A Simian Human Immunodeficiency Virus with a Nonfunctional Vpu ([Delta] vpuSHIVKU-1bMC33) Isolated from a Macaque with NeuroAIDS Has Selected for …

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Virology 282, 123–140 (2001)doi:10.1006/viro.2000.0821, available online at http://www.idealibrary.com on

A Simian Human Immunodeficiency Virus with a Nonfunctional Vpu (DvpuSHIVKU-1bMC33) Isolatedfrom a Macaque with NeuroAIDS Has Selected for Mutations in Env and Nef

That Contributed to Its Pathogenic Phenotype

Dinesh K. Singh,* Coleen McCormick,† Erik Pacyniak,* Kathi Lawrence,* Steven B. Dalton,† Dave M. Pinson,‡Francis Sun,§ Nancy E. J. Berman,* Meredith Calvert,i Robert S. Gunderson,i

Scott W. Wong,i and Edward B. Stephens*,1

*Department of Anatomy and Cell Biology; †Department of Microbiology, Molecular Genetics and Immunology; ‡Department of LaboratoryMedicine and Pathology; §Department of Laboratory Animal Resources, University of Kansas Medical Center,

Kansas City, Kansas 66160; and iOregon Regional Primate Center, Beaverton, Oregon

Received September 21, 2000; returned to author for revision November 14, 2000; accepted December 28, 2000; published online March 6, 2001

Previous studies have shown that passage of nonpathogenic SHIV-4 through a series of macaques results in the selectionof variants of the virus that are capable of causing rapid subtotal loss of CD41 T cells and AIDS within 6–8 months followinginoculation into pig-tailed macaques. Using a pathogenic variant of SHIV-4 known as SHIVKU-1bMC33, we reported that a mutantof this virus with the majority of the vpu deleted was still capable of causing profound CD41 T cell loss and neuroAIDS inpig-tailed macaques (McCormick-Davis et al., 2000, Virology 272, 112–116). In this study, we have analyzed the tissue-specificchanges in the env and nef in one macaque that developed neuroAIDS (macaque 50 O) and in three macaques that developedonly a moderate or no significant loss of CD41 T cells and no neurological disease (macaques 50 Y, 20220, 20228) followinginoculation with DvpuSHIVKU-1bMC33. Sequence analysis of the gp120 region of env isolated from lymphoid tissues (lymph nodeand spleen) of macaques 50 Y, 20220, and 20228 revealed no consensus amino acid substitutions. In contrast, analysis ofthe gp120 sequences isolated from lymphoid and CNS tissues (parietal cortex, basal ganglia, and pons) of macaque 50 Orevealed numerous amino acid substitutions. The significance of the amino acid substitutions in gp120 was supported byneutralization assays which showed that the virus isolated from the lymph node of macaque 50 O was neutralization resistantcompared to the parental SHIVKU-1bMC33. Analysis of changes in the nef gene from macaque 50 O revealed in-frame deletionsin Nef that ranged from 4 to 13 amino acids in length, whereas the nef genes isolated from the other three macaques revealedno deletions or consensus amino acid substitutions. Inoculation of the virus isolated from the lymph node of the macaquewhich developed neuroAIDS, SHIV50OLNV, into four pig-tailed macaques resulted in a severe loss of the circulating CD41 T cellswithin 2 weeks postinoculation, which was maintained for up to 20 weeks postinoculation, confirming that this virus hadindeed become more pathogenic in pig-tailed macaques. Taken together, these observations suggest that DvpuSHIVKU-1bMC33

has a low pathogenic phenotype in macaques but that individual pig-tailed macaques can select for additional mutations

within the Env and Nef which can compensate for the lack of an intact Vpu and ultimately increase its pathogenicity. © 2001

Academic Press

INTRODUCTION

The development of pathogenic simian-human immu-nodeficiency viruses (SHIV) containing the tat, rev, vpu,and env of HIV-1 in a genetic background of SIVmac239has permitted analysis of the role of HIV-1-specific geneproducts in a pathogenic macaque model. PathogenicSHIVs expressing the envelope glycoproteins fromT-tropic or dualtropic isolates of HIV-1 have now beendeveloped in several laboratories (Joag et al., 1996;Reiman et al., 1996; Shibata et al., 1997; Joag et al., 1998;Harouse et al., 1999; Luciw et al., 1999). Macaques inoc-ulated with these pathogenic SHIVs generally develop arapid decline in the number of circulating CD41 T cells,

1

To whom correspondence should be addressed. Fax: (913) 588-710. E-mail: [email protected].

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increased susceptibility to opportunistic infections, andgenerally succumb to their infections with 6–8 months(Joag et al., 1997). In addition to the severe immunedysfunction, some pathogenic SHIVs also cause otherclinical syndromes such as neurological disease that ischaracterized by perivascular cuffing, microglial nodules,giant cells, and demyelination (Raghavan et al., 1997; Liuet al., 1999; McCormick-Davis et al., 2000) and a ne-phropathy that is characterized by glomerulosclerosis,elevated serum urea nitrogen levels, and microprotein-uria (Liu et al., 1999; Stephens et al., 2000).

Recently, we reported that a SHIV containing a trun-cated, nonmembrane-bound Vpu (DvpuSHIVKU-1bMC33) wasstill capable of causing a rapid decline in the circulatingCD41 T cells and neuroAIDS following inoculation into

pig-tailed macaques (McCormick-Davis et al., 2000). Be-cause previous studies had shown that following inocu-

0042-6822/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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124 SINGH ET AL.

lation with the parental SHIVKU-1 and SHIVKU-1b, pig-tailedmacaques developed severe CD41 T cell loss in theabsence of neurological disease (Raghavan et al., 1997;

arayan et al., 1999), it suggested that the virus in thisacaque may have evolved genotypic and phenotypic

roperties that differed from the parental virus used tonoculate these macaques. In this report, we have ana-yzed the sequence changes in the tat, rev, vpu, env, andef regions in one macaque that developed neuroAIDSnd have compared the phenotypic characteristics to theiruses recovered from other macaques that developed

ow or moderate CD41 T cell loss without neuroAIDS.Our results indicate that the Env protein of this virus fromthe macaque that developed neuroAIDS has accumu-lated significant amino acid substitutions and small in-frame deletions in the gp120 region that probably ac-counted for its neutralization-resistant phenotype. Per-haps more interesting was that in certain regions of theCNS, the majority of the amino acid substitutions in thegp120 were reversions to the parental T-tropic HXB2envelope glycoprotein, suggesting that the input env ofthis dualtropic env had evolved toward a T-tropic pheno-type. Since previous studies have shown that infectingM-tropic HIV-1 can evolve toward a T-tropic phenotypeduring later stages of the disease, this study furthershows the utility of the SHIV/macaque model in studyingescape variants of Env.

RESULTS

CD41 T cell counts and virus burdens in macaquesnoculated with DvpuSHIVKU-1bMC33

We previously reported on the circulating CD41 T cellevels and virus burdens in two macaques (50 O and 50) that were inoculated with DvpuSHIVKU-1bMC33 (McCor-

mic-Davis et al., 2000). We have since inoculated twoadditional macaques with this virus (20220 and 20228).The circulating CD41 T cells counts and virus burdensare shown in Fig. 1. Both macaques 20220 and 20228had initial bursts of virus replication. Virus was intermit-tently recovered from macaque 20220 and virus wasrecovered from macaque 20228 throughout the course ofits infection. The circulating CD41 T cell counts in thesetwo macaques were stable throughout the course of the7-month study.

Rationale for sequence analysis

In previous studies, pig-tailed macaques inoculatedwith pathogenic variants of SHIV-4 (SHIVKU-1, SHIVKU-1b)

id not develop SHIV-associated neurologic diseaseRaghavan et al., 1997; Narayan et al., 1999). Thus, when

a pig-tailed macaque 50 O developed severe CD41 T cellloss and SHIV-associated encephalitis following inocu-

lation with a variant of this virus (DvpuSHIVKU-1bMC33), it

uggested that amino acid changes had occurred in this

irus that were not seen in previously derived pathogenicHIVs. In addition, our previous studies suggested that

he most amino acid substitutions were observed in thenv and nef genes of the virus. Therefore, we concen-

trated on the analysis of changes that occurred in the envand nef genes isolated from two lymphoid tissuesspleen and lymph node) and three regions of the CNSpons, basal ganglia, and occipital cortex). As expected,equence analysis revealed that the large deletion within

he vpu region from macaque 50 O, 50 Y, 20220, and0228, which reflected the deletion in the originalvpuSHIVKU-1bMC33 used to inoculate these macaques,

was maintained throughout the course of infection (Fig.1). No other changes were detected in the truncated vpu

hen compared to the input DvpuSHIVKU-1bMC33 virus.

The nef gene of the virus isolated from macaque 50O contains a series of in-frame deletions

We next analyzed the sequence of the nef genesamplified from the same tissues from the four macaques.As shown in Fig. 2, the nef genes amplified from thevarious tissues of macaque 50 O had two consensusamino acid changes. A threonine to alanine at position110 (in 17 of 25 clones analyzed) and an alanine tothreonine at position 185 (19 of 25 clones analyzed). Mostinteresting, however, was the finding that 72% of the nefgenes isolated (18 of 25 clones analyzed) from macaque50 O had in-frame deletions in nef. These deletions werebetween amino acid residues 37 and 55 and varied from4 to 13 amino acids in length. Of these deletions found inthe nef clones, the 4 amino acid deletion found at posi-tion 45–48 was the most prominent (16 of 18 clones withdeletions). In contrast, the amplified nef genes isolatedfrom the spleen and lymph node of macaque 50 Y, 20220,or 20228 revealed neither any consensus amino acidsubstitutions nor any deletions (data not shown).

The deletions in nef occurred following the severedepletion of CD41 T cells in macaque 50 O

Because in previous studies we had observed aminoacid substitutions in Nef, but never any Nef deletionsduring the pathogenic adaptation of SHIV-4 to causedisease in pig-tailed and rhesus macaques, we deter-mined when this deletion first appeared following inoc-ulation with DvpuSHIVKU-1bMC33. This was accomplished byisolating DNA from PBMC obtained at different timesafter inoculation (1, 2, and 3 months) and amplification ofthe region the nef gene by PCR followed by sequenceanalysis. As shown in Fig. 3, we first observed the dele-tions in this region of nef at 2 months postinoculation.Since the severe CD41 T cell depletion had occurred inmacaque 50 O prior to this time point, it indicated that the

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CD4 T cell loss was not associated with the appear-ance of the deletions in nef.

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125GENOTYPIC AND PHENOTYPIC CHARACTERIZATION AND NEUROPATHOGENIC SHIV

Macaque 50 O selected for significant amino acidsubstitutions in the Env

Because the clinical course of disease in macaque 50O was much different from macaques 50 Y, 20220, and20228 (severe CD41 T cell loss/neuroAIDS versus mod-erate to no CD41 T cell loss) following inoculation with

vpuSHIVKU-1bMC33, it suggested that these macaques mayhave selected for different viral gp120 variants. We ex-amined the viral gp120 sequences from two lymphoidtissues (spleen and lymph node) and three regions of theCNS (parietal cortex, basal ganglia, and pons) by PCRamplification, cloning, and sequence analysis. These re-sults shown in Fig. 4 and Table 1 indicate that thepercentage of amino acid substitutions in the gp120

FIG. 1. The levels of CD41 T cells (A) and numbers of cells producinoculation with DvpuSHIVKU-1bMC33.

compared to the input DvpuSHIVKU-1bMC33 in various 50 Otissues varied from 3.3% in the basal ganglia to 1.4% in

the spleen and pons. When compared to the parentalSHIV-4 (HXB2) Env, the consensus lymph node andspleen sequences had a 6.7 and 4.7% change in theamino acid sequence versus 3.7% for the gp120 ofDvpuSHIVKU-1bMC33. When we compared the consensusgp120 sequences from three regions of the CNS (parietalcortex, basal ganglia, and pons) we found that the gp120sequences from these regions also had a signifi-cant number of amino acid substitutions varying from7 substitutions (1.4% change from DvpuSHIVKU-1bMC33)in the pons to 18 substitutions (3.3% change fromDvpuSHIVKU-1bMC33) in the basal ganglia. Our analysis ofthe gp120 sequences from the CNS revealed that of the13 consensus amino acid substitutions in the gp120 of

ctious virus (B) in macaques 50 O, 50 Y, 20220, and 20228 following

the parietal cortex, 8 of these substitutions (61.5%) werereversions to the HXB2 sequence of SHIV-4. Amino acid

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126 SINGH ET AL.

FIG. 2. Comparison of the consensus amino acid sequence of Nef from molecularly cloned DvpuSHIVKU-1bMC33, and clones isolated from the lymphode (LN), spleen (SP), parietal cortex (PC), basal ganglia (BG), and pons (PONS) from macaque 50 O, 50 Y, 20220, and 20228. Shown above the

equences is the sequence of SIVmac239 Nef (Regier and Desrosiers, 1990) with Nef corrected at codon 92. Dashes (—) represent amino acid identity

and (x) represent deletions.

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127GENOTYPIC AND PHENOTYPIC CHARACTERIZATION AND NEUROPATHOGENIC SHIV

reversions were also observed in the lymph node,spleen, and basal ganglia (2 each; Table 1). In contrast,our results indicate that the gp120 sequences isolatedfrom macaque 50 Y, 20220, and 20228 had no consensusamino acid substitutions or deletions (summarized inTable 1). Thus, when compared to the Env of the originalnonpathogenic SHIV-4, lymphoid tissues appeared to beevolving away from the parental SHIV-4 sequencewhereas in other tissues (i.e., the parietal cortex of theCNS) appeared to be reverting back to the originalSHIV-4 sequence.

The virus isolated from the lymph node of macaque50 O was antibody neutralization resistant comparedto the parental virus

Because the predicted amino acid sequences of thegp120 genes isolated from macaque 50 O were divergentfrom the parental DvpuSHIVKU-1bMC33, we examined theneutralization properties of SHIV-4, DvpuSHIVKU-1bMC33,and the virus recovered from lymph node cells isolatedfrom macaque 50 O (SHIV50OLNV). In order to more easilyanalyze the neutralization properties of the viruses withdifferent envelope glycoproteins, we developed SHIVvectors (with the SHIV-4, SHIVKU-1bMC33, and SHIV50OLNV

gp160) that stably expressed the enhanced greenfluorescent protein (EGFP). Plasma obtained from ma-

FIG. 2

caque 50 O at necropsy was found to neutralizeDvpuSHIVKU-1bMC33 at a dilution of 1:64, suggesting that the

antibodies in this plasma effectively neutralized the pa-rental virus used to inoculate these macaques. Plasmasamples from macaques 50 Y, 20220, and 20228 wereable to neutralize DvpuSHIVKU-1bMC33 at dilutions thatranged from 1:8 to 1:64. These same plasmas failed toeffectively neutralize (titer of #2) the infectivity of virussolated from the lymph node of macaque 50 OSHIV50OLNV). The positive control plasma used in these

studies, a plasma pool from several macaques that hadlong-term infections with SHIV, was found to neutralizeSHIV-4 and DvpuSHIVKU-1bMC33 at a dilution of 1:64 andSHIV50OLNV at a dilution of 1:128 (Table 2). Taken together,hese results indicate that macaque 50 O had selectedor a virus (SHIV50OLNV) that was resistant to neutralization

by plasmas isolated at necropsy from all four macaquesinoculated with DvpuSHIVKU-1bMC33 and thus has the phe-notype of an antibody neutralization escape virus.

The SHIV50OLNV enters and replicates in cells withkinetics similar to DvpuSHIVKU-1bMC33 and has retainedCXCR4 coreceptor usage

Stocks of SHIV-4, SHIVKU-1bMC33, DvpuSHIVKU-1bMC33, andHIV50OLNV were prepared and titered in C8166 cells.

Equal amounts of virus were used to inoculate C8166cultures and assessed for virus replication by assayingculture supernatants for the presence p27 antigen. Our

inued

results indicate that SHIV50OLNV and DvpuSHIVKU-1bMC33 rep-licated with similar kinetics in C8166 cells (data not

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128 SINGH ET AL.

shown). We examined whether the SHIV50OLNV virus usedthe same coreceptor(s) for entry as the virus used toinoculate macaque 50 O (DvpuSHIVKU-1bMC33). The results

hown in Table 3 indicate that SHIV-4, SHIVKU-1bMC33,DvpuSHIVKU-1bMC33, and SHIV50OLNV all used CXCR4 as theircoreceptor for entry. We also determined if the kinetics

FIG. 3. Comparison of the consensus amino acid sequence of Nef frof macaque 50 O at 1, 2, and 3 months after inoculation with DvpuSHIVK

and Desrosiers, 1990) with Nef corrected at codon 92.

of virus entry into cells expressing CD4 and CXCR4was different among the four viruses. In this assay,

GHOST-CD4 cells expressing CXCR4 coreceptorswere infected with various SHIVs (SHIV-4, SHIVKU-1bMC33,DvpuSHIVKU-1bMC33, SHIV50OLNV) for various periods oftime, and the number of fluorescent cells was deter-mined at 48 h. As shown in Fig. 5, DvpuSHIVKU-1bMC33,SHIV50OLNV, and SHIVKU-1bMC33 infected GHOST/X4 cells

ecular clone DvpuSHIVKU-1bMC33, and nef clones isolated from the PBMCShown above the sequences is the sequence of SIVmac239 Nef (Regier

m mol

with approximately the same kinetics (i.e., the slopeswere approximately the same) while the SHIV-4 virus

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infected the same cell type at a somewhat slower rate.These results indicate that while all three of the patho-genic SHIVs were more efficient at infecting cells thannonpathogenic SHIV-4, significant differences werenot observed in the efficiency of infection by the threepathogenic SHIVs examined. Taken together, theseresults suggest that the virus isolated from the lymphnode of macaque 50 O did not bind and enter cells ata faster rate than the parental DvpuSHIVKU-1bMC33, andutilized the same coreceptor for entry.

SHIV50OLNV causes severe CD41 T cell depletion inpig-tailed macaques

We determined if the virus derived from the lymphnode of 50 O (SHIV50OLNV) was capable of causing thesevere CD41 T cell loss following inoculation into pig-tailed macaques. Four macaques were inoculated intra-venously with 104 TCID50 of SHIV50OLNV and followed for 20weeks. As shown in Fig. 6A, all four macaques devel-oped a severe loss in CD41 T cells within 2 weeks (38 to152 circulating CD41 T cells/ml). This was followed by atransient rebound in the CD41 T cell numbers at 4weeks, and then a sustained loss of circulating CD41 Tcells that has been maintained for up to 20 weeks post-inoculation in three of four macaques. The massive lossof CD41 T cells was accompanied by high numbers ofCD41 T cells producing infectious, cytopathic virusthroughout the 20-week period (Fig. 6B), which is typi-cally observed for pig-tailed macaques inoculated withpathogenic SHIVs (Joag et al., 1997). These results indi-

FIG. 3

cate that SHIV50OLNV is more pathogenic for pig-tailedmacaques than the parental DvpuSHIVKU-1bMC33.

DISCUSSION

In our previous studies on the evolution of the env fromthe parental nonpathogenic SHIV-4 (containing theT-tropic HXB2 env sequence) we have shown that thisvirus evolved from a T-tropic virus to one that was dual-tropic, capable of replicating both in macaque macro-phage cultures and in established T cell lines (Stephenset al., 1996, 1997; Narayan et al., 1999; Liu et al., 1999). In

time course study that analyzed the molecular changesn the vpu, env, and nef as the virus became pathogenicor pig-tailed macaques, there was an association be-ween reversion of the vpu to a functional open read-ng frame and increased amino acid substitutions innv and Nef (McCormick-Davis et al., 1998). This sug-ested that the Vpu protein may have a role in theHIV-induced CD41 T cell loss observed in these

pig-tailed macaques. To test this hypothesis, we con-structed a SHIV (DvpuSHIVKU-1bMC33) in which the major-ty of the vpu was deleted (McCormick-Davis et al.,000). When inoculated into macaques, we observed

hat one of four macaques (macaque 50 O) developedsevere loss in its circulating CD41 T cells and

neuroAIDS. This suggested that this macaque mayhave selected for additional changes in the virus ge-nome that accounted for its pathogenic phenotype.The results in this study indicate that macaques inoc-ulated with DvpuSHIVKU-1bMC33 (50 Y, 22020, and 22028),which developed only moderate or no significant CD41

T cell loss during a 8-month infection, selected for

inued

minimal amino acid substitutions in the gp120 regionof Env and Nef when compared to the parental

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FIG. 4. Comparison of the consensus amino acid sequence of gp120 from molecular clone DvpuSHIVKU-1bMC33, and clones isolated from the lymphode (LN), spleen (SP), parietal cortex (PC), basal ganglia (BG), and pons (PONS) clones isolated from macaque 50 O, 50 Y, 20220, and 20228. Shown

bove the sequences is the sequence of HXB2 gp160. Dashes (—) represent amino acid identity and (x) represent deletions. For comparativeurposes, brackets corresponding to the variable regions V1, V2, V3, V4, and V5 were placed above the gp120 sequences.

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131GENOTYPIC AND PHENOTYPIC CHARACTERIZATION AND NEUROPATHOGENIC SHIV

DvpuSHIVKU-1bMC33. The fact that macaque 50 Y devel-1

FIG. 4

ped a moderate loss of CD4 T cells suggests thatthe DvpuSHIVKU-1bMC33 virus, despite the lack of a func-

ional Vpu, was still capable of causing CD41 T cell

inued

loss. In contrast, analysis of the gp120 sequencesfrom the one macaque that developed severe CD41 T

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132 SINGH ET AL.

cell loss and neuroAIDS revealed the accumulation of

FIG. 4

significant numbers of amino acid substitutions whencompared to parental DvpuSHIVKU-1bMC33. The percent-

age of amino acid substitutions in gp120 genes iso-

inued

lated from the lymph node had increased from 3.7% inDvpuSHIVKU-1bMC33 to 6.7% at necropsy in macaque 50

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133GENOTYPIC AND PHENOTYPIC CHARACTERIZATION AND NEUROPATHOGENIC SHIV

O, when compared to the original SHIV-4. In addition tothe lymph node, the gp120 genes isolated from thespleen and three regions of the brain of this macaquealso had a significant number of amino acid substitu-tions. These changes correlated well with the pheno-typic properties of the virus isolated from the lymphnode of this macaque, which was more neutralizationresistant compared to DvpuSHIVKU-1bMC33. This neutral-zation-resistant phenotype of the SHIV50OLNV appeared

to be broad based since this virus was not effectivelyneutralized by plasmas from the other three macaquesinoculated with the same virus. This finding is some-what different than that found for the derivation ofSHIVKU-1bMC33, which was found to be a neutralization-esistant variant only in the context of the macaquerom which it was derived (Narayan et al., 1999). Neu-

tralization-resistant variants have also been isolatedfrom a laboratory worker that was accidentally in-fected with the IIIB strain of HIV-1 (Reitz et al., 1994).

In previous studies on the adaptation of SHIV-4 to

T

Summary of Consensus Sequence Changes in gp120 G

No. of consensusAA changes from

HXB2

No. of consensus AAchanges from

KU1bMC33 gp120

DvpuSHIVKU-1bMC33 19 NA50 O lymph node 34 1650 O spleen 24 750 O parietal cortex 16 1350 O basal ganglia 37 1850 O pons 26 750 Y lymph node 19 050 Y spleen 19 020220 lymph node 19 020220 spleen 19 020228 lymph node 19 020228 spleen 19 0

Note. DNA was extracted from various lymphoid organs or regionsHIVKU1bMC33 gp120 region of env, and analyzed by DNA sequence ana

TABLE 2

Neutralization Titers of Plasma Samples Derived from Macaques0 O, 50 Y, 20220, and 20228 against SHIV-4, DvpuSHIVKU-1bMC33, and

SHIV50OLNV

Virus

Plasma samplea

50 O 50 Y 20220 20228 a-SHIV Negative

SHIV-4 32 2 8 8 64 ,2DvpuSHIVKU-1bMC33 64 8 16 64 64 ,2SHIV50OLNV ,2 ,2 ,2 2 128 ,2

a Plasma samples were collected at necropsy.

cause disease in pig-tailed and rhesus macaques (Joaget al., 1996; Stephens et al., 1996, 1997; Raghavan et al.,1997), sequence analysis had revealed amino acid sub-stitutions but no deletions in nef. Thus, the observationthat a high percentage of the nef clones isolated fromive tissues of macaque 50 O had in-frame deletions of 4o 13 amino acids (with the 4 amino acid deletion beingredominant) is novel to the evolution of this SHIV from

he nonpathogenic SHIV-4. It was of interest that in two ofhe nef clones examined, the deletions included one ofhe putative SH-2 domains (Y39xxS) in SIV nef (Du et al.,995, 1996). The ability to amplify nef genes with sucheletions suggests that the virus can replicate effectively

n the absence of this domain. These results correlateell with recent site-directed mutagenesis studies that

howed that the tyrosine residues at positions 28 and 39ere not important to SIV nef function (Carl et al., 2000).

solated from Macaques 50 O, 50 Y, 22020, and 22028

ge fromMC33120

% Change fromHXB2 gp120

% Consensuschanges that

were reversionsto HXB2 gp120

No. of consensusAA changes that

were reversions toHXB2 gp120

A 3.7 NA NA.1 6.7 12.5 2.4 4.7 28.6 2.5 3.1 61.5 8.3 7.2 11.1 2.4 5.1 0 0

3.7 0 03.7 0 03.7 0 03.7 0 03.7 0 03.7 0 0

CNS, used in nested PCR with oligonucleotides which amplified the

TABLE 3

Coreceptor Use by SHIV50OLNV

Virus

Replication in the presence of the followingcoreceptor

CCR1 CCR2 CCR3 CCR5 CXCR4

SHIVKU-1bMC33 2 2 2 2 1111DvpuSHIVKU-1bMC33 2 2 2 2 1111SHIV50OLNV 2 2 2 2 1111SHIV-4 2 2 2 2 111SIVmac239 2 2 2 1111 2

Note. Ability to replicate in GHOST-CD4 cells expressing variouscoreceptors. Replication was recorded as Gag production with 2,

ABLE 1

enes I

% ChanKU1b

gp

N31231000000

,100 pg/ml; 1, 100 pg–1 ng/ml; 11, 1 to 5 ng/ml, 111, 5 to 15 ng/ml;1111, .15 ng/ml.

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134 SINGH ET AL.

The appearance of these deletions did not coincide withthe severe CD41 T cell loss in this macaque but ratheroccurred later in the disease (i.e., 2 months) course inthis animal. Thus, the significance of these deletions andwhether the Nefs containing these deletions are func-tional are unknown. The appearance of in-frame dele-tions has also been reported for patients infected withHIV-1 (Brambilla et al., 1999). These investigators wereable to detect small in-frame deletions of 6–15 nucleo-tides in the nef genes isolated from both long-term non-progressors (LTNP) as well as from progressors, al-though they found that chimeric viruses expressing Nefswith in-frame deletions replicated to lower titers whencompared to the same virus with an intact Nef (Brambillaet al., 1999). Taken together, the results of the sequenceanalysis indicate that in those macaques which had lowor moderate CD41 T cell loss, expansion of the viralquasispecies swarm did not occur and the macaquesessentially controlled their infection. In contrast, se-quence analysis of viral sequences amplified from differ-ent tissues (lymph node, spleen, and three regions of thebrain) revealed an expansion of the viral quasispecies inmacaque 50 O, in which one or more of these viralspecies was probably pathogenic for this macaque. Theexpansion of the viral quasispecies in this macaque issimilar to what we found during the derivation of patho-genic virus variants from the nonpathogenic SHIV-4 (Mc-Cormick-Davis et al., 1998).

FIG. 5. The kinetics of viral entry for SHIV-4, DvpuSHIVKU-1MC33, andnfectious units of each virus for various periods of time (0, 5, 15, 30, a8 h. At 48 h, the numbers of EGFP expressing cells were quantitatedeparate experiments. SHIV-4 (E), DvpuSHIVKU-1MC33 (Œ), and SHIV50OLNV

Because these results suggested that compensatingchanges in Env and Nef may have contributed to its

increased pathogenicity in macaque 50 O, four pig-tailedmacaques were inoculated with the virus isolated fromthe lymph node (SHIV50OLNV) of this animal. Our resultsindicate that four macaques developed a severe loss ofcirculating CD41 T cell levels within 2 weeks after inoc-ulation and in three of four macaques the circulatingCD41 T cells have remained low for the first 20 weeksfollowing inoculation, which is characteristic of otherpathogenic SHIVs (Joag et al., 1996; Luciw et al., 1999;Igarashi et al., 1999; Liu et al., 1999; McCormick-Davis etal., 2000). Virus containing these deletions in nef were

pparently a major quasispecies in the SHIV50OLNV stock(and not impaired in replication) since inoculation ofSHIV50OLNV into four additional macaques resulted in theisolation of virus containing these nef deletions at 4months postinoculation as well as severe CD41 T cellloss (unpublished observations). While it is known thatHIV and SIV are able to infect astrocytes in the CNS,infection is generally nonproductive, with only theearly viral proteins such as Nef and Tat being ex-pressed (Kort et al., 1998; Ludwig et al., 1999; Guille-min et al., 2000). It is unknown if these Nef deletionshave conferred a unique structural property that mayalter signaling within the infected cell or more efficientdownregulation of CD4 from the cell surface. Whetherthese in-frame deletions in Nef, with or without thesignificant amino acid substitutions in Env, confer aunique property to this virus (i.e., a neuropathogenic

LNV into GHOST cells. Cells were inoculated with a similar number ofmin), washed three times to remove inoculum, and then incubated forlotted as a function of time. The curves represent the mean of threed SHIVKU-1bMC33 (‚).

SHIV50O

nd 60

phenotype) is currently under investigation. These re-sults also show that SHIV50OLNV was more pathogenic

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135GENOTYPIC AND PHENOTYPIC CHARACTERIZATION AND NEUROPATHOGENIC SHIV

(4 of 4 macaques with severe CD41 T cell loss to ,200ells/ml) than the parental DvpuSHIVKU-1bMC33 (only 1 of

4 macaques with severe CD41 T cell loss). Theseresults indicate that the phenotype of DvpuSHIVKU-1bMC33

is probably one of a low pathogenic phenotype butfollowing inoculation of an outbred species such asmacaques (in this case macaque 50 O), unrestrictedvirus replication resulted in the selection of viruseswith significant nucleotide substitutions in certain viralgenes (such as but not necessarily limited to env andnef) that compensated for the lack of a functional Vpu.Previous studies have suggested that the function of

FIG. 6. The levels of CD41 T cells (A) and numbers of cells producnoculation with SHIV50OLNV.

Vpu may be redundant since the Env and Nef proteinsof HIV-1 are also involved in downregulation of CD4

from the surface of infected cells (Piquet et al., 1999).The results from this study indicate that a functionalVpu is important to the pathogenicity caused by SHIV(i.e., the severe loss of CD41 T cells within weeks afterinoculation) but would suggest that the function of Vpuis somewhat redundant and can be compensated withamino acid substitutions in other viral proteins Envand Nef, both of which are involved in downregulationof CD4 from the surface of virus-infected cells.

The results from this study demonstrate for the first timein the SHIV/macaque model system that following inocula-tion into macaques, the virus that enters the central ner-

ectious virus (B) in macaques AT99, AT9J, AX5H, and AX5G following

vous system (CNS) is able to evolve region-specific quasi-species in different regions of the brain that are apparently

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136 SINGH ET AL.

different from those in lymphoid organs. The independentevolution of virus in different regions of the brain from thismacaque shares similarities with a recent study that foundindependent evolution of HIV-1 in different regions of thebrain from three patients with neuroAIDS (Shapshak et al.,1999), although the results presented here involve the useof a molecularly cloned virus. While it appeared that theevolution of region-specific sequences had occurred inmacaque 50 O, there was no correlation between variationin the five variable regions (V1–V5) of gp120 and neurolog-ical disease in this macaque. The inability to find signaturesequence changes in the macaque with neuroAIDS agreeswith previous studies on HIV-1 isolates from AIDS dementiapatients (Donaldson et al., 1994; Reddy et al., 1996; Di

tefano et al., 1996).It is generally accepted that non-syncytia-inducing

NSI) CCR5 utilizing macrophage-tropic HIV-1 probablynters the CNS soon after infection (Zhu et al., 1993; Annd Scaravilli, 1997). However, despite presumably per-isting in the brain for many years without neurologicalisease, infection of macrophages and microglial cellsy non-syncytia-inducing HIV-1 is considered to be in-trumental for the development of AIDS dementia com-lex (ADC) (Cheng-Meyer et al., 1989; Gabuzda andang, 1999). The long period of time between neuroin-

asion and onset of HIV-1-induced neurological diseaseuggests that other viral and/or host factors may be

nvolved in the neuropathogenesis of HIV-1. The involve-ent of T cell tropic variants, which generally evolve

uring the latter stages of disease progression and at aeriod when HIV-1 neurological disease occurs, has noteen well studied in relation to the neurological mani-

estations of HIV-1 infection. It is well known that T-tropicariants of HIV-1 utilize CXCR4 as their coreceptor forntry into susceptible cells (Berger et al., 1996, 1999;

Hoffman et al., 1998, 1999). This coreceptor is alsopresent on the surface of astrocytes, microglia, and neu-rons of the CNS (Ohtani et al., 1998; Bagetto et al., 1999;

lein et al., 1999). In a recent study, the ability of mac-ophage-tropic (ADA, JR-FL, Bal, MS-CSF, and DJV), du-ltropic (89.6), and T-tropic (MN, IIIB, and Lai) strains ofIV-1 to affect intracellular signaling and apoptosis ofeurons, astrocytes, and monocyte-derived macro-hages was examined (Zheng et al., 1999). These inves-

igators found that T-tropic strains of HIV-1 elicited theost astrocytic and neuronal damage whereas the mac-

ophage-tropic strains produced the least neural dam-ge. These results suggest that T-tropic strains may haven important role in the neurological disease caused byIV-1. The sequence analysis presented here indicates

hat in one of three regions of the brain of macaque 50 Onalyzed (the parietal cortex), the majority of the aminocid substitutions in the gp120 were reversions back to

he gp120 sequence of the parental, nonpathogenic,

-tropic SHIV-4 virus. These results underscore the com-lexity of the viral quasispecies evolving in the brain

ollowing inoculation with the dual-tropic SHIVKU-1bMC33.Whether the CNS represent a sanctuary for “less fit”viruses that would normally be eliminated by neutralizingantibodies present outside the CNS is currently beingexamined in the SHIV/macaque model. Precedent forthis comes from the SIVmac/macaque model, in whichchimeric virus constructed with the env isolated from thebrain of a macaque with severe encephalitis was highlysensitive to antibody-mediated neutralization and not aspathogenic (Joag et al., 1995; Zhu et al., 1997).

MATERIALS AND METHODS

Cells, plasmids, and viruses

C8166 cell line was used as the indicator cells tomeasure infectivity and cytopathicity of the viruses usedin this study. C8166 cells were maintained in RPMI 1640,supplemented with 10 mM HEPES buffer, pH 7.3, 2 mMglutamine, 50 mg/ml gentamicin, and 10% fetal bovineserum (R10FBS). The derivation of SHIVKU-1b and

vpuSHIVKU-1bMC33 has been previously described(Narayan et al., 1999; McCormick-Davis et al., 2000).

Macaques analyzed in this study

Two pig-tailed macaques (50 O and 50 Y) were inoc-ulated intravenously with 1 ml of undiluted supernatantfrom C8166-grown stocks of DvpuSHIVKU-1bMC33 containingapproximately 104 TCID50/ml as previously described(McCormick-Davis et al., 2000). Macaque 50 O devel-oped severe CD41 T cell loss and signs of neuroAIDS at35 weeks postinoculation whereas macaque 50 Y devel-oped moderate CD41 T cell loss and was euthanized atWeek 39 postinoculation in apparent good health. Twoadditional macaques (20220 and 20228) were subse-quently inoculated with the same virus stock and devel-oped no significant loss of CD41 T cells over the courseof 6-month study period. At 8 months, these macaqueswere euthanized in good health and tissues examinedfor virus content.

Four pig-tailed macaques (AT99, AT9J, AX5H, andAX5G) were inoculated intravenously with approximately104 TCID50 of SHIV50OLNV grown in macaque PBMC. Bloodwas collected weekly in EDTA for the first 4 weeks, thenat 2-week intervals for the next months, and thereafter atmonthly intervals. All aspects of the animal studies wereperformed according to the institutional guidelines ofanimal care and use at University of Kansas MedicalCenter and the Oregon Regional Primate ResearchCenter.

PCR amplification and sequence analysis of tat, rev,vpu, env, and nef sequences

Because previous studies indicated that the majority

of the sequence changes occurred in env and nef genesduring the derivation of a pathogenic SHIV from non-

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137GENOTYPIC AND PHENOTYPIC CHARACTERIZATION AND NEUROPATHOGENIC SHIV

pathogenic SHIV-4, we compared the env and nef se-quences of this virus with the sequences from the mo-lecularly cloned DvpuSHIVKU-1bMC33 and original nonpatho-genic SHIV-4. Visceral organs and various regions of theCNS were collected and DNA was extracted as previ-ously described (McCormick-Davis et al., 2000). For anal-ysis of vpu and gp120 sequences, oligonucleotide prim-ers 59-CCTAGACTAGAGCCCTGGAAGCATCC-39 and 59-

GTGCTTCCTGCTGCTCCCAAGAACCC-39 were used,hich are complementary to nucleotides 5845 and 5870nd 7781 to 7810 of the HIV-1 (HXB2) genome (Ratner etl., 1985), respectively. One microgram of genomic DNAas used in the PCR (Saiki et al., 1985, 1988) containing

.4 mM MgSO4, 200 mM each of the four deoxynucleotidetriphosphates, 100 pM of each oligonucleotide primerand a mixture of Taq and Pyrococcus species GB-Dpolymerases (Elongase, GibcoBRL). The template wasdenatured at 94°C for 2 min and PCR amplification per-formed with an automated DNA Thermal Cycler (Perkin-Elmer Cetus) for 35 cycles using the following profile:denaturation at 94°C for 30 s, annealing at 65°C for 1min, and primer extension at 68°C for 5 min. Amplifica-tion was completed by incubation for 10 min at 68°C.Three separate PCRs were performed for each region ofviral genome that was studied. The amplified productsfrom three separate reactions were separated by elec-trophoresis in a 1% agarose gel, isolated, and molecu-larly cloned into the pGEM-T Easy vector according tothe manufacturer’s instructions. Cycle sequencing reac-tions using the BigDye Terminator cycle sequencingReady Reaction Kit with AmpliTaq DNA polymerase, FS(PE Applied Biosystems, Foster City, CA) and sequencedetection was conducted with an Applied Biosystems377 Prism XL automated DNA sequencer and visualizedusing the ABI Editview program.

For amplification of nef, oligonucleotide primers 59-CCACATACCTAGAAGAATAAGACAGGG-39 (sense) and59-ACATCCCCTTGTGGAAAGTCCCTGCTGTTT-39 (anti-sense) were used, which are complementary to nucleo-tides 8746 to 8772 of the HIV-1 (HXB2) genome (Ratner etal., 1985) and 9868 to 9898 of the SIVmac239 genome(Regier and Desrosiers, 1990), respectively. The condi-tions for amplification, cloning, and sequencing of thePCR products were the same as described above.

Virus neutralization assays

To more easily analyze the neutralization properties ofSHIV variants, we constructed a SHIV-based virus thatexpressed the enhanced green fluorescent protein(Zhang et al., 1996; Lee et al., 1997; Alexander et al.,1999). In order to relieve the packing constraints causedby the insertion of EGFP into SHIV, we first deleted thenonessential vpu sequences prior to the initiation codon

of Env. A unique NsiI site was introduced at the AUG ofVpu in the plasmid ptrvSHIVKU-1bMC33 (McCormick-Davis et

al., 2000) using the Quick Change site-directed mutagen-esis kit (Stratagene) with the primer 59-GCAGTAAGTAG-

ACATGTAATGCATCCTATACCAATAGTAGCAATAG-39and its complement 59-CTATTGCTACTATTGGTATAGGAT-GCATTACATGTACTACTTACTGC-39 according to themanufacturer’s directions. The resulting plasmid was di-gested with NsiI and BbsI to release a 155-bp fragment,reated with Klenow, and ligated. The ligated product

as used to transform E. coli (strain XL-1) and a plasmidas selected for the absence of a NsiI site. This plasmidas digested with SphI and KpnI and purified by elec-

rophoresis in a 1% agarose gel and extracted throughMillipore column. The 380-bp fragment was ligated

nto p39SHIVKU-1bMC33, which was also digested withSphI and KpnI to release a 535-bp fragment. Theligated product was used to transform E. coli (strainXL-1) and plasmids were selected for a 380-bp frag-ment when digested with SphI and KpnI. In the result-ing plasmid, p39novpuSHIVKU-1bMC33, 155 bp of vpu prioro env has been deleted without disrupting the initia-ion AUG of Env. A NcoI site was introduced at the 39

end of the EGFP gene in the plasmid pEGFP (Clon-tech), using the Quick Change site-directed mutatgen-esis kit (Stratagene) with primer 59-GCCGGTCGCTAC-CATGGCCAACTTGTCTGGTG-39 and its complement59-CACCAGACAAGTTGGCCATGGTAGCGACCGGC-39.A plasmid was selected for the presence of two NcoI

ites, as there is already a NcoI site located in theulticloning region of pEGFP. This plasmid was then

ubjected to another mutagenesis step, using primer9-GGTCGCCACCATGGATGAGCAAGGGCG-39 and its

complement 59-CGCCCTTGCTCATCCATGGTGGCGACC-39to introduce an additional adenosine such that the read-ing frame of the EGFP gene would be conserved whenintroduced into p39novpuSHIVKU-1bMC33. The resulting plas-mid was then digested with NcoI to release a 769-bpfragment consisting of the EGFP gene. This fragmentwas ligated into p39novpuSHIVKU-1bMC33 which had alsobeen digested with NcoI. There is a unique NcoI sitelocated within the nef gene. The ligated product wastransformed into E. coli (strain XL-1). A plasmid,

39novpuSHIVEGFP, was selected for the presence of a769-bp fragment when digested with NcoI. All plasmidswere sequenced at each step to verify the site-directedmutagenesis. In order to generate virus, the plasmidsp59SHIV-4 and p39novpuSHIVEGFP were digested withSphI, ligated using T4 DNA ligase (NEB), and transfectedinto C8166 cells by the DEAE-dextran method (Milmanand Herzberg, 1981). Stocks were prepared and titered inC8166 cells. Despite multiple passages, the expressionof the EGFP marker protein was found to be stable.

In order to compare the ability of various gp120s to beneutralized by serum from macaques 50 O and 50 Y, wegenerated a series of interviral recombinants in which

sequenced gp160 genes from SHIV-4 (HXB2) and a rep-resentative env clone from the lymph node and parietal

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138 SINGH ET AL.

cortex were inserted into the p39novpuSHIVEGFP. Thegp160 clones were digested with KpnI and RsrII, and theenv gene was purified and inserted into the same sites ofthe p39novpuSHIVEGFP. The resulting plasmids,

39novpuSHIV-4EGFP and p39novpuSHIV50OLNVEGFP wereused to generate virus stocks as described above. Theviruses, SHIV-4EGFP and SHIV50OLNEGFP, were used in neu-tralization assays described below.

A standard neutralization assay method was used withC8166 cells as the indicator cell type (Joag et al., 1993).All four macaques developed robust antibody responsesto the viral Env as determined by immune precipitationassays (data not shown). Serial twofold dilutions ofplasma in RPMI 1640 medium were prepared in quadru-plicate in 96-well plates, 100 TCID50 of each virus wasadded to each well, and samples were incubated for1.5 h at 37°C. Following this incubation, 1 3 104 indicatorcells (in 100 ml) were added to each well and culturesncubated at 37°C incubator in 5% CO2 for 7 days. Cul-ures were observed daily by phase-contrast microscopyor development of syncytial cytopathic effects (CPE) andy fluorescence microscopy for expression of EGFP. We

ound that there was a perfect correlation between theresence of CPE and the expression of EGFP by fluores-ence microscopy. That is, in the absence of virus neu-

ralization (as evidenced by the presence of CPE), EGFPxpression was readily detected, and vice versa. The0% neutralization end point was calculated using thearber method (Lennette, 1969). The pooled plasma fromeveral macaques that were infected with SHIV-4 forver 1 year was used as a positive control. This serumad a neutralization titer of 1:64 against SHIVKU-1bMC33 and

a titer of 1:64 against SHIV-4 in C8166 cells. Plasma froman SHIV-seronegative macaque (NRP) was used as neg-ative control in all the neutralization assays.

Viral entry assays

We determined if the coreceptor use of SHIV50OLNV wasimilar to the parental virus used to inoculate macaque0 O or if its coreceptor had been altered with changes

n Env. Use of CXCR4, CCR5, CCR3, CCR2b, and CCR1oreceptors was determined using human osteosar-oma (GHOST)-CD4 cells expressing individual corecep-

ors as previously described (Zhang et al., 2000). In allases, the levels of p27 Gag in the culture supernatantsf GHOST-CD4 cells transfected with various corecep-

ors were subtracted from that produced from controlHOST-CD4 cells.The kinetics of virus entry of different strains of SHIV,

HIV-4, DvpuSHIVKU-1bMC33, and SHIV50OLNV were deter-mined using human osteosarcoma cells (GHOST)-CD4cells expressing CXCR4, obtained from the AIDS Re-search and Reference Reagents Program (Division of

AIDS, National Institutes of Allergy and Infectious Dis-eases, National Institutes of Health, from Nathaniel

Landau). Virus stocks were first titrated on GHOST-CD4/X4 cells by preparing a series of threefold dilutionsfollowed by inoculation of cells in 96-well plates. Cellswere then incubated for 48 h and cultures examined forthe expression of green fluorescent protein (GFP) aspreviously described (Lee et al., 1997). For kinetic as-

ays, cells grown in 6-well plates were inoculated withirus for various periods of time (0, 5, 15, 30, and 60 min),ashed three times to remove any unbound virus inoc-lum, and then incubated at 37°C for 48 h. At 48 h, theells were examined under a Nikon Ophiphot invertedhase microscope equipped with an Optronics Mag-afire imaging system. The number of fluorescent cellser field (using the 20X objective; 5 fields examined) wasnumerated, and the average taken and then plotted asfunction of time. Presented are the average of triplicate

xperiments.

rocessing of blood and tissue samples

The peripheral blood mononuclear cells (PBMC) wererepared by centrifugation on Ficoll-Hypaque gradientss described previously (Joag et al., 1996). Tenfold dilu-

tions of PBMC (106 cells/ml) were inoculated into repli-ate cultures of C8166 cells in 24-well plates and theocultures examined for development of cytopathic ef-

ects as previously described (McCormick-Davis et al.,000). Infectivity titers in plasma were reported asCID50/ml and cells producing infectious cytopathic virus

in the blood reported as the number of infected cells/106

PBMC. Alterations in CD41 and CD81 T lymphocytesafter experimental inoculations were monitored sequen-tially by FACS analysis (Becton Dickinson). T lymphocytesubsets were labeled with OKT4 (CD4; Ortho Diagnos-tics Systems, Inc.), B9.11 (CD8, Coulter Immunology), andSP34 (CD3; Pharmingen) or FN18 (CD3; Biosource Inter-national) monoclonal antibodies.

ACKNOWLEDGMENTS

The work reported here is supported by NIH Grants MH61230 andDK49516 (E.B.S.). S.W. was supported by the PHS Grant RR00163.

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