Gene Expression of Herpes Simplex Virus - Journal of Virology

JOURNAL OF VIROLOGY, June 1980, p. 604-6140022-538X/80/06-0604/11$02.00/0

Gene Expression of Herpes Simplex VirusII. UV Radiological Analysis of Viral Transcription Units

ROBERT L. MILLETTE* AND ROSEMARIE KLAIBER

Department ofImmunology and Microbiology, Wayne State University School ofMedicine, Detroit,Michigan 48201

The transcriptional organization of the genome of herpes simplex virus type 1was analyzed by measuring the sensitivity of viral polypeptide synthesis to UVirradiation of the infecting virus. Herpes simplex virus type 1 was irradiated withvarious doses of UV light and used to infect xeroderma pigmentosum fibroblasts.Immediate early transcription units were analyzed by having cycloheximidepresent throughout the period of infection, removing the drug at 8 h postinfection,and pulse-labeling proteins with [3S]methionine. Delayed early transcriptionunits were analyzed in similar studies by having 9-fi-D-arabinofuranosyladeninepresent during the experiment to block replication of the input irradiated genome.The viral polypeptides were separated by gel electrophoresis and quantitated bydensitometry of the gel autoradiograms. The following results were obtained. (i)The UV target sizes for the viral transcription units analyzed ranged from 1.44 to5.65 kilobase pairs. This implies that the corresponding primary transcripts haveminimum molecular weights ranging from 0.46 x 106 to 1.82 x 106. (ii) The genesfor the four viral proteins, 165, 145, 116, and 71 (molecular weight x 103), exhibitedUV target sizes that agree with their calculated gene size or measured mRNA sizeor both and thus must reside in promoter-adjacent positions. (iii) The transcrip-tion units for the remaining genes analyzed showed target sizes that range from0.42 to 2.59 kilobase pairs greater than needed to encode the respective proteins.This probably is a reflection of their distances from promoters or the presence ofintervening sequences or both. It further suggests that these genes are transcribedas precursor RNA molecules that are larger than their mRNA's. (iv) The resultsindicate that none of the immediate early genes analyzed can be cotranscribed,whereas some of the delayed early genes might be cotranscribed. No evidencewas found for the existance of large, multigene transcription units.

Previous studies on the characterization ofviral transcripts produced in cells infected withherpes simplex virus type 1 (HSV-1) demon-strated that virus-specific transcripts found inthe nuclei of infected cells range from 10S togreater than 60S, whereas those found in thecytoplasm range from about 10S to 35S (31).This prompted the speculation that HSVmRNA's are synthesized as high-molecular-weight precursors that are processed into lower-molecular weight cytoplasmic mRNA's (31).Subsequent sedimentation studies on HSV-1transcripts selected by liquid hybridization in-dicated that the nuclear transcripts have only a

somewhat larger average size than the cytoplas-mic transcripts (28). However, more recent elec-trophoretic analyses of viral RNA on methyl-mercury-agarose gels have shown that early nu-

clear and polyribosomal RNAs have similar sizedistributions, whereas late nuclear RNA has a

considerably larger average size than the poly-somal RNA (9). Nevertheless, the true size of

the HSV-1 primary transcripts remains in ques-tion since these studies have neither proven aprecursor-product relationship between thelarger nuclear and smaller cytoplasmic HSVRNAs nor taken into account possible rapidprocessing of larger nuclear precursors.

Other recent studies have been directed to-ward physically mapping the viral mRNA's andpolypeptide coding sequences in the viral ge-nome. These have utilized liquid (13) and blothybridizations of RNA to HSV DNA restrictionfragments (3, 4, 9), R-loop mapping (29), andbiochemical analysis of HSV-1 x HSV-2 geneticrecombinants (18, 19, 21). Although these stud-ies have lead to the mapping of a large numberof viral polypeptides, RNAs, and functions, theyhave not yet fully established the number andprecise location of promoters, the polarity ofmost transcription units, the existence of com-mon transcription units and intervening se-quences, or the size and genome map coordinatesof the viral transcription units.

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RADIOLOGICAL ANALYSIS OF HSV TRANSCRIPTION 605

To answer some of these questions regardingthe transcriptional organization of the HSV-1genome, we utilized the UV mapping techniquedeveloped by Sauerbier and co-workers (23, 25).The principle of this method is that the sensitiv-ity of expression of a gene to UV irradiation is afunction of the distance of that gene from itstranscriptional promoter. The effect of UV ir-radiation on the relative rate of gene expressionmay be quantitated either directly, by measuringthe synthesis of specific mRNA's, or indirectly,by measuring the synthesis of the specific poly-peptides. From the UV sensitivity of expressionof a given gene, one can calculate the UV targetsize, in base pairs, of its transcription unit. Thismethod has been used successfully to analyzethe transcriptional organization of a variety ofprocaryotic, eucaryotic, and viral genomes (forreview, see reference 24).By using host cells lacking excision repair

activity, xeroderma pigmentosum (XP) fibro-blasts, to prevent the repair of the input irradi-ated viral genome, we have been able to analyzethe sensitivity of synthesis of a large number ofHSV-1 polypeptides to UV irradiation of theinfecting virus. The results of these studies pro-vide values for the muiimum sizes of viral tran-scription units and their primary transcripts andthe distances of viral genes from their promotersand place limitations on which genes may becotranscribed. Moreover, they show that certainviral genes are promoter adjacent, whereas oth-ers are considerably removed from their pro-moters and therefore must be transcribed aslarger precursors.

MATERIALS AND MEETHODSVirus and celis. HSV-1, Fl strain (7), was propa-

gated in HEp-2 (human epidermoid carcinoma) cellsby a modification of previously described methods (26,27). Confluent HEp-2 cell monolayers in roller bottleswere infected at a multiplicity of 0.02 PFU/cell inphosphate-buffered saline containing 0.1% glucose and1% inactivated calf serum (PBS-gs). After adsorptionat 37°C for 2 h, the medium was replaced with medium199 containing 1% inactivated calf serum, and incuba-tion was continued for 2 to 3 days at 34°C. The cellswere scraped, centrifuged, and resuspended in medium199 plus 1% inactivated calf serum, using 2.5 to 3 ml ofmedium per roller bottle. The cells were disrupted bysonication for 2 min in an ice bath with an MSEUltrasonic Power Unit at maximum power. The celldebris was removed by centrifugation for 30 min at2,000 x g. Virus was purified from the cell extract bycentrifugation through linear gradients of 10 to 50%(wt/vol) sucrose in VB (0.15 M NaCl-0.02 M Tris-hydrochloride [pH 7.5]) for 1 h at 25,000 rpm and 4°Cin a Spinco SW27 rotor. The virus band was collected,diluted threefold in VB, and centrifuged for 1 h at25,000 rpm and 4°C in an SW27 rotor to pellet thevirus. The virus, dissolved in a small volume of PBS-

gs, had a titer of 4 x 109 to 6 x 10i PFU/ml. Virus wasplaque assayed on Vero (African green monkey kid-ney) cells and XP cells with an overlay medium of 1%methylcellulose in Dulbecco-modified Eagle medium(DME) plus 1% inactivated calf serum. Virus andHEp-2 and Vero cells were obtained from B. Roizman,University of Chicago, Chicago, Ill.XP cells (American Type Culture Collection, Rock-

vile, Md.; no. CRL 1223, XP 12BE) were used for allUV mapping experiments. They were propagated inDME plus 10% inactivated fetal bovine serum (FlowLaboratories Inc., Rockville, Md.) and passaged at a 1:3 or 1:4 dilution every 3 or 4 days. HEp-2 cells werepropagated in medium 199 plus 10% inactivated fetalbovine serum in 32-ounce (ca. 0.946-liter) bottles or inmedium 199 plus 5% calf serum in roller bottles; Verocells were propagated in DME plus 10% inactivatedfetal bovine serum. Both kinds of cells were passagedevery 3 to 4 days at a 1:7 to 1:9 dilution.UV irradiation of virus. Mixtures consisting of

1.8 x 109 to 5 x 109 PFU of sucrose gradient-purifiedHSV-1 and 9 x 10'0 to 12 x 1010 PFU of bacteriophageT7 (included as a dose indicator) in 2.0 to 2.5 ml ofPBS-gs were irradiated with constant stirring in a 12-cm watch glass on ice. A UV lamp (Gelman InstrumentCo., Ann Arbor, Mich.) that provided an output of0.38 to 0.54 J/m2 per s at a peak wavelength of 254 nmand distance of 34 cm was used. The virus was irradi-ated with doses varying from 0 to 67 J/m2. Samples of20 to 50 Id were removed at various times and dilutedinto 1 to 2 ml of PBS-gs on ice. The UV inactivationof phage T7 was monitored by removing 10- to 20-ulsamples and titrating on Escherichia coli B., (a re-pair-minus strain from W. Sauerbier, University ofMinnesota, St. Paul, Minn.). This served as an internaldose standard with 1.8 J/ m2 giving one T7 lethal hit(1).

After UV irradiation, all operations involving irra-diated virus and infected XP cells were performedunder yellow light or in darkness until termination ofthe experiments to avoid photoreactivaton of the UV-damaged DNA.

Infecting, labeling, and harvesting cells. Mono-layers of XP cells, 85 to 99% confluent, were preparedin 9.6-cm2 cluster dishes. The cells were infected withunirradiated or UV-irradiated HSV-1 at 20 to 50 PFU/cell in 0.4 ml of PBS-gs for 1 h at 37°C. Virus wasremoved, and the cells were overlaid with 2 ml ofDME plus 1% inactivated calf serum containing eitherno inhibitors, 50 Ag of cycloheximide (Calbiochem, LaJolla, Calif.) per ml, or 100 to 150 AM 9-.8-D-arabino-furanosyladenine (ara-A) and 1 jg of Covidarabine (2-deoxycoformycin) per ml (both were gifts from H.Machamer, Parke, Davis & Co., Detroit, Mich.). At 2to 8 h postinfection (times after addition of virus tocells), the cells were rinsed three times withDME plus1% inactivated calf serum (when cycloheximide wasused) and two times with MEM-1/100 Met-1% DCS(minimal essential medium plus 1% dialyzed calfserumand containing 1/100 the usual amount of methionine)and then labeled with 0.4 ml of MEM-1/100 Metcontaining [3S]methionine (Amersham Corp., Arling-ton Heights, Ill.) at 20 to 30 1Ci/ml for 45 min at 37°C.The labeling medium was then removed, and the cellswere rinsed two times with ice-cold phosphate-

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606 MILLETTE AND KLAIBER

buffered saline, lysed with 0.2 or 0.3 ml of lysing buffer(2% sodium dodecyl sulfate, 0.05M Tris-hydrochloride[pH 7.0], 0.7 M 8-mercaptoethanol, 5% sucrose, andbromophenol blue), and frozen at -90°C until electro-phoresis.Polyacrylamide gel electrophoresis. Infected

cell lysates were thawed and removed from the culturedishes with the aid of a scraper, sonicated briefly toreduce the viscosity, and heated for 2 min at 100°C todenature proteins. Polyacrylamide slab gels (140 by160 by 1.3 mm) were prepared essentially by themethod of Laemmli (15) but with 0.36% N,N'-diallyl-tartardiamide (Eastman Organic Chemicals, Roches-ter, N.Y.) as a cross-linker (8). A running gel of 8.5%acrylamide and a stacking gel of 3.5 or 4% acrylamidewere used. Protein samples containing 8,000 to 50,000cpm of 3S in 20 to 50 pl were applied and subjected toelectrophoresis for 6.5 to 7.5 h at 200 V and 12 to 16mA. Gels were stained for 4 h with 0.25% Coomassiebrilliant blue (Sigma Chemical Co., St. Louis, Mo.) inethanol-acetic acid-water (5:1:5), destained with wa-ter-acetic acid-isopropanol (8:1:1), dried by heatingunder vacuum on Whatman 3MM paper, and exposedto Kodak X-Omat RP film for 3 to 20 days for auto-radiography. Polypeptides were quantitated by scan-ning films with a Zienah soft-laser scanning densitom-eter (Biomed Instruments, Inc., Chicago, Ill.) andmeasuring peak heights. Studies with labeled proteinstandards run on gels showed that peak heights weredirectly proportional to the quantity of protein labelunder the conditions used.

Molecular weights of viral polypeptides were deter-mined from mobilities relative to protein standards(Escherichia coli RNA polymerase, bovine serum al-bumin, ovalbumin, immunoglobulin G, E. coli DNaseI, papain, trypsin, chymotrypsin, and pepsin). Viralpolypeptides were numbered according to their molec-ular weights in thousands and related to ICP (infected-cell polypeptide) numbers of Honess and Roizman (10,11).

Calculations. The percentage of the amount ofeach polypeptide synthesized relative to the unirra-diated control sample was calculated and plotted on asemilog plot as a function of UV dose to the infectingvirus in joules per square meter. Lines giving the bestfit to the equation ln(Rd/Ro) = -kd were calculatedby the curve-fitting program on a Hewlett-Packard(HP 97) calculator. In this expression, Rd is the rate ofsynthesis of the individual polypeptide at UV dose din joules per square meter, Ro is the rate of synthesisof the polypeptide at a zero UV dose, and k is the first-order rate constant or UV inactivation cross section insquare meters per joule. The slopes of the lines wereequal to the UV inactivation cross sections, k; thereciprocal of k provided the UV dose yielding one hit,or 37% inactivation of polypeptide synthesis. The UVtarget size of transcription units in base pairs wascalculated by using the value of 2.30 x 10-2 m2/J asthe UV inactivation cross section for 1,000 base pairsofDNA under our conditions. This value was obtainedfrom a plot of the UV inactivation cross sections, k,versus the sizes of the genes for the individual poly-peptides in base pairs (see below, Fig. 5). The numberof base pairs required to encode each polypeptide was

J. VIROL.

calculated from the following relationship: number ofbase pairs = molecular weight of polypeptide x 3/115,where 115 equals the average molecular weight of anamino acid.

RESULTSUV radiological analysis of early viral

genes. In the first series of UV mapping exper-iments, we analyzed the sensitivity of synthesisof HSV-1 immediate early (IE) polypeptides toUV irradiation of the virus. The mRNA for theIE proteins accumulates in the cytoplasm ofinfected cells during a cycloheximide block, andit can be assayed by its translation into polypep-tides immediately after removal of the drug (11,14). The virus was irradiated with increasingdoses of UV light and then used to infect XPcells. Cycloheximide was present from 1 h afteraddition of virus and throughout the period ofinfection. At 7 or 8 h postinfection , the drug wasremoved, the cells were pulse-labeled with[3S]methionine, and the labeled polypeptideswere analyzed by polyacrylamide gel electropho-resis and autoradiography. Six IE viral polypep-tides can readily be distinguished by the sensi-tivity of their synthesis to UV irradiation of theinfecting virus (Fig. 1). These are designated asviral polypeptides 165, 145, 123, 86, 71, and 55 interms of their molecular weights x 103 as deter-mined from gel mobilities. According to the Ho-ness and Roizman nomenclature (10, 11), thesepresumably correspond to the a and ,B polypep-tides ICP 4, 6, 0, 20, 22, and 27. In some experi-ments, a delayed early (DE) polypeptide, 116,probably the ft polypeptide ICP 10, was detectedand analyzed. This protein usually does not ap-pear when cycloheximide is added with or beforethe addition of virus. However, to achieve abetter host turnoff in these experiments, thedrug was added at the time of virus removal, 1h postinfection (30).When the relative amount of each viral pro-

tein synthesized was determined by densitome-try of the autoradiograms and its logarithm wasplotted against the UV dose given to the infect-ing virus, a series of first-order relationships wasobtained (Fig. 2). The lines represent the best-fit plots for the datum points as determined bycomputer analysis. It is apparent that the UVsensitivity of synthesis of each polypeptide isnot a direct function of its molecular weight aswould be expected if the mRNA for each proteinwere transcribed from a continuous promoter-adjacent sequence.The slopes of the lines yield the first-order

rate constants for UV inactivation, or the UVinactivation cross sections, k, which are a meas-ure of the relative UV sensitivity of the expres-

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UV Dose (J.m-2)U C 0 7.46 14.9 224 29.8

165

145-123-

116- ;

86 S£*

71 - Op"-

i .

55 -

FIG. 1. Effect of UV irradiation on HSV-1 early gene expression. HSV-1 was irradiated with UV light atthe indicated doses and used to infect XP cells. Cycloheximide (50 pg/ml) was present from 1 to 8 hpostinfection. The drug was removed, and theproteins were labeled with [3S]methionine for 45 min. Proteinswere analyzed by gel electrophoresis and autoradiography. .Viralpolypeptides are numbered in thousands ofdaltons. (U) Uninfected cell proteins; (C) control, infected cells not treated with cycloheximide.

sion of each gene. From these values, using theintrinsic dose-response factor of 2.30 x 10-2 M2/J (see below, Fig. 5), we calculated the targetsizes in base pairs of the transcription units ofthe early genes analyzed (Table 1). Since theUV sensitivity assay does not take into accounttranscription "downstream" from the gene beinganalyzed (unless these sequences would be re-quired for the production of functional mRNA),the values shown represent minimum sizes forthe early transcription units. In Table 1, thetranscription unit target sizes are compared withthe number of base pairs calculated to encodeeach polypeptide. Valid data were not obtainedfor polypeptide 71 since it comigrates with a hostprotein.From these data we conclude the following.

(i) The minimum target sizes for the early genetranscription units analyzed range from 3.05 to

5.35 kilobase pairs. (ii) The genes for proteins116 and 165 have UV target sizes that corre-spond closely to their predicted gene sizes andthus most likely reside in promoter-adjacent po-sitions. (iii) The genes for viral proteins 145, 123,86, and 55 show UV target sizes that are 0.46 to2.59 kilobase pairs greater than needed to encodetheir respective polypeptides. This could be areflection of their distances from promoters orthe presence of intervening sequences or bothand indicates that their primary transcripts are

considerably larger than the size required fortheir mRNA.UV radiological analysis of DE HSV-1

genes. In this series of experiments, we allowedviral proteins to be synthesized from the onsetof infection, but to prevent the production ofnonirradiated progeny DNA molecules, we in-hibited viral DNA replication with ara-A. As

373 44.8

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1001O XL1 231 165

3 ~~~~~~~~~~~~~~~145

10 20 30 10 20 30

UV DoW (J-m2)

FIG. 2. Relative rates of early protein synthesis as a function of UV dose. Relative rates of synthesis weredeternined by densitometry of several gel autoradiograhzs, including that shown in Fig. 1. Lines representcalculated best-fit first-order plots from the averages of several experiments. The different kinds of datumpoints indicate separate experiments. For a better comparison, all lines were normalized so as to intersect theordinate at 100%. Viral polypeptide numbers are given in the right-hand margins.

TABLE 1. UV sensitivity and target sizes of earlyHSV-I polypeptides

UV inacti- BaseViral vationa Target pairs'poly-

crs e- SZb required Differencepeptide tion,k (base to en- (base(mol t tion2k bas code pairs)x 10-3) (xl0-2) pairs) pe°yd

(x102) ~~peptide165 9.66 4,200 4,300 -100145 12.3 5,350 3,780 1,570123 8.44 3,670 3,210 460116 7.02 3,050 3,030 2086 11.8 4,830 2,240 2,59055 7.57 3,290 1,430 1,860

a k values were determined by computer from theslopes of the UV inactivation plots. The values repre-sent averages of three or four separate experiments.

b Target sizes were calculated by dividing the UVinactivation cross section values, k, by the intrinsiccalibration factor, 2.3 x 10-2 m2/J per 1,000 base pairs.'Number of base pairs = molecular weight of poly-

peptide x (3/115).

previously shown by Drach and Shipman (6),this drug, in conjunction with the adenosinedeaminase inhibitor convidarabine, will prefer-entially and effectively block HSV-1 DNA syn-thesis. Futhermore, we have found that at con-centrations of these drugs that quantitativelyblock HSV-1 DNA synthesis, all but the lateclasses of viral proteins are synthesized in in-fected XP cells (Pedersen et al., submitted forpublication).Monolayers of XP cells were infected with

virus that had been irradiated with various dosesof UV light and then further incubated withmedium containing ara-A and covidarabine. Theinfected cells were pulse-labeled for 45 min with[3S]methionine at 2 to 5 h postinfection, andthe viral polypeptides were analyzed by polyac-rylamide gel electrophoresis and autoradiogra-phy.A large number of viral polypeptides can be

identified by correlating their mobilities withpreviously published gel data (10, 11) and fromthe sensitivity of their synthesis to UV irradia-tion of the infecting virus (Fig. 3). From thekinetics of synthesis of these polypeptides (Ped-ersen et al., submitted for publication), mostappear to belong to the DE, or fl, polypeptideclass (11). Since ara-A also causes a delayedturnoff of many of the early proteins (Fig. 3 andPedersen et al., submitted for publication), sev-eral IE polypeptides also appeared in these anal-yses. In addition, a late polypeptide, 154, wasconsistently observed.The "survival" curves for the UV sensitivity

of synthesis of these polypeptides are plotted inFig. 4. As in the preceding IE gene studies, onecan again observe that no direct correlation be-tween UV sensitivity and polypeptide size exists.From the kinetic data, we calculated the UVinactivation cross sections and transcription unittarget sizes for the viral genes expressed in thepresence of ara-A (Table 2). The results may besummarized as follows. (i) All transcription unitsanalyzed in the ara-A experiments have mini-mum target sizes that range from 1.44 to 5.65

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UV Dose (J.m-2)U 0 9.5 19 2&5 38 475 57 66.5 U.~~~~~~ w

1e"_t_S.

Ins. ~ ~ ~ f

-~ ~ -

1P#.usar -S

n - -.--

32 34-

FIG. 3. Effect of UV irradiation on HSV-1 early and late polypeptides synthesized in the presence of ara-A. HSV-1 was irradiated with LW light at the doses indicated and used to infect XP cells. Ara-A (150 gA)and covidarabine (1 pg/ml) werepresent from I to 5.75 hpostinfection. Cells were labeled with [35rJmethioninefrom 5 to 5.75 h postinfection. Proteins were analzed by gel electrophoresis and autoradiography. Viralpeptides are designated by molecular weight x 10- . (U) Uninfected cell controls.

~~~~~~~~~~~~~~~~~~A

ae~~~~~~~~~~

10 20 30 10 20 30 10 20 0

V Dor (j m2)

FIG. 4. Relative rates of synthesis of viral polypeptides, synthesized in the presence of ara-A, as a functionof UV dose. Lines plotted represent computer-determined, best-fit first-order plots of data from two or threeexperiments such as the one shown in Fig. 3. The different symbols represent different experiments. Viralpolypeptide numbers are indicated in the right margins.

kilobase pairs. (ii) The UV target sizes for theexpression of most genes analyzed are consider-ably larger than the number of base pairs cal-

culated to encode their respective polypeptides.The data indicate that these genes are removedfrom their transcriptional promoters by about

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145 154-126 130:116 119=

86 92-

71-61-55-

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TABLE 2. UV sensitivity and target sizes of earlyand late HSV-1 genes expressed in the presence of

ara-A a

UV in-Viral activa- Tat Base pairmpoly- tion sairzge required Difference

peptide cross (b to encode (base(mol wt section, ase poly- pairs)x 10-3) k (m2/J) pairs) peptide

(x10-2)

165 7.97 3,465 4,300 -840154 13.0 5,650 4,020 1,630145 10.5 4,570 3,780 790130 10.2 4,430 3,390 1,040126 10.4 4,520 3,290 1,230119 9.61 4,180 3,100 1,080116 7.07 3,070 3,030 4092 6.48 2,820 2,400 42086 6.94 3,020 2,240 78071 4.24 1,850 1,830 2061 7.21 3,130 1,590 1,54055 3.31 1,440 1,430 1034 7.63 3,320 890 2,43032 7.74 3,370 840 2,530

a For calculations, see Table 1 and text. The valuesshown represent averages of four or five separateexperiments.

0.42 to 2.53 kilobase pairs and are, therefore,most likely transcribed as larger precursor RNAmolecules. (iii) The genes for viral proteins 116,71, and 55, however, show a UV sensitivity com-mensurate with their predicted gene size in basepairs. Thus these genes most likely reside inpromoter-adjacent positions.

It should be noted that the UV target sizes forseveral IE genes were about 450 to 1,800 basepairs smaller in these studies than in the preced-ing cycloheximide experiments. From kineticanalyses of the polypeptides synthesized in thepresence and absence of ara-A ( Pedersen et al.,submitted for publication) and from studies onthe partial proteolytic peptides of several of theIE polypeptides (S. Talley-Brown, unpublisheddata), it seems that we were observing the sameIE polypeptides in both experiments. There aretwo likely explanations for this apparent shift inthe UV sensitivity of these genes: (i) expressionof the DE genes might allow transcription of theIE genes from a more proximal promoter; and(ii) since DE gene expression is required for theturnoff of the IE genes (11, 12), UV inactivationof the DE genes might cause a decreased rate ofturnoff and a lower apparent UV sensitivity ofthe IE genes. With the present data, we cannotdistinguish between these possibilities. How-ever, it is clear from these considerations andfrom the higher level of IE gene expression aftercycloheximide reversal that the more valid dataon the IE genes come from the cycloheximideexperiments.

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Conversion ofUV inactivation cross sec-tions into transcriptional distances. To con-vert UV inactivation cross sections into tran-scriptional distances, ideally, one should have asa standard an internal transcription unit ofknown size that is expressed under the sameexperimental conditions. Lacking a well-definedviral transcription unit in this system, we estab-lished an intrinsic calibration by using several ofthe genes analyzed. This is based on the as-sumption that genes having minimum ratios ofUV cross section to gene size reside in promoter-adjacent positions. Thus, in a plot of UV inacti-vation cross section versus RNA size in basepairs or molecular weight, the genes adjacent topromoters will establish a line of intrinsic cali-bration for the UV dose response under theseexperimental conditions (for a discussion of cal-ibration methods, see reference 24). In such aplot with the present data, the genes for fourviral proteins, 55, 71, 116, and 165, establish anintrinsic calibration line (Fig. 5). Further supportfor this calibration is obtained if we use theactual measured sizes of several IE mRNA'sinstead of the gene sizes calculated from poly-peptide molecular weights. By electrophoresis indenaturing gels, we found that the mRNA's forviral proteins 165 and 145 have sizes of 3.8 and5.2 kilobases, respectively (S. Talley-Brown andR. Millette, submitted for publication). Usingthese values in the calibration plot (open sym-bols, Fig. 5), we found that the UV inactivationcross sections for these genes fall very near theintrinsic calibration line. This line shows that1,000 base pairs of HSV-1 DNA have a UVinactivation cross section of 2.30 x 10-2 m2/Junder these conditions. In other words, it takes43.5 J/m2 to produce one transcription-termi-nating lesion per 1,000 base pairs of HSV-1DNA. We used this standard cross section tocalculate the transcriptional distances.

DISCUSSIONIn these studies we have demonstrated that

XP cells that lack detectable DNA excision re-pair activity can be used effectively to performUV mapping experiments on HSV gene expres-sion. These cells offer several advantages forsuch experiments. (i) They allow UV mappingstudies to be carried out at the translationallevel, providing that viral DNA replication andpossible virus-induced DNA repair can beblocked. To obtain valid results by this method,however, the rate of polypeptide synthesis mustbe proportional to the amount of mRNA syn-thesized. This seemed to be the case in thepresent experiments since the UV inactivationcurves showed first-order inactivation kineticsto a 15% survival level or less in most cases. (ii)

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-1

II

'I

UV 1noc*tvtion Cross Section, k x102

(m2/J)FIG. 5. Plot of gene size versus UV inactivation

cross section. The datum points are taken from Ta-bles 1 and 2. The line represents an intrinsic calibra-tion for relating UV dose to transcription-terminat-ing lesions produced by UV irradiation in the viralDNA, where k = 2.3 x 10-2 m2/Jper 1,000 basepairsofDNA. Symbols: 0, datum points from the cyclohex-imide experiments; A, datum points from the ara-Aexperiments; 0 and A, datum points for the genes ofproteins 145 and 165 replotted by using the measuredsizes of their mRNA's.

In contrast to UV mapping studies in whichinfected cell complexes are irradiated, studieswith XP cells can be performed by irradiatingonly the free virus. This assures that the meas-

ured UV effects are a direct result of UV lesionsin the viral genome and allows a more precisemeasurement of the actual UV dose delivered tothe DNA. (iii) XP cells permit UV mappingstudies to be performed under essentially DNArepair-free conditions. With other cell lines, thiscondition can be approximated only by pulse-labeling the infected cell complexes immediatelyafter irradiation. However, this again involvesdose measurement problems and precludes UVmapping studies at the translational level.The main conclusions derived from these ex-

periments are the following. (i) The UV targetsizes for the transcription of all HSV-1 genesanalyzed range from 1.43 to 5.65 kilobase pairs.This implies that the corresponding primary

transcripts have minimum sizes that range fromabout 0.46 x 106 to 1.82 x 106 daltons of RNA.(ii) Several of the genes studied, those for pro-teins 165, 146, 116, and 71, exhibit UV targetsizes that coincide with their calculated genesizes or measured mRNA sizes or both. Thissuggests that these genes are promoter adjacentand do not have extensive intervening se-quences. (iii) The UV target sizes for the tran-scription of most of the genes studied are largerthan calculated to code for their respective poly-peptides. Assuming that the transcription of se-quences downstream from a gene is not requiredfor the production of its mRNA, the excess basepairs probably reflect the distances from pro-moters or the presence of intervening sequencesor both. (iv) A comparison of the measured UVtarget sizes with the number ofbase pairs neededto code for the corresponding polypeptides (Ta-bles 1 and 2) reveals that few, if any, of the HSV-1 genes analyzed could reside in common tran-scription units. However, overlapping transcrip-tion units, such as those observed for adenovirusand simian virus 40, cannot be ruled out.Absence of HSV DNA repair and repli-

cation. To successfully apply the UV mappingtechnique to the analysis of viral gene expres-sion, it is essential that the irradiated viral ge-nome be neither repaired nor replicated duringthe experiment. The following considerationsshow that these requirements have been satis-fied.

(i) Host cell DNA repair is negligible in thissystem. The XP cell line used in the studies(CRL 1223, XP 12BE) belongs to the A comple-mentation group, the most UV sensitive of theXP cell lines. Studies showing that these cellshave less than 2% of the rate of DNA repairfound in normal human fibroblasts (22) havebeen verified by S. Talley-Brown in our labora-tory with repair assays in cell lysates by themethod of Ciarrochi and Linn (2) (unpublisheddata). Furthermore, studies on the prolongedsurvival of XP cells after UV irradiation byMaher et al. (17) and on the repair of UV-irra-diated adenovirus by Day (5) have shown thatthere is virtually no DNA repair activity in thesecells.

(ii) Viral DNA repair was not detectable inthe presence of cycloheximide. In the experi-ments on IE viral gene expression, the presenceof cycloheximide prevented synthesis of theHSV-1 DNA polymerase and potential virus-specified repair enzymes. After removal of thedrug, the brief 45-min pulse-labeling period al-lowed mainly the IE proteins to be synthesized.In experiments not shown, the UV sensitivity ofIE polypeptide synthesis was determined afterthe irradiated viral genomes had been in the

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cells for 4, 8, or 12 h in the presence of cyclohex-imide. For any given IE gene, no significantchange occurred in the slope of the UV inacti-vation curve with increasing times postinfection(data not shown). Thus, there was no detectablerepair or replication of viral DNA under theseconditions.

(iii) Viral DNA repair and replication werenot detectable in the presence of ara-A. To studythe UV inactivation of DE viral genes, we usedara-A to block viral DNA replication. Underthese conditions, viral DNA synthesis was neg-ligible (less than 0.15% of the control levels[Pedersen et al., submitted for publication]). Inadditional UV experiments, viral polypeptideswere pulse-labeled for 45 min after 2 to 12 h ofinfection in the presence of ara-A. During thefirst 5 h after infection, the UV inactivation crosssections for each of the DE genes remainedconstant (data not shown). This indicates thatthere was no detectable loss of transcription-terminating lesions from the viral DNA duringthis period.Implications in the mapping of HSV-1

genes. The measured UV target sizes place anumber of limitations on the transcriptional or-ganization of the HSV-1 genome. A comparisonof the UV target sizes of the IE genes with thenumber of nucleotides needed to code for theirpolypeptides (Table 1) indicates that it is notpossible for any of the IE genes that we analyzedto be contranscribed. This conclusion would bevalid even if we overestimated the magnitude ofthe dose-response calibration factor.Mapping data from several laboratories have

shown that the gene for IE protein 165 (ICP 4)maps in the terminal and internal repeat se-quences (TRs and IRs) bracketing the smallunique sequence (Us) and that the gene forprotein 123 (ICP 0) maps in the repeats (TRLand IRL) adjoining the large unique (UL) DNAsequence (3, 18-20). The data presented hereeliminate the possibility of contranscription ofthe genes across the IRL-IRs junction and alsopreclude transcription of the IE genes from asingle promoter region on a circular template, aswas proposed recently by Clements et al. (3).A comparison of the UV target sizes with the

number of base pairs needed to code for the DEpolypeptides (Table 2) reveals that there areonly a few possibilities for contranscription. Al-though the data are compatible with the genefor protein 71 being promoter proximal to thatof 32 or 34 and the gene for protein 55 lyingpromoter proximal to either 154, 61, 34, or 32,additional physical and UV mapping data willbe needed to prove any cotranscriptions. Fur-thermore, the measured UV target sizes provide

no indication for the existence of large multigenetranscription units such as that found for ade-novirus late transcription.UV inactivation of IE genes does not af-

fect the UV senstivity data ofthe DE genes.There is now considerable evidence that at leastone of the IE proteins is required for the expres-sion of the DE and late viral polypeptides (11,12, 16, 20). It is conceivable, therefore, that theUV inactivation curves observed for the DEgenes represent a summation of two effects: theinactivation of one or more of the IE genes andthe inactivation of the individual DE genes. Ifthis were the case, one can envision severalpossible modes of action of the IE gene prod-uct(s) on DE transcription.

(i) An IE gene product is required in verysmall or catalytic amounts for the turn on ofDEgenes. Let us assume for this discussion that thisis an IE gene having an average UV target size,gene 165. Since we infected the cells at a multi-plicity of 30 and inactivated the gene for poly-peptide 165 to about 10% of the control at themaximum UV dose, there remained at this levelof inactivation approximately three good copiesof gene 165 product per cell. According to Pois-son distribution, the fraction of cells receivingno intact copies in this case would be e-&, or 0.05.This would cause, at most, only a slight down-ward curvature to the observed UV inactivationcurves at the higher doses. The values at a dosegiving a 90% reduction in polypeptide synthesis(28 J/m2) would be lowered by only about 5%. Ifthe expression of three different IE genes wererequired, assuming for simplicity that each hasthe UV sensitivity of gene 165, the DE UVinactivation curves at 28 J/m2 would be loweredby only 15%. This does not appear to be the casesince no significant downward curvatures wereobserved in the DE UV inactivation curves.

(ii) A certain threshold level of the IE geneproduct is required for maximum DE geneexpression; below that level the amount of DEexpression is proportional to the amount of IEgene expression. In this case our observed DEgene inactivation curves would be biphasic, firstshowing a slope intrinsic to the UV inactivationof the DE gene and then exhibiting a slope thatis the sum of that of the DE gene and that ofthe IE gene. Such a mechanism is very unlikelysince biphasic curves for the UV inactivation ofDE gene expression were not observed, and thetarget sizes for the inactivation ofmost DE genesare not large enough to include additional IEgene target sizes.

(iii) The amount of DE gene expression isdirectly proportional to the amount of the IEgene expression. In this most extreme case we

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would expect to see first-order UV inactivationcurves for the DE genes, but their slopes wouldbe equal to the sum of the k values for the IEplus the DE gene. This would yield apparenttarget sizes for the DE genes that would be atleast 4,200 base pairs (the average target size foran IE gene) in excess of their coding require-ments. In the case in which more than one IEgene product would be required for DE expres-sion, the apparent DE gene target sizes wouldbe even larger. The results given in Table 2 forthe DE genes show that this was not the case.From these considerations, we must concludethat the UV inactivation parameters that weobserved for the DE and late HSV-1 genes arenot significantly altered by the UV sensitivity ofthe IE genes.

In conclusion, we have shown that by usingexogenously irradiated virus with XP cells, onecan analyze the transcriptional organization ofthe HSV-1 genome by UV mapping. The datagenerated by this method have provided valuesfor the minimum sizes of viral transcription unitsand distances of genes from their promoters andhave placed restrictions on which genes may becotranscribed. This method should be equallyapplicable for studying the transcription of otheranimal viruses, as long as viral gene expressioncan be restricted to the input, unrepaired paren-tal genome. The transcriptional distances ob-tained by this method should complement thoseobtained by physical and genetic mapping inthat they should include all nontranslated se-quences required for gene expression. Futher-more, they can provide direct evidence for co-transcription, polarity of transcription, and pro-moter shifts.

ACKNOWLEDGMENTSWe are grateful to Sue Talley-Brown and Margaret Ped-

ersen for enlightening discussions, invaluable assistance in thisresearch, and many hours devoted to reading manuscripts anddrawing figures. Special thanks are due Walter Sauerbier,University of Minnesota, for his helpful criticisms and discus-sions of this work.

This investigation was supported by Public Health Servicegrant CA 21065 awarded by the National Cancer Institute.

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2. Ciarrochi, G., and S. Linn. 1978. A cell-free assay meas-uring repair DNA synthesis in human fibroblasts. Proc.Natl. Acad. Sci. USA 75:1887-1891.

3. Clements, J. B., J. McLauchlan, and D. J. McGeoch.1979. Orientation of herpes simplex virus type 1 imme-diate early mRNA's. Nucleic Acids Res. 7:77-91.

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17. Maher, V. M., D. J. Dorney, B. Konze-Thomas, A.Mendrala, and J. J. McCormick. 1979. DNA excisionrepair processes in human cells can eliminate the cyto-toxic and mutagenic consequences of ultraviolet irradia-tion. Mutat. Res. 62:311-323.

18. Marsden, H. S., N. D. Stow, V. G. Preston, M. C.Timbury, and N. M. Wilkie. 1978. Physical mappingof herpes simplex virus-induced polypeptides. J. Virol.28:624-642.

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25. Sauerbier, W., R. L. Millette, and P. B. Hackett, Jr.1970. The effects of ultraviolet irradiation on the tran-scription of T4 DNA. Biochim. Biophys. Acta 209:368-386.

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29. Stringer, J. R., L. E. Holland, and E. K. Wagner. 1978.Mapping early transcripts of herpes simplex virus type1 by electron microscopy. J. Virol. 27:56-73.

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31. Wagner, E. K., and B. Roizman. 1969. RNA synthesisin cells infected with herpes simpex virus. II. Evidencethat a class of viral mRNA is derived from a highmolecular weight precursor synthesized in the nucleus.Proc. Natl. Acad. Sci. U.S.A. 64:626-633.

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Gene Expression of Herpes Simplex Virus - Journal of Virology

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