesicles of the Golgi

apparatus (Sanchez et al., 2000). Both tegumentation and re-

envelopment are driven by multiple specific protein-protein

interactions to secure the integrity of the viral particle

(Mettenleiter, 2002). These mature particles are retained

within the vesicles and transported to the cell surface via

the Golgi network, which is enlarged due to the accumula-

tion of nucleocapsids and DB. The Golgi alterations during

the late replication stages create inclusions around the

nucleus that result in its characteristic kidney-like appear-

ance (Pass, 2001). Progeny virus accumulates in the cyto-

plasm, and infectious virus is released into the extracellular

compartment beginning at 72 hr p.i. In the very late stages,

however, a substantial number of viral particles are still

associated with the cell.

3. Epidemiology of human cytomegalovirus infection

HCMV is one of the most successful parasites. It is found

in both the developed industrial societies and in isolated

aboriginal groups (Griffiths, 2000; Pass, 2001). Following

infection, it is excreted in body fluids (urine, saliva, tears,

semen, milk, and cervical secretions) for months to years.

Infection is usually mild and subclinical. The unsuspecting

host is thus able to spread the virus both vertically and

horizontally. Virus can appear following primary infection,

reinfection, or reactivation. About 10% of infants are

infected by the age of 6 months, following transmission

from their mothers via the placenta, during delivery, and by

breast feeding (Pass, 1985; Stagno et al., 1986). Trans-

placental infection can occur both in women infected for the

first time during pregnancy and those infected long before

conception (recurrent infection). Primary infection during

pregnancy as a source of fetal infection was suggested by

the observation that transplacental transmission ranges from

20% to 40% when primary infection varies from 0.7% to

4.1% (Pass & Boppana, 1999). In the case of recurrent

infections, high rates of congenital infections are observed

in populations with higher rates of maternal seropositivity,

suggesting that most congenital infections are caused by


reactivation of latent virus (Stagno et al., 1973; Yeager &

Martin, 1977). Stagno et al. (1977, 1982), for example,

found an incidence of � 2% of congenital infections among

births from mothers seropositive long before conception (as

documented by virus shedding and serum abs).

Infection during delivery is the outcome of shedding

from the vagina or cervix, followed by ingestion of infected

secretions by the offspring. Shedding close to the time of

delivery has been noted in from 2% to 28% of the mothers

(Reynolds et al., 1973). About 50% of the newborns may be

infected in this way, and they usually begin to shed the virus

themselves when they are 6 weeks old.

Breast-feeding is the most common route of transmis-

sion. Transmission is a function of its duration and the

relatively low virus load in the milk (Dworsky et al., 1983).

It has been shown, for example, that infants nursed for < 1

month do not become infected compared with almost 40%

of those nursed longer. Moreover, 69% of infants are

infected when the virus can be isolated from the milk,

whereas only 10% are infected when the mother is sero-

positive, but negative for virus in the milk. Polymerase

chain reaction (PCR) studies have also demonstrated a

strong relationship between the presence of viral DNA in

milk and transmission to the infant (Vochem et al., 1998).

The precise route of infection throughout childhood is not

known, although close contact is required. Infection increases

when people live in crowded, unhygienic conditions, and

hence is most common in socially disadvantaged countries.

Most children from populations with low socioeconomic

backgrounds, in fact, are infected at the onset of puberty,

whereas < 40% of adolescents from the industrial countries

are infected, followed by an increase of � 1% per year

(Griffiths, 2000; Pass, 2001). Infection is life long. Latent

CMV may reactivate and produce infectious virions that are

shed in the saliva, urine, and other body fluids. Reactivation

is usually asymptomatic, and enables the virus to spread

horizontally and vertically. The incidence of congenital

infection is highest in poor communities, since most women

are infected before puberty. Reactivation is thus a more

frequent cause of congenital infection than primary maternal

infection, although the latter presents a greater risk to the fetus

than recurrent infection. An epidemiological distinction is

often drawn between reinfection and reactivation as opposed

to latent infection. In clinical practice, however, the term

‘‘recurrent’’ is used to indicate both possibilities.

4. Infection routes

Primary infection does not usually result in a clinical

illness, and cannot be identified in pregnant women. Its

status as a risk factor for fetus infection is thus uncertain.

Intrauterine infection occurs in only one-third of pregnant

women with primary infection. The ways in which the fetus

escapes infection are unknown, although macrophages in the

placenta may constitute a barrier (Burton & Watson, 1997).

Ingestion of infected maternal in genital secretions or

breast milk is the main perinatal route of infection. ‘‘Copi-

ous’’ quantities of genital secretions containing high virus

titers as a result of recurrent maternal infection surround the

fetus during delivery, and upon ingestion, may result in

virus transmission to the newborn (Stagno et al., 1982).

Virus titers are usually low in breast milk, but long-term

feeding results in the build-up of an effective inoculum.

Newborns from women with infected breast milk who fed

with formula milk are not infected. Once ingested, the virus

infects the mucosa of the oropharynx, esophagus, or the

upper airways (Stagno et al., 1982).

The absence of symptoms following postnatal infection

makes it impossible to determine the transmission routes.

Molecular analysis of CMV isolates has greatly increased

our understanding of CMV epidemiology. PCR amplifica-

tion of selected segments of the genome and hybridization

of the variable junction region of the CMV genome require

only small amounts of virus for diagnosis and allow faster

sequencing of viral DNA. Chou and Dennison (1991)

compared envelope glycoprotein gene sequences and found

that clinical and laboratory strains can be grouped into

four gB genotypes, although epidemiologic and clinical

significance of the gB genotype distribution in immuno-

compromised patients remains unclear.

Primary infection with HCMV via blood transfusions was

noted in the mid-1960s, although epidemiologic studies have

demonstrated that this is an uncommon route. Attempts to

culture HCMV from fresh donor blood have rarely been

successful. It is thus assumed that the virus is latent in the

blood cells of healthy donors and is reactivated following

transfusion when they encounter an allogeneic stimulus. The

nature of the leukocytes carrying latent virus is unknown,

although attention is being increasingly focused on the

monocytes/macrophages (Plachter et al., 1996; Soderberg-

Naucler et al., 1997, 2001).

HCMV is a significant post-allograft pathogen. Several

studies have shown that seronegative recipients of an organ

from a seropositive donor are at risk of acquiring a primary

infection and that � 60%–80% develop a more severe

disease than seropositive recipients of seropositive organs,

suggesting that acquired immunity modulates infection

(Peterson et al., 1980; Pollard, 1988). The contribution

of immunity to resistance to HCMV disease, however,

decreases as the level of immunosuppression increases.

Variables that contribute to post-transplant infection include

donor and recipient serological status, type of immunosup-

pression, source of allograft, HLA matching of donor and

recipient, and type and amount of blood products used

(Rubin et al., 1985; Miller et al., 1991). Since the main

determinant of transmission via blood is the presence of

leukocytes, reduction of the cell contamination of blood

products has dramatically decreased the incidence of trans-

fusion-acquired infection in allograft recipients (Miller et al.,

1991). The effects of infection extend well beyond the

immediate post-transplant period. It appears that HCMV


contributes to graft loss independently of graft rejection.

Following kidney transplantation, a histologically distinct

glomerulopathy may result from infection of the graft and

may significantly alter its survival (Richardson et al., 1981;

Herrera et al., 1986). Following liver transplantation, a

distinct bile duct sclerosis syndrome has been related to

active HCMV infection. HCMV may perhaps increase the

expression of MHC antigens in the graft and may favor its

rejection (Donaldson et al., 1987; Vierling & Fennel, 1985),

although the way in which this could take place has not been

defined. The role of HCMV infection in rejection is most

convincing in the case of cardiac allografts. Accelerated

development of coronary atherosclerosis associated with

post-transplant HCMV infection has been reported and

found to be independent of reports of age, lipid levels, and

immunosuppression regimen (Loebe et al., 1990). Interac-

tions between HCMV IE proteins and p53 in vessel smooth

muscle cells have also been noted, and the virus may thus

contribute to the formation of atherosclerotic plaques in the

heart vessels (Streblow et al., 2001).

The incidence of HCMV infection following allogenic

bone marrow transplant (BMT) ranges from 32% to 70%,

with an average of 50%, regardless of the prior serological

status of the recipient and donor. Typing of strains has

shown that infection is due to a virus derived from the

recipient (Ruutu et al., 1990; Rubie et al., 1993; Winston et

al., 1993). Its incidence in seronegative recipients receiving

a seropositive marrow is lower than in seropositive recipi-

ents receiving a seropositive marrow, suggesting a transfer

of adoptive immunity from donor to recipient (Griffiths,

2000). Thus, the most critical event is reactivation of a latent

virus in seropositive BMT recipients, whereas in seroneg-

ative recipients, transmission mostly occurs through the

larger quantities of blood products that they receive com-

pared with solid organ transplant recipients. Increasing

evidence indicates that apart from the serological status of

donor and recipient, administration of blood products carries

a significant risk of exposure to HCMV infection following

BMT.

Another major risk factor is the immunosuppressive

regimen. Reactivation of infection ranges between 20%

with cyclosporine and 60% with OKT3 administration.

Hematologic, hepatic, and gastrointestinal abnormalities

are relatively common. Uncertainty, however, persists with

regard to both the role of virus replication in the onset of

graft versus host disease and the toxicity of the pretransplant

therapy. The most significant HCMV-associated syndrome

in BMT is viral pneumonia, with an incidence ranging

between 10 and 15%. Its pathogenesis is unknown, although

several mechanisms, such as uncontrolled replication lead-

ing to organ damage and respiratory failure or immunologic

destruction of the lungs triggered by HCMV, have been

suggested.

HCMV interaction during the induction and progression

of HIV infection has not been definitively established. Even

so, HCMV is a leading opportunistic agent in acquired

immunodeficiency syndrome (AIDS). Autopsies have

shown that 90% of patients develop active HCMV infection

and 40% display sight- and/or life-threatening diseases. The

increased survival of HIV-infected individuals following

more effective prophylaxis and treatment of bacterial and

protozoal infections, accompanied by virtual inactivity of

their immune system, has augmented the importance of

HCMV infection in these patients. Invasive HCMV infec-

tion, in fact, increases rapidly as CD4+ lymphocyte counts

fall (Gallant et al., 1992; Webster et al., 1992; Leach et al.,

1993). Three major organ systems are clinically affected: the

CNS, the lungs, and the gastrointestinal tract. Retinitis is the

most frequent CNS infection directly attributable to HCMV

replication, and the most sight-threatening (Vinters et al.,

1989; Sison et al., 1991; Pertel et al., 1992; Faber et al.,

1992). Autoptic immunohistochemical analysis has shown

HCMV presence in all layers of the retina, without under-

lying choroidal involvement. Absence of virus in the endo-

thelium of the retinal microvasculature indicates that its

hematogenous spread does not follow replication in these

cells, but that perhaps the virus breaks through the capillary

endothelium.

5. Pathogenesis and pathology

Histopathologic and immunohistochemical examination

of necropsy tissues has indicated that the virus initially

enters via the epithelium of the upper alimentary, respiratory,

or genitourinary tracts. However, since infection is readily

established by transfusion and transplantation, initial infec-

tion of epithelial cells does not seem essential (Sinzger &

Jahn, 1996). Cytotrophoblasts form a barrier between the

maternal and fetal circulation, but readily allow HCMV

replication in vitro, suggesting that the fetus is infected

hematogenously (Halwachs-Baumann et al., 1998; Hem-

mings et al., 1998). Leukocytes and vascular endothelial

cells aid the spread of HCMV. During acute infection,

HCMV can be isolated from buffy coat preparations of

leukocytes, but not from the plasma, and is presumably

carried by one or more cell subsets. Viral antigens are found

mainly in neutrophils and monocytes in acute infection

(Sinzger & Jahn, 1996) and transmission through blood

products from seropositive donors can be prevented by

removal of leukocytes (Pass, 2001). Moreover, immunohis-

tochemical double-labeling has shown that macrophages

infiltrating organ tissues support virus replication, whereas

granulocytes show no signs of viral late gene expression

(Sinzger & Jahn, 1996). Lastly, virus encoded chemokines

may facilitate dissemination of CMV from the initial

replication site by attracting neutrophils and monocytes

(Penfold et al., 1999). Saederup et al. (1999), in fact, have

demonstrated that mouse CMV-encoded b-chemokine pro-

motes monocyte-associated viremia, whereas viruses with

deletions in the gene for this b-chemokine have significantly

reduced levels of viremia. Endothelial cells detached from


the basal membrane as a result of HCMV infection are also

involved in hematogenous spreading of the virus. However,

the molecular mechanisms responsible for the interaction

between these cell populations and the microvascular sys-

tem, and subsequent transmission of the virus to the target

organs, have not been determined.

Hematogenous spreading is typically followed by infec-

tion of ductal epithelial cells. Fibroblasts are rarely in-

volved, despite the fact that the virus can replicate in such

cells in vitro. CMV is recognized histologically by its

characteristic ‘‘owl’s eye’’ intranuclear inclusions with a

surrounding halo and marginated chromatin. Typical cyto-

megalic cells are found in the salivary gland, bile duct,

bronchial and renal tubular epithelium, islet cells, epithelial

cells of the inner ear, the capillary endothelium, astrocytes,

and neurons (Griffiths, 2000; Pass, 2001). In cases of severe

disseminated disease, evidence of HCMV involvement can

be found in all organs. The present review, however, will be

confined to those most commonly involved (for further

details, see Ho, 1991 and Becroft, 1981). Salivary gland

involvement is probably chronic and probably the outcome

of subclinical congenital and perinatal infection, since it is

more frequent in infants and young children and diminishes

with age. In contrast, viruria is a constant feature in all age

groups as the result of replication in the genitourinary tract

(Plachter et al., 1996). In the kidneys, infected cells are

found in the proximal tubules, Henle’s loop, and the

collecting tubules, but seldom in the glomeruli. As already

mentioned in Section 4, infection is directly involved in

renal transplant dysfunction and rejection, whereas it rarely

leads to dysfunction in normal individuals. Elevated liver

enzyme levels indicative of subclinical hepatitis are fre-

quently associated with HCMV infection in immunocompe-

tent individuals. Cytomegalic cells are usually found in the

bile duct epithelium, less frequently in the capillary endo-

thelium, and rarely in the parenchyma. Together with

cytomegalic cells, areas of inflammatory infiltrates are a

dominant feature in histological sections, possibly suggest-

ing that liver dysfunction might result directly from virus

cytopathogenicity or indirectly from the inflammatory reac-

tion to HCMV infection (Phillips et al., 1977).

HCMV infection in HIV patients often affects other parts,

or even the entire digestive system (Rene et al., 1988). The

colon is the most common replication site, followed by the

esophagus, rectum, and small intestine. The direct effects of

infection range from punctate, superficial ulcers to deep

ulcerations, with extensive necrotizing involvement and gut

perforation.

HCMV pneumonia is infrequent in immunocompetent

individuals, but often is severe in immunosuppressed

patients, especially BMT and heart-lung recipients. Infected

cells are mainly observed in the alveolar and bronchial

epithelium, along with inflammatory infiltrates (Apperley

& Goldman, 1988; Wreghitt et al., 1988). The range of

clinical presentation, from severe pulmonary dysfunction

and limited focal infection, together with similar findings in

animal models, suggests that dysfunction may be due to

virus-associated dysregulation of the immune response.

CNS damage is a frequent feature of congenital infec-

tion (Fowler et al., 1992). Symptoms include mental

retardation, seizures, hypotonia, and hearing loss due to

various alterations in the form of many small, necrotic foci

surrounded by an inflammatory infiltrate. In histological

sections, inclusion-bearing cells positive for viral antigens

have been found in neurons, glia, ependyma, choroid

plexus, meninges, and vascular endothelium. These cells

are also widespread in the semicircular canals, vestibular

membrane, and cochleae. CNS involvement was once

regarded as infrequent, except in congenital infection.

Today, however, CNS tissues are the major target organs

in AIDS patients, where HCMV replicates efficiently in

various brain structures and in specialized organs, such as

the retina and cochlea. Histopathologic alterations include

microglial nodules, focal parenchymal necrosis, necrotizing

ventriculitis, hemorrhages, astrocytic proliferation, and

perivascular inflammation. Acute retinal infection fre-

quently leads to blindness, probably as the outcome of

direct lysis of the retinal pigment epithelial cells during

virus replication.

In conclusion, organ dysfunction seems to coincide with

HCMV replication, although the frequent association of an

inflammatory reaction indicates that other pathogenetic

mechanisms are also operative.

6. Host defences

HCMV infections are kept under control by the immune

system. Total HCMV clearance, however, is rarely achieved,

and the viral genome remains at selected sites in a latent

state (Reddehase et al., 1994). Initiation of productive

replication results in transient virus shedding and recrudes-

cent disease (Mocarski, 1996). Since severe infection is

usually restricted to individuals with impaired cell-mediated

immunity, it is evident that this arm of the immune response

provides the most protection. Even so, the supportive role of

the humoral system in keeping CMV loads below critical

thresholds must not be overlooked.

6.1. Cell-mediated immunity

Mouse models have been employed to determine the role

of specific components of the immune response and their

recognition of viral proteins (Koszinowski et al., 1990).

Early studies demonstrated that a CMV-specific cytotoxic T

lymphocyte (CTL) response was required for recovery from

CMV infection. Suppression of CTLs caused reactivation

and dissemination of natural infection. Further studies

showed that both natural killer (NK) cells and CD8+

cytotoxic T-cells are of primary importance in the preven-

tion of recurrence (Jonjic et al., 1990; Polic et al., 1996;

Hengel et al., 1998). Adoptive transfer of CD8+ cells


protected mice from lethal challenge independent of CD4+

T helper cells. CD8+ T-cell clones specific for both struc-

tural and nonstructural proteins were identified. However,

CD8+ depletion with abs or in mouse mutants deficient in

MHC Class I expression revealed additional antiviral activ-

ities mediated by CD4+-derived cytokines not seen when

CD8+ cells are present. Lastly, experiments with mice

deficient for perforin and granzyme- or Fas-mediated cyto-

toxicity have shown that replication is controlled by NK and

CD8+ cells via the perforin and granzyme-mediated path-

way, whereas the Fas-Fas ligand system is not critical (Riera

et al., 2000, 2001).

As already mentioned in Section 4, since infection (apart

from congenital forms) is most severe in patients with

dramatically impaired cell-mediated immunity, such as

BMT recipients, and those with AIDS, it is evident that this

arm of the immune response provides the most protection

(Pass et al., 1983; Zanghellini et al., 1999). Its investigation

in studies of lymphocyte proliferation in response to CMV

antigens has shown that in most seropositive immunocom-

petent adults, this usually takes the form of CD4+ cell

proliferation in response to envelope glycoproteins gB and

gH, the lower matrix protein pp65, and IE proteins, in

addition to other proteins (Liu et al., 1993; Davignon et

al., 1995; He et al., 1995; Beninga et al., 1995). The function

of proliferating CD4+ cells in resistance to HCMV infection

is not clear. Studies in mice point to several antiviral effector

functions, such as the production of cytokines and CD4+-

mediated cytotoxicity. The virus-encoded proteins that are

targets of CTLs include a collection of structural and non-

structural forms (Boppana & Britt, 1996; Kern et al., 1999).

Comparison of CTL precursor frequencies for envelope gB

or the 72-kDa IE1 protein revealed a low frequency of gB-

specific CTLs ( < 6%), whereas 18–58% of CTLs recog-

nized IE1-expressing target cells. The high frequencies for

pp65 and IE1 proteins found in subsequent studies show that

they are the chief targets of the CTL-mediated immune

response (Pass, 2001).

Although a direct role for virus-specific CTL responses

in the resolution of infection has yet to be defined, in vivo

evidence of the control of HCMV infection by CTLs has

been obtained through the adoptive transfer of virus-spe-

cific clones. Passive administration of autologous HCMV-

specific CD8+ CTLs at set intervals after BMT to sero-

negative recipients of marrow from seropositive donors

generated a vigorous CMV-specific CTL response without

onset of viremia or CMV disease (Walter et al., 1995).

These studies provided the first evidence for the role of

CTLs in controlling human CMV infection, and opened

new therapeutic perspectives for CMV treatment in organ

transplantation.

6.2. Humoral immunity

During primary infection, immunocompetent individuals

produce anti-HCMV immunoglobulin (Ig)M class abs that

persist for 3–4 months, followed a few weeks later by IgG

class abs that persist for life. Experimental and clinical

findings show that the humoral response is beneficial.

Mice immunized against murine CMV gB were protected

against a lethal challenge (Rapp et al., 1992), and immun-

ization of pregnant guinea pigs against guinea pig CMV

envelope glycoprotein protected their fetuses in the same

way (Harrison et al., 1995). Intrauterine infection is less

severe when transmitted by means of recurrent rather than

primary maternal infection (Griffiths, 2000). During prim-

ary infection, women who transmit the virus in utero have

high levels of total IgG characterized by low avidity and

neutralizing activity. Correlation of an enhanced ab

response with a poor fetal prognosis suggests that fetal

ab is responsible for immunopathology. Primary infection

is more frequent and more severe when the renal transplant

recipient is seronegative and the donor seropositive. Alle-

viation of this severity by preimmunization with the

attenuated Towne strain (Plotkin et al., 1984) or adminis-

tration of high-titer anti-CMV Igs (Snydman et al., 1987)

also shows that humoral immunity is beneficial. Many

CMV proteins are recognized by the humoral immune

system (Landini & Michelson, 1988; Landini et al.,

1988). The envelope glycoproteins (mainly gB and gH)

are the targets of virus-neutralizing abs in both human and

mouse models (Pass, 2001). The predominance of gB as a

target is best explained by its dominant immunogenicity

and abundance compared with other components of the

envelope. Anti-gH abs have a potent, complement-inde-

pendent, but minor neutralizing activity. Viral tegument

components, including pp28 (UL99), pp65 (UL83), and

pp150 (UL32), trigger an intense and long-lasting ab

response that provides an indirect measure of viral

replication and correlates with the clinical outcome. How-

ever, these abs are unable to react with the surface of virions

and infected cells, and are of limited importance in a

protective response.

6.3. Immune evasion by human cytomegalovirus

A characteristic feature of infection in the normal host

is persistence of the viral genome in a nonproductive form

at specific anatomical sites for months or even years

(Hengel et al., 1998). This ability to avoid elimination

by the immune system is the result of (1) induction of a

latent state of infection, (2) exploitation of immunologi-

cally privileged tissues for replication (i.e., epithelial cells

of the salivary glands expressing an insufficient number of

MHC Class I molecules to trigger clearance by CD8+ cell,

and (3) expression of genes that interfere with the immune

response (Mocarski, 2002). Table 2 summarizes the main

mechanisms exploited by HCMV to evade the immune

response. Escape from CD8+ cells is mediated by several

mechanisms, all of which block the expression of MHC

Class I molecules complexed with a potentially important

CTL target, the 72-kDa IE1 phosphoprotein, or which

Table 2

HCMV genes associated with immune evasion

Immune function Virus genes Mechanisms

NK cell activity UL 40 Up-regulation of HLA expression:

binding of inhibitory NK cell receptors

MHC Class I

antigen

US 2 Export of MHC Class I heavy

chains from the ER VIA Sec61

presentation/

processing

US 3 Retention of MHC Class I

complexes in the ER

US 6 Inhibition of TAP-mediated peptide

translocation into the ER

US 11 Dislocation of MHC Class I

heavy chains into cytosol

UL 83 Inhibition of 72-kDa IE1 antigen

presentation to CD+ T-cells

MHC Class II

antigen

presentation

US 2 Degradation of MHC Class II

antigen by down-regulating

antigen presentation

to CD4+ T-cells

Modulation of US 27/28 b-Chemokine-binding proteins

chemokine UL 78 Putative GPCR homologue

activity UL 144 Putative transferrin receptor

homologue


degrade MHC Class II molecule proteins, and, thus,

prevent presentation of viral antigen to CD4+ lympho-

cytes. HCMV deletion mutants have been used to map two

gene regions whose products are associated with the down-

regulation of MHC Class I complex formation; namely,

US11 and that spanning US2 and US5 (Machold et al.,

1997). Both US2- and US11-encoded proteins lead to the

accumulation of MHC Class I heavy chains in the cytosol

of infected cells, where they are degraded by the proteo-

some, thus shortening their half-life and preventing their

expression on the cell surface. Binding of peptides to

MHC Class I molecules depends on transport across the

ER membrane by a specific transporter complex desig-

nated TAP 1/2 (transporter associated with antigen pro-

cessing) (Sadasivan et al., 1996). Only after their

introduction into the ER can peptides form heterodimers

with MHC Class I molecules and b2-microglobulin and be

recognized by CD8+ CTLs. Despite a significant increase

in the expression of TAP molecules in infected cells, the

HCMV glycoprotein encoded by the US6 gene inactivates

the TAP system by interfering with formation of the TAP1-

TAP2-MHC Class I-b2-microglobulin complex. The find-

ing that lysis of HCMV-infected fibroblasts by IE-specific

CTL is usually low, but increases in fibroblasts infected

with a mutant HCMV lacking the UL83 encoding the

matrix pp65, suggests that phosphorylation of the 72-kDa

IE1 protein by pp65 interferes with its antigenic processing

and presentation to CD8+ cells (Wiertz et al., 1996; Gilbert

et al., 1996; Jones & Sun, 1997).

Experiments have demonstrated that NK cells are

involved in the control of CMV infection. Beige mice

(genetically deficient in functional NK cells) are more

susceptible to murine CMV (Shellam et al., 1981). In

humans, MHC Class I HLA–E molecules protect target

cells from NK cells by binding to inhibitory receptors on

their surface. A convincing mechanism for escape from NK

cell attack stems from the observation that the HCMV UL40

gene encodes for a glycoprotein containing a nine amino

acid sequence homologous to the MHC Class I sequence

that up-regulates the expression of Class I HLA-E molecules

(Tomasec et al., 2000).

Lastly, mechanisms interfering with chemokine-driven

inflammation are exploited by HCMV to evade the immune

response. HCMV-infected cells express viral G-protein-

coupled receptor (GPCR) homologues encoded by US27,

US28, UL33, and UL78, which are needed for virus

replication in vitro (Chee et al., 1990b; Bodaghi et al.,

1998). The US28-encoded GPCR binds to and sequesters

several b-chemokines and, thus, enables the virus to evade

the immune response.

6.4. Persistence and release from the host

Cells in bone marrow and peripheral blood are the chief

reservoirs for latent CMV infection. CMV DNA is found in

a small percentage of peripheral blood monocytes, and

gene expression is limited to the E genes. These findings

and the demonstration that tissue macrophages differenti-

ated from circulating monocytes also express E and L

genes have led to the formulation of the following model:

bone marrow precursors of blood monocytes are the site of

CMV latency and provide a means of dissemination upon

differentiation into circulating monocytes (Pass, 2001).

Differentiation of latently infected monocytes into macro-

phages leads to reactivation and productive infection.

Support for this model is provided by the reactivation of

CMV by allogeneic stimulation in vitro of peripheral blood

mononuclear cells from seropositive healthy donors (Soder-

berg-Naucler et al., 1997). Moreover, it has been found that

a small percentage (close to 0.01%) of CD33+/CD14+ or

CD33+/CD15+ bone marrow mononuclear cells from sero-

positive human donors express transcripts associated with

latency. In contrast, T lymphocytes, B lymphocytes, and

CD33- mature granulocytes and macrophages are negative

(Hahn et al., 1998).

Recurrent infection can be defined as indefinite, but

intermittent, excretion of the virus from single or multiple

sites. It should be distinguished from the prolonged excre-

tion typical of primary infection and also infection in the

immunocompromised host (Pass, 2001), where productive

infection, as measured by viral excretion, is markedly

increased. In allograft recipients and AIDS patients, the

incidence of virus excretion from multiple sites approaches

100%.

During primary, recurrent, or persistent infection, CMV

is shed in multiple body fluids, such as saliva, urine, tears,

cervicovaginal fluid, breast milk, and semen, probably due

to virus replication in glandular epithelial cells, accompan-

ied by virus release into excretions.

Table 3

Clinical syndromes associated with HCMV infection in the immunocom-

promised patient

Syndromes AIDS Solid transplant BMT

Esophagitis + + +

Gastritis + + +

Enterocolitis + + +

Hepatitis + + + +

Pancreatitis � + + �Pneumonitis � + ++

Retinitis + + + +

Encephalopathy + � �Polyradiculopathy + � �


7. Clinical features associated

with human cytomegalovirus infection

7.1. Infection in normal hosts

HCMV infection in normal immunocompetent hosts is

generally subclinical, but nonetheless, is thought to account

for 8% of all the cases of mononucleosis (Nesmith & Pass,

1995). Clinical manifestations of mononucleosis due to

HCMV are very similar to that induced by the more

common Epstein-Barr virus. Persistent fever, myalgia, head-

ache, cervical lymphadenopathy, splenomegaly, and non-

specific constitutional symptoms are common, and may

persist for weeks. Rash is also present in � 30% of patients.

Uncommon complications include pneumonia, myocarditis,

hemolytic anemia, retinitis, gastrointestinal ulceration, hep-

atitis, CNS involvement (Guillain-Barre syndrome), and

peripheral neuropathy. Common laboratory findings include

a negative heterophil ab response (Paul-Bunnel test), atyp-

ical lymphocytosis, and elevated liver aminotransferases.

Slight elevation of serum bilirubin and jaundice are occa-

sional findings.

7.2. Congenital infection

From 5% to 10% of congenitally infected newborns

display cytomegalic inclusion disease, whose symptoms

include intrauterine growth retardation, jaundice, hepatos-

plenomegaly, thrombocytopenia, petechiae, chorioretinitis,

and hepatitis, along with CNS involvement in the form of

microcephaly, encephalitis, seizures, and focal neurological

signs. Most of the non-CNS (liver and blood-forming

organs) manifestations are self-limiting and resolve without

therapy in the vast majority of cases. In contrast, the neuro-

logical damage is permanent and accounts for the long-term

morbidity and poor prognosis of cytomegalovirus inclusion

disease (Boppana et al., 1992). Long-term prospective

studies indicate that 80% of infants with symptomatic

congenital infection will display serious life-long neuro-

logical abnormalities. In 11–20%, damage caused by the

virus leads to such a severe life-threatening organ dysfunc-

tion that the patient dies during infancy.

Long-term follow-up shows that � 15% of the asympto-

matic infected infants at birth (90–95% are asymptomatic)

may develop hearing defects or impaired intellectual per-

formance. Differences in the reported incidence of hearing

defects are probably attributable to the use of different

evaluation methods.

7.3. Cytomegalovirus infection

in the immunocompromised host

HCMV is a significant opportunistic pathogen in immu-

nocompromised patients. Primary infection, reactivation of

latent virus, and reinfection are possible and are often

clinically silent. The onset of infection is marked by

spiking pyrexia, which may resolve in a few days (Pass,

2001). Its severity is roughly parallel with the level of

immunosuppression, and is greatest in BMT recipients and

AIDS patients with low CD4+ T-cell counts. Solid organ

transplant recipients, patients receiving immunosuppressive

chemotherapy, and subjects with congenital immunodefi-

ciencies may also be symptomatic. Table 3 summarizes the

major clinical diseases related to the type of immunocom-

promised host. Some patients may develop a CMV syn-

drome associated with pneumonitis. This has a poor

prognosis (up to 90% mortality). Retinitis may be present

alone following virus dissemination. Replication of CMV

in the gut may be asymptomatic or associated with

extensive ulceration due to erosion of the neighboring

blood vessels and hemorrhages. Replication in the CNS

of AIDS patients produces some of the symptoms

observed in congenital infection, and is often followed

by encephalopathy and/or peripheral polyradiculopathy. It

is not certain whether CMV infection and disease are

simply markers of the immune dysfunction that follows

HIV replication or whether CMV infection itself promotes

HIV progression, although it is clear that highly active

antiretroviral therapy has reduced HIV-1 viral load, along

with a reduction in CMV viremia and disease (Gerna et al.,

1998a; O’Sullivan et al., 1999). Clinical signs of HCMV

infection in transplant recipients may be absent or severe,

although severe infection is now less frequent as a result of

better prophylaxis. HCMV is initially localized in the

transplanted organ, i.e., hepatitis occurs generally in liver

transplant recipients, pancreatitis in pancreas transplant

recipients, but then spreads throughout the gastrointestinal

tract and to the retina, skin, endometrium, lungs, and CNS

(Rubin, 1994). HCMV disease is more difficult to treat in

BMT compared with solid organ transplant recipients, and

HCMV pneumonia has a high mortality rate, despite the

recent introduction of specific antiviral drugs (Reed et al.,

1988). Lastly, an immunosuppressive syndrome often

related to HCMV infection in the late post-transplant

period is characterized by superinfection with bacteria,

fungi, and protozoa, perhaps due to disturbance of both

the humoral and cellular immune response by HCMV.

Fig. 5. HCMV antigenemia assay. This picture was kindly provided by

Rossana Cavallo.


8. Diagnosis

Virological and molecular detection of HCMV and sero-

logical demonstration of a specific immune response are

used for diagnosis, although acute infection is usually

diagnosed through detection of the virus in body fluids.

8.1. Virus detection

HCMV can be isolated from several body fluids, such as

urine, saliva, vaginal secretions, amniotic fluid, and blood,

following primary or recurrent infections (Gerna et al.,

1990; Revello et al., 1999). Moreover, HCMV-infected cells

can be identified in biopsy samples (e.g., liver, lungs),

owing to ‘‘owl’s eye’’ inclusions or, more efficiently, by

immunohistochemistry, in situ PCR, or nucleic acid hybrid-

ization. Electron microscopy is employed to detect the virus

in the urine from congenitally or perinatally infected infants

since, unlike other human herpesviruses, the HCMV virions

are present in high titers. However, it is generally agreed

that acute HCMV infection is best diagnosed by detection of

the virus in the blood since the level of viremia correlates

with the clinical picture and the prognosis. The most widely

used assays for detection and quantitation of HCMV load in

blood include tests for viremia, antigenemia, DNAemia, and

IE and L mRNAemia.

8.1.1. Viremia

Viremia is measured by an assay that allows the detec-

tion and the quantification of infectious viral particles in the

blood. Conventional methods are the inoculation of low

passage ( < 25) human embryonic lung or foreskin fibro-

blasts with peripheral blood leukocytes (PBL) and iden-

tification of the characteristic focal CPE (Fig. 2). This

usually develops very slowly, and the cultures must be

carefully observed for at least 21 days before being

reported as negative. This expensive and time-consuming

Fig. 4. HCMV viremia assay. This picture was kindly provided by Rossana

Cavallo.

technique has been replaced by the ‘‘shell vials’’ assay,

which retains the specificity and sensitivity of virus isola-

tion, but provides results within 24 hr (Gleaves et al., 1984;

Griffiths et al., 1984). A human fibroblast monolayer is

inoculated with 2� 105 PBL and fixed 18 hr later. The

nuclei of the infected fibroblasts are stained by immuno-

fluorescence using a monoclonal ab reactive with the MIE

protein p72 (Fig. 4). The assay is based on the assumption

that each stained cell was infected by a single PBL carrying

an infectious virus. Several studies indicate that trans-

planted patients with HCMV viremia in the range of 10–

100 infected fibroblasts following inoculation with 2� 105

PBL have a high risk of developing symptomatic HCMV

diseases (Gerna et al., 1995; Grossi et al., 1995) and should

be treated with antiviral drugs (Gerna et al., 1991). More-

over, viremia is a rapid way to monitor the efficacy of an

antiviral treatment since it decreases sharply 1 or 2 days

after the beginning of an effective regimen (Gerna et al.,

1993), whereas persistent or increasing viremia levels

predict the emergence of a drug-resistant HCMV strain

(Gerna et al., 1992a).

8.1.2. Antigenemia

Antigenemia is measured by the quantitation of leuko-

cyte nuclei positive, in an immunofluorescence assay, for

the HCMV lower matrix phosphoprotein pp65 in a cyto-

spin preparation of 2� 105 PBL (van der Bij et al., 1988a,

1988b; Revello et al., 1989; Gerna et al., 1992b) (Fig. 5).

This rapid assay provides a result in a few hours. Anti-

genemia is an indirect marker of disseminated infection,

since pp65 is transferred to uninfected leukocytes by

infected cells after transient fusion of plasma membranes

and exchange of cytoplasmic material (Gerna et al.,

2000b). By comparison with the test for infectious virus,

the test for viral antigen gives a positive result earlier after

the onset of infection and becomes negative later in the

late phase of a systemic infection. Since high levels of


antigen are frequently found in patients with HCMV

disease and low levels correlate with asymptomatic infec-

tions, the test for antigenemia is sensitive enough to

monitor infection and antiviral treatment in immunocom-

promised patients (van den Berg et al., 1989; Grossi et al.,

1995).

8.1.3. DNAemia

HCMV DNA in clinical samples is currently detected

and quantitated by qualitative or quantitative PCR. Qual-

itative PCR detects viral DNA in the amniotic fluid

(Revello et al., 1995) and the aqueous humor of AIDS

patients with HCMV retinits (Gerna et al., 1994a), whereas

quantitative PCR provides more clinically useful informa-

tion concerning the virus load in blood. PCR measurement

of DNA levels produces good prognostic information in

both transplant recipients and HIV-positive individuals

(Kidd et al., 1993; Gerna et al., 1994b, 1998b; Bowen et

al., 1997).

8.1.4. RNAemia

This assay is a good marker for monitoring active

HCMV replication and disease, since it detects mRNAs

transcribed from IE or L genes in PBL or whole blood

(Gozlan et al., 1996; Lam et al., 1998). Reverse transcrip-

tion-PCR was the first technique employed to detect viral

transcripts. Its major drawbacks, namely, instability of the

RNA and the difficulty of discriminating PCR products

from a DNA or RNA template when working with

unspliced viral transcripts, cause both false negatives and

false positives. However, these difficulties have now been

circumvented by nucleic acid sequence-based amplification,

which allows specific amplification of unspliced viral

mRNAs in a DNA background, and is a highly promising

technique for monitoring HCMV infection in transplant

recipients (Blok et al., 1998; Aono et al., 1998; Gerna et

al., 1999, 2000a) and for the early diagnosis of both

pregnant women with primary infection and congenitally

infected newborns (Revello et al., 2001).

8.2. Detection of the immune response

Serological determination of a past or recent HCMV

infection in immunocompetent individuals rests on detection

of virus-specific IgG or IgM abs. Occurrence of a primary

infection is conventionally deduced from seroconversion

from IgG ab negative to IgG ab positive in the interval

between two serological assays. A specific IgM response

may be serologic evidence of a recent primary infection.

Detection of IgM abs, however, is not reliable because false

positives may be induced by rheumatoid factor, antinuclear

abs, and other cross-reactive factors not yet identified.

Moreover, true-positive tests detect IgM persistence in

patients with a past HCMV infection. Interpretation of a

positive IgM assay in pregnant women is assisted by

determination of the avidity of HCMV-specific IgG, since

low-avidity IgG is produced early in infection and high-

avidity IgG is a marker of past or recurrent infection

(Blackburn et al., 1991; Grangeot-Keros et al., 1997;

Bodeus et al., 1998; Lazzarotto et al., 1999). Detection of

anti-HCMV IgG abs is a useful way of documenting a past

infection, and they are also a marker of potential infectivity,

since reactivation of a latent infection may occur. Serologic

assays are thus of great clinical importance in donor

selection, since HCMV can be transmitted by blood dona-

tion and organ transplantation.

9. Prevention of human

cytomegalovirus infection and disease

Steps to prevent infection in groups at risk have been

devised in light of what is known about the transmission

routes for HCMV. In solid organ transplant recipients, for

example, the risk of a primary infection could be reduced by

matching seronegative donors and recipients, although this

approach is hampered by the scarcity of donor organs

(Stratta et al., 1990). Prophylaxis with antiviral agents and

pre-emptive therapy (see Section 10) have proved useful in

transplant patients. Transmission to immunocompromised

individuals, pregnant women, and premature newborns

through donor blood can be avoided by using HCMV-

seronegative, filtered, or leukocyte-deprived blood products.

This method places a greater burden on blood transfusion

centers, but should be used whenever possible, since it

significantly reduces HCMV transmission (Gilbert et al.,

1989; Sayers et al., 1992). Administration of an HCMV

vaccine to secure pre-exposure immunization would be

another way of protecting groups at risk.

9.1. Human cytomegalovirus vaccines

Development of a vaccine to prevent congenital HCMV

infection is a priority concern, since the virus is a leading

cause of CNS damage in children (Pass, 1996). The poten-

tial benefit of vaccine-induced immunity has been estimated

at 40-fold reduction with respect to intrauterine infection

and 25- to 30-fold reduction with respect to decrease in CNS

damage (Britt, 1996). However, no vaccine is available and

live-virus vaccines have not obtained official approval (Elek

& Stern, 1974; Plotkin et al., 1994). Both humoral and cell-

mediated immunity are needed to prevent HCMV disease.

Many studies indicate that gB and pp65 are absolutely

necessary vaccine components to secure the induction of

neutralizing abs and CTL responses (Britt et al., 1990;

Gonczol et al., 1991; Riddell et al., 1991; Marshall et al.,

1992; Wills et al., 1996). Moreover, gH and gN for

neutralizing abs and IE1 and pp150 for CTL responses

could increase the protective immune response (Kern et al.,

1999; Gyulai et al., 2000). There are a variety of options for

the elaboration of a safe and effective vaccine: attenuated

live virus vaccines (Towne strain), recombinant live vac-


cines using chimeric viruses comprising genomic portions

of the virulent strain Toledo and the attenuated strain

Towne, canarypox-HCMV recombinants expressing gB

and pp65, vaccination with HCMV DB, subunit gB vaccine;

pp65 peptide-based vaccine, and DNA vaccines encoding

the gB and pp65 proteins. The attenuated Towne strain, the

recombinant gB, and the canarypox gB and pp65 vaccines

already have been tested in clinical trials.

The attenuated Towne vaccine has been tested both in

healthy volunteers and in transplant recipients (Plotkin et

al., 1975, 1976, 1989, 1991; Adler et al., 1995, 1998). It

induced neutralizing abs and an CTL response and reduced

HCMV disease in seronegative renal transplant recipients,

but did not prevent infection. However, there was no

evidence of its efficacy in a placebo-controlled trial in

seronegative mothers of HCMV-infected children. In con-

clusion, it is immunogenic, but the protection it offers is less

effective than that seen after natural infection. In addition, a

live virus is a potential risk factor in transplant recipients,

and all live attenuated herpesvirus vaccines are overshad-

owed by their possible oncogenicity. To circumvent these

risks, poxviruses with limited potential for replication in

humans (canarypox) have been tested as vehicles for the

expression of recombinant gB and pp65 (Adler et al., 1999;

Berencsi et al., 2001). A subunit vaccine composed of a

modified gB protein was evaluated after combination with a

new powerful adjuvant, MF59, based on an oil-in-water

emulsion of squalene. Results of a trial of 46 seronegative

adults established safety, immunogenicity, and optimal anti-

gen dose (Pass et al., 1999). Whether the neutralizing abs

elicited by this vaccine will be sufficient to prevent recur-

rence or primary HCMV infection is still an open question

(Drulak et al., 2000). Other promising preclinical ap-

proaches include vaccination with HCMV DB (Pepperl et

al., 2000) and administration of DNAvaccine encoding viral

immunogenic proteins to elicit a humoral and a CTL

response (Endresz et al., 1999, 2001).

10. Antiviral treatment

10.1. Currently available drugs

Treatment of HCMV infections is difficult because there

are few options. To date, the nucleoside analog GCV, the

nucleotide analog cidofovir [CDV; (S)-1-(3-hydroxy-2-

phosphonylmethoxypropyl) cytosine], and the pyrophos-

phate analog foscarnet (PFA) have been licensed for serious

or life-threatening HCMV infections in immunocompro-

mised individuals. These drugs have produced clinical

improvement in many patients, but suffer from poor

oral bioavailability, low potency, development of resist-

ance in clinical practice, and dose-limiting toxicities, and

hospitalization is sometimes required.

GCV is a competitive inhibitor of viral DNA polymerase

(UL54), and its antiviral activity requires monophosphor-

ylation in the infected cell by the phosphotransferase

encoded by the UL97 gene of HCMV (Littler et al.,

1992), followed by diphosphorylation by cellular kinases.

It is administered by intravenous infusion, and is usually the

front-line drug for the treatment of HCMV infections. An

oral formulation that is effective for prophylaxis in immu-

nocompromised patients has also been approved (Spector et

al., 1996). However, its side-effects include potentially

dose-limiting neutropenia and the emergence of GCV-

resistant strains due to UL97 and UL54 gene mutations,

mainly in AIDS patients during prolonged maintenance

therapy (Erice et al., 1989; Lurain et al., 1994; Chou et

al., 1995; Baldanti et al., 1995, 1998), and less frequently in

solid organ transplant recipients (Limaye et al., 2000, 2002).

PFA is a noncompetitive inhibitor of the pyrophosphate-

binding site of HCMV DNA polymerase, which does not

require prior activation by a virally encoded enzyme. It is

administered intravenously as an alternative to GCV in the

event of GCV resistance or severe side-effects, although its

application is limited by nephrotoxicity and disturbance of

the electrolyte balance (Vogel et al., 1998; Chrisp &

Clissold, 1991). Long-term exposure to PFA may lead to

the emergence of resistant strains due to UL54 mutations

(Knox et al., 1991; Baldanti et al., 1996).

CDV is a competitive inhibitor of the HCMV DNA

polymerase, and has been approved for the treatment of

HCMV retinitis (De Clercq, 1998). One of its advantages

compared with GCV and PFA is its long intracellular half-

life (Ho et al., 1992; Aduma et al., 1995). Its intermittent

intravenous administration is also possible. Toxicology

evaluations in animals have demonstrated that nephrotox-

icity is its major side-effect, although this can be reduced by

its less frequent administration, coupled with a high pro-

benecid dose and saline hydration (Cundy et al., 1994).

Cross-resistance between GCV and CDV could become a

concern since they share the same target, the product of the

UL54 gene. The emergence of GCV-resistant strains to

GCV through UL54 mutations in vitro or in AIDS patients

is well documented (Smith et al., 1997; Cherrington et al.,

1998). Double resistance to GCV and PFA in strains from

AIDS patients has been reported (Baldanti et al., 1996), and

high-level GCV-resistant strains with both UL97 and UL54

mutations were found to be cross-resistant to CDV (Smith et

al., 1997). At present, however, CDV is effective against

most of the GCV-resistant isolates studied, as well as PFA-

resistant viruses generated in vitro.

An antisense oligonucleotide against the HCMV IE2

mRNA (ISIS2922, fomivirsen) has also been developed

(Azad et al., 1993; Anderson et al., 1996) for intravitreal

application in patients with retinitis (De Smet et al., 1999)

who do not respond to conventional management.

A promising new class of anti-HCMV compounds is

formed of benzimidazole ribosides, which do not inhibit

the viral DNA polymerase. The benzimidazole analog

1263W94 has many characteristics that make it an attractive

candidate for development; namely, high potency in vitro,


selectivity, good oral bioavailability, and lower toxicity than

current therapies. Initial clinical trials have provided

encouraging results, including good tolerability and linear

pharmacokinetics over a wide dose range (Chulay et al.,

1999).

10.2. Therapeutic approaches

The antiviral agents described in the previous section are

used to treat and prevent HCMV infections in immunocom-

promised patients. In the immunocompetent host, infections

normally resolve spontaneously and antiviral therapy is not

indicated. Three major therapeutic approaches are currently

employed to manage HCMV infections and diseases: pro-

phylaxis, pre-emptive therapy, and treatment of an estab-

lished disease.

10.2.1. Prophylaxis

In prophylaxis, the antiviral drug is administered before

active HCMV infection is detected to prevent its occurrence.

The therapeutic value of an antiviral prophylaxis in AIDS

patients at high risk for HCMV disease is controversial. A

randomized clinical trial of oral GCV versus placebo found

that prophylaxis significantly reduced disseminated disease

and the incidence of retinitis (Spector et al., 1996). In

contrast, Brosgart et al. (1998) found no significant reduc-

tion in the occurrence of HCMV disease. Moreover, it is still

not clear whether oral GCV prophylaxis improves survival

and sight preservation compared with pre-emptive therapy

(Masur et al., 1996).

GCV prophylaxis after allogenic BMT in seropositive

recipients was significantly protective in two studies (Good-

rich et al., 1993; Boeckh & Bowden, 1995), but failed in a

third (Winston et al., 1993) and did not reduce overall

mortality (Goodrich et al., 1993; Winston et al., 1993;

Boeckh & Bowden, 1995), probably because drug-induced

neutropenia led to fatal bacterial and fungal infections.

In one study of heart transplant recipients, GCV prophy-

laxis exhibited a protective effect in seropositive patients, but

not in primary infection (Merigan et al., 1992), but the

opposite result was obtained in a different study (Macdonald

et al., 1995). For renal transplant recipients, prophylaxis is

recommended when the donor or recipient is seropositive

and if the recipient is subjected to an immunosuppressive

regimen, including an antilymphocyte treatment, or for a

seronegative recipient of a graft from a seropositive donor

(Jassal et al., 1998). In conclusion, antiviral prophylaxis may

be useful in some cases, but it is a high-cost strategy that

exposes all patients to drug toxicity (marrow suppression and

renal toxicity), the selection of antiviral resistance, and the

risk of late-onset HCMV infection and disease (Emery,

2001).

10.2.2. Pre-emptive treatment

Pre-emptive treatment was first described in the early

1990s (Rubin, 1991) as a means of reducing the incidence of

HCMV disease by withholding antiviral drugs until they

would be maximally effective. Antiviral treatment is initi-

ated when HCMV positivity is detected in the blood or

broncoalveolar lavage fluid using sensitive methods, such as

PCR, nucleic acid sequence-based amplification, and tests

for viral antigen. The advantages for the management of

HCMV infection include the targeting of antiviral therapy to

those most at risk for future disease, reducing the number of

patients exposed to antiviral toxicity, lowering the risk of

drug resistance, and maximizing the cost:benefit ratio. In the

BMT setting, pre-emptive GCV administration reduced

HCMV disease, and was literally life saving (Goodrich et

al., 1991; Einsele et al., 1995) in contrast to its lack of effect

on survival when used prophylactically (Goodrich et al.,

1993; Winston et al., 1993; Boeckh & Bowden, 1995). The

pre-emptive approach also prevented HCMV diseases in

liver transplant recipients (Singh et al., 1994). A prospective

study of the predictive value of PCR for CMV in asympto-

matic kidney transplant recipients indicated that while a

substantial number of recipients become positive for

HCMV-PCR after transplantation, only a minority would

develop CMV disease. Negative CMV-PCR assay is an

accurate negative predictor for CMV disease, but the value

of HCMV-PCR as a guide for pre-emptive anti-CMV

therapy in kidney transplant recipients is limited (Benedetti

et al., 1998).

10.2.3. Treatment of an established disease

It is generally agreed that it is better to prevent HCMV

diseases with pre-emptive approaches than to treat an

established disease, since an ongoing virus infection may

induce pathological phenomena unresponsive to antiviral

drugs and extensive tissue damage that may result in target

organ failure.

Clinical trials on AIDS patients with retinitis showed that

intravenous GCV, PFA, and CDV slow the progression of the

disease and lead to remission (Palestine et al., 1991; Spector

et al., 1993; Polis et al., 1995; Chavez-de la Paz et al., 1997;

Lalezari et al., 1998). In one study, PFA showed a higher

survival benefit than GCV (Anonymous, 1992). Retinitis can

also be treated with intravitreal injections of antiviral

compounds or by inserting an intraocular implant that

slowly releases the drug into the vitreous fluid (Masur et

al., 1996; Jacobson, 1997–1998). All antiviral compounds

suppress active replication of the virus, but do not eliminate

it. Unless immune reconstitution occurs as a consequence of

highly active antiretroviral therapy, chronic suppressive

maintenance anti-HCMV management is required, and will

be equally effective when administered intravenously, per os,

or locally (Drew et al., 1995). However, while oral and local

administration avoid the inconveniences and risks associated

with the intravenous route, they are less effective in prevent-

ing disease in the contralateral eye (Jacobson, 1997–1998;

Cheung & Teich, 1999). Maintenance therapy may result in

the appearance of resistant and refractory strains (Drew et al.,

1991; Cherrington et al., 1996; Jabs et al., 1998a, 1998b),


whose estimated emergence rate is � 25–33% after 9

months. These rates are similar for GCV, PFA, and CDV

(Jabs et al., 1998a, 1998b). The efficacy of the antisense

oligonucleotide fomivirsen in treating HCMV retinitis in

AIDS patients has been demonstrated recently (The Vitra-

vene Study Group, 2002a, 2002b).

GCVor PFA regimens have enjoyed some success in the

treatment of symptomatic gastrointestinal (esophagitis,

enterocolitis), respiratory (pneumonia), and neurologic

(encephalitis, peripheral neuropathy, polyradiculoneurop-

athy) HCMV diseases in AIDS (Dieterich et al., 1993;

Blanshard et al., 1995; Cheung & Teich, 1999).

No antiviral agents have been approved as yet for the

treatment of congenital HCMV infections.

Acknowledgments

The authors’ research is supported by grants from

MURST-CNR Biotechnology program L. 95/95, MURST

(40% and 60%), from the AIDS Research Project, and by

grants from the Ricerca Sanitaria Finalizzata (Regione

Piemonte).

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