JOURNAL OF VIROLOGY, May 1993, P. 2575-25820022-538X/93/052575-08$02.00/0Copyright © 1993, American Society for Microbiology

Open Reading Frames UL44, IRS1/TRS1, and UL36-38 AreRequired for Transient Complementation of HumanCytomegalovirus oriLyt-Dependent DNA Synthesis

GREGORY S. PARI,1 MARILYN A. KACICA,2 AND DAVID G. ANDERS*

Laboratory of Virology, The Wadsworth Center for Laboratories and Research, New York State Department ofHealth,and Department ofBiomedical Sciences, SUNY School ofPublic Health, Albany, New York 12201-0509,1 and

Department ofPediatrics, Albany Medical College, Albany, New York 122012

Received 18 November 1992/Accepted 19 January 1993

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oniLyt are replicatedafter transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G.Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now usedoriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfectedcosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replicationwas oniLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMVlytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes

that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequencecomparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependentDNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplexvirus type 1 replication genes. We further used the assay to define essential regions in two of the requiredcosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemicalevidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition,three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNAsynthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activateUIA4 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IEl and IE2. Combined,these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus,it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performinglytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in humanfibroblasts.

The set of human cytomegalovirus (HCMV) genes thatcarry out lytic-phase DNA replication has not been estab-lished, largely because of difficulties in performing geneticstudies in this system. Therefore, much of our understandingofHCMV DNA replication has been gleaned by comparisonwith the prototype herpesvirus, herpes simplex virus type 1(HSV-1). Challberg and colleagues defined seven HSV-1genes as necessary and sufficient for origin-dependent DNAreplication by using a transient cotransfection assay to testthe ability of cloned fragments of HSV DNA to support thereplication of an origin-containing plasmid (9-11, 63). Thesegenes encode a DNA polymerase (UL30) and an associatedpolymerase accessory protein (UL42), a single-stranded-DNA-binding protein (UL29), a heterotrimeric helicase-primase complex (UL5, UL8, and UL52), and an origin-binding protein (UL9) (reviewed in references 10 and 11).Their results proved to be a nexus for extensive genetic andbiochemical studies (e.g., references 8, 24, 42, 44, 50, 51,and 61; reviewed in reference 60). Candidate HCMV ho-mologs of four of these HSV-1 replication genes, including a

DNA polymerase (UL54), a single-stranded-DNA-bindingprotein (UL57), and two subunits of the helicase-primasecomplex (UL105 and UL70), were identified by sequencesimilarity (12). In addition, HCMV UL44 was identified as a

candidate polymerase accessory protein on the basis of

* Corresponding author.

biochemical studies of the expressed protein (18, 19).HCMV open reading frames (ORFs) positionally equivalentto HSV-1 UL8 and UL9 are not detectably similar insequence (12). Protein products of ORFs UL57, UL54, andUL44 have been purified and characterized to various de-grees (1, 3, 18, 19, 31, 34, 35), but except for the DNApolymerase, there has been little direct evidence that thesecandidate homologs are required for viral DNA synthesis invivo.

It is important to better define all of the components ofHCMV replication, because they confer the unique range ofsensitivities exhibited for several antiviral agents. In addi-tion, these components may provide useful targets for therational design of new anti-HCMV chemotherapeutic agents.There may be significant differences between the compo-nents of HCMV DNA replication and those of other herpes-viruses, despite the identification of candidate HCMV ho-mologs of several of the HSV-1 replication genes andobserved similarities in the DNA replication process. Forexample, one conspicuous gap is the failure to identify bysequence similarity an HCMV homolog of the HSV-1 origin-binding protein (12). This absence is particularly intriguing inthe light of the distinctive structure of an HCMV origin oflytic-phase DNA replication, oriLyt (2, 4, 29, 43). In thisregard, it was recently suggested that the gammaherpesvirusEpstein-Barr virus (EBV) lacks an equivalent of HSV-1 UL9(20). Finally, some essential HCMV replication genes may

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be unique in that other herpesviruses do not encode ho-mologs.Our laboratory (2, 4) and others (29, 43) recently described

HCMV oniLyt. HCMV onLyt replicates autonomouslywhen transfected into permissive cells if essential trans-acting factors are supplied by HCMV infection, which hasfacilitated its characterization (2, 4, 43). This finding sug-

gested to us that the cotransfection strategy described byChallberg (9) could be used to locate essential HCMVreplication genes. In this report, we describe a transientcomplementation assay in which trans-acting factors neces-

sary for HCMV lytic-phase DNA replication are provided bya set of overlapping cosmids. We further describe the use ofthis assay to show that HCMV UL44, a proposed homologof the HSV-1 polymerase accessory protein (18), is requiredfor complementation of origin-dependent DNA replication.Moreover, IRS1/TRS1, a recently described transcriptionactivator that acts cooperatively with HCMV immediate-early (IE) proteins to upregulate the UL44 promoter (55),also was indispensable, as were two other regions encodingpreviously described HCMV transcriptional activators, themajor IE (MIE) regions 1 and 2, and ORFs UL36-38.

MATERIALS AND METHODS

Cells and virus. Human foreskin fibroblast (HFF) cellswere used for all experiments and were cultured in Dulbec-co's modified Eagle's medium (DMEM) supplemented with10% (vol/vol) fetal calf serum. HCMV strain AD169 (Amer-ican Type Culture Collection) was maintained as frozenstocks. Infection was done at an approximate multiplicity ofinfection of 10. Phosphonoformic acid (PFA; Sigma Chemi-cal Co., St. Louis, Mo.) was used at a final concentration of200 jig/ml and added either at 24 h posttransfection or at thetime of infection, as indicated.

Plasmid constructs. Construction of the replication re-

porter pSP50 was described previously (2); it containsHCMV oriLyt as a 5.6-kbp PvuII-to-KpnI subfragment ofHindIII-D. HCMV cosmid clones (21) were kindly suppliedby A. Colberg-Poley with the permission of B. Fleckenstein.Subclones of these cosmids all were constructed inpGEM7ZF(-) (Promega, Madison, Wis.) by using standardprocedures (52). pSVH contains HCMV IEl and IE2 (56)and was a gift of R. Stenberg. A clone containing strainAD169 TRS1 (pXEXX-6.1) was obtained from T. Jones (36),and plasmids containing strain Towne TRS1 (pON2334) andTRS1 with a frameshift mutation (pON2336) were suppliedby E. Mocarski (55). Additional plasmid constructs were

made as detailed below.(i) Subclones of cosmid pCM1052. Plasmid pZP1 was made

by excising from a low-melting-point (LMP) agarose gel the20-kbp HindIII G fragment derived from pCM1052 andligating it into pGEM7Zf(-). Plasmid pZP2 was made byexcising the 12.6-kbp EcoRI F fragment from an LMP geland ligating it into pGEM7Zf(-). Plasmid pZP3 was made byligating the 3.6-kbp SmaI fragment extending from nucle-otides (nt) 189500 to 193108 into pGEM7Zf(-).

(ii) Subclones of pCM1017. Plasmid pZP4 was made byligating into pGEM7Zf(-) the 10.3-kb HindIII M fragment.pZP5 was made by excising the 7.0-kb Sacl fragment ex-

tending from nt 52260 to 59219 from an LMP gel and ligatingit into pGEM7Zf(-). pZP6 was made by excising the 8.0-kbpBglII fragment extending from nt 47366 to 56170 from an

LMP gel and ligating it into pGEM7Zf(-). pZP7 was madeby ligating the 2.6-kbp KpnI fragment extending from nt54118 to 56701 into pGEM7Zf(-). pAZP7 is pZP7 with a

582-bp HinclI deletion extending from nt 54231 to 54813 thatremoves 429 nt of UL42 and 432 nt of UL43. pZP8 was madeby ligating the 11-kbp KpnI fragment extending from nt42045 to 53104 into pGEM7Zf(-).

Transfection and replication assay. HFF cells were platedonto 6-cm-diameter dishes at a cell density of 105 per plate 24h before transfection. Approximately 4 h prior to transfec-tion, the media were replaced with 3 ml of fresh DMEM.DNA was transfected by using the procedure described byChen and Okayama (14). Briefly, 4 Rg of pSP50, 1 ,ug each ofcosmids pCM1017, pCM1029, pCM1039, pCM1035,pCM1052, pCM1058, and pCM1015, and 1 ,ug of pSVH wereadded to a 5-ml sterile test tube. To this DNA mixture wasadded 0.5 ml of 0.25 M CaCl2 followed by 0.5 ml of 2x BBS[50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid (BES), 280 mM NaCl, 1.5 mM Na2HPO4, (pH 6.95)].The solution was vortexed briefly and allowed to stand atroom temperature for 20 min; 0.5 ml of this cocktail wasadded dropwise to each plate, and the dishes were incubatedat 37°C in a 3% Co2 incubator for approximately 20 h.Dishes then were washed twice with DMEM, 3 ml of freshDMEM containing 10% fetal calf serum was added, and cellswere further incubated at 37°C in a 5% CO2 incubator for upto 120 h. All transfections were done in duplicate. A typicalpositive control for these experiments, not usually depictedin the results presented, was a parallel transfection with thecomplete set of cosmids plus pSP50, followed by infection 24h after transfection.

Total cell DNA was collected by removing the media,washing cells once with phosphate-buffered saline, andlysing cells directly on the dish with 400 [lI of cell lysis buffer(2% sodium dodecyl sulfate, 10 mM Tris-HCl [pH 8.0], 10mM EDTA). Cell lysates were transferred to a 1.5-mlmicrocentrifuge tube, proteinase K was added to a finalconcentration of 200 ,ug/ml, and the lysates were incubatedat 50°C for 3 h. Sodium acetate (pH 5.4) was added to a finalconcentration of 0.3 M; the cell lysates were then extractedonce with phenol-chloroform-isoamyl alcohol (25:24:1) andonce with chloroform-isoamyl alcohol (24:1) and ethanolprecipitated. DNA was resuspended in 100 ,I of Tris-EDTA(pH 8.0), and one-quarter of the total was treated with 25 Uof EcoRI and 10 U of DpnI in a volume of 50 RI at 37°C for4 to 16 h. Cleaved DNA was electrophoresed through a 0.8%agarose gel, transferred to a Zeta-probe (Bio-Rad, Rich-mond, Calif.) nylon membrane, and probed with random-primer 32P-labeled pGEM. Hybridizations were performedaccording to manufacturers' instructions. Southern blotswere then exposed to X-Omat AR X-ray film (Kodak) at-80°C for 24 h.

RESULTS

HCMV cosmid clones can direct lytic-phase DNA replica-tion when cotransfected with a plasmid which expresses IEIand IE2. In developing a transient complementation assayfor HCMV DNA replication, our strategy was to introducelarge overlapping cloned segments of the HCMV genomeinto permissive HFF cells together with a plasmid containingHCMV oniLyt. Available cosmid libraries (21) providedconvenient sets of overlapping genomic fragments, and theoniLyt-containing plasmid SP50 (4) served as an assayablereporter of oriLyt-mediated DNA synthesis; replication ofpSP50 was detected by treatment of transfected-cell DNAwith DpnI, which cleaves only unreplicated dam-methylatedinput DNA. Initial experiments tested various combinationsof available cosmids, plus or minus added pSVH, which

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TRANSIENT COMPLEMENTATION OF HCMV DNA SYNTHESIS 2577

pCM1052

pCM1035 pCM1017 pCM1075 pCM1 058

pCM1015 pCM1029 pCM1 039 pCM1035

v v z = D. .,X IiFIvI

- aA.VA-X,X/'OR1I

B 1 2

>. * -Dpnl-resistant

F-DpnI-cleaved

- + pSVH

FIG. 1. Ability of cotransfected cosmids to complement replication of cloned HCMV oriLyt. (A) Physical maps of the HCMV genome andlocations of the cosmids used for the described experiments. (B) Evidence that cosmid complementation requires pSVH. Cotransfection wasdone as described in Materials and Methods. Cotransfection mixtures contained 4 pg of pSP50 and 1 ,ug each of cosmids pCM1015, pCM1017,pCM1029, pCM1039, pCM1058, pCM1052, and pCM1035 (lane 1) or the same set of recombinants plus 1 ,ug of pSVH (lane 2). Shown is an

autoradiogram of the resulting Southern transfer. The arrowhead indicates the position of the pGEM7Zf(-)-containing EcoRI fragment of thereporter plasmid pSP50.

contains the MIE regions 1 and 2 (56), and compared severaltransfection methods, including standard CaPO4, DEAE-dextran and lipofection protocols. Although all of the trans-fection protocols were efficient enough for standard trans-fection experiments in which only onLyt was transfectedand necessary trans-acting factors were supplied by infec-tion, we found that only the BES-buffered, modified CaPO4procedure described by Chen and Okayama (14) yieldedcomplementation (47). Positive results, measured by conver-

sion of onLyt to DpnI resistance, first were obtained whenthe seven cosmids pCM1015, pCM1017, pCM1029,pCM1035, pCM1039, pCM1058, and pCM1052 were cotrans-fected with a roughly 20-fold molar excess ofpSVH (Fig. 1B,lane 2). Thus, these seven partially overlapping cosmidsexpressed all of the proteins needed to direct detectableoniLyt replication. Together they comprise almost the entireHCMV genome (Fig. 1A), including all of the describedhomologs of HSV-1 replication genes. Without addedpSVH, the same set of cosmids reproducibly failed tocomplement DNA replication (e.g., Fig. 1B, lane 1), eventhough pCM1058 contains a functioning MIE region (47). Inthis and other experiments, only oriLyt-containing plasmidswere replicated. While it is difficult to interpret these resultsquantitatively, we note that in typical experiments, comple-mented replication produced a DpnI-resistant signal roughly50- to 100-fold lower than that found for the parallel infectedcontrol (e.g., compare lanes 0 and 1 in Fig. 4C).Two additional control experiments were done to test

whether the detected DpnI-resistant products resulted fromHCMV lytic-phase DNA synthesis. PFA treatment, whichselectively inhibits the virus-encoded DNA polymerase, wasused to determine whether the oniLyt replication resultingfrom cosmid cotransfection required HCMV DNA poly-merase activity. HFF cells were cotransfected with theindicated set of clones and then incubated with or withoutPFA for up to 144 h (Fig. 2A). Replication of oniLyt wasdetected from 72 h after transfection in untreated cells (lanes4, 6, 8, and 10). However, in PFA-treated cells, no DpnI-resistant product was detected at any time point (lanes 1, 3,5, 7, and 9). This finding is consistent with results fromearlier experiments showing the PFA sensitivity of onLyt-mediated DNA replication (4). We also examined the struc-tures of replication products resulting from cosmid cotrans-fection. Replication products of oniLyt-containing plasmidsin the transient transfection-plus-infection assay are tandemarrays (4). Thus, partial digestion with an enzyme thatcleaves the vector at a unique site produces a ladder of

fragments representing 1N, 2N, 3N, etc., copies of thereporter plasmid. Similar patterns were seen when onLytreplication products from HCMV-infected (Fig. 2C) andcosmid-cotransfected (Fig. 2B) HFF cells were compared bypartial digestion. Together, these results argue that oriLytreplication directed by cotransfected cosmids is equivalentto that of the transient transfection-plus-infection assay andaccurately reproduces events of HCMV lytic-phase DNAreplication.

Five cosmids are needed for complementation of HCMVorigin-dependent DNA replication. Once it was establishedthat the cotransfected cosmids directed authentic lytic-phasereplication of oniLyt, we next identified which of the cosmidscontained genes essential for transient complementation byeliminating one cosmid at a time from transfection mixtures.When cosmids pCM1015 and pCM1035 were omitted fromcotransfection cocktails (Fig. 3, lanes 2 and 5, respectively),onLyt replication was unaffected. HindlIl fragments I, H,

A1 2 3 4 5 6 7 8 910

- - - - - -. 1- - PFA48 7 2 96 20 144

B C

Co-transfected HCMV-infectedIi

5 4 32 1 1 23 4 5

ww_w --4N- __,wiF -3N-

O. ' -2N- 4 "

- 1N- - -I

Increasing enzyme dilution

FIG. 2. Evidence that replication of oriLyt following cotransfec-tion is authentic HCMV lytic-phase DNA synthesis. (A) Comple-mentation is PFA sensitive. HFF cells were cotransfected with a

mixture containing cosmids pCM1017, pCM1029, pCM1039,pCM1058, and pCM1052 plus plasmids pSVH and pSP50 and thenincubated for the indicated time with (lanes 1, 3, 5, 7, and 9) orwithout (lanes 2, 4, 6, 8, and 10) 200 ,ug of PFA per ml of medium.DNA was extracted and digested as described in Materials andMethods. Only that portion of the resulting Southern transfercontaining the DpnI-resistant products is shown. (B and C) Com-plemented DNA synthesis produces tandem arrays of pSP50. Par-allel sets of dishes were cotransfected with a replication-competentset of cosmids plus pSP50. One set of dishes was infected (C), andthe other set was left uninfected (B); the dishes were incubated for96 h, and total DNA was prepared as described in Materials andMethods. DpnI-resistant DNA was cleaved partially with HindIII bythe serial dilution method.

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2 In r-- m m oi a 7~1

O--° Lo LOc) E jE j j jj -Omitted cosmids

'0 )0-<: CL Cf a 9- a_ 9 L a

-w C)-Dpnl-resistant

F- Dpn l-cleaved

1 2 3 4 5 6 7 8

FIG. 3. Evidence that five cosmids are needed for complemen-tation of HCMV origin-dependent DNA replication. Transient com-

plementation assays were performed as described in Materials andMethods. All transfections contained pSP50 and pSVH plus eitherthe complete set of seven cosmid clones (pCM1015, pCM1017,pCM1029, pCM1035, pCM1039, pCM1052, and pCM1058; lane 1) orthe complete set of cosmid clones minus the indicated cosmid (lanes2 to 8). Shown is the resulting Southern transfer. The position ofDpnI-resistant replication products is indicated.

and 0 are unique to these cosmids (Fig. 1A); therefore thesefragments do not express genes essential for origin-depen-dent HCMV DNA replication. However, no replicationproducts were detected when any of the four cosmidscontaining previously identified candidate replication genes,pCM1017, pCM1029, pCM1039, and pCM1058, were omit-ted from the transfection cocktails (Fig. 3, lanes 3, 4, 6, and8). In addition, cosmid pCM1052, which does not contain an

identified homolog of an HSV-1 replication gene, also wasrequired (Fig. 3, lane 7). Thus, five cosmids (pCM1017,pCM1029, pCM1039, pCM1052, and pCM1058) plus pSVHwere needed to complement DNA replication in our tran-sient assay.IRS1/TRS1 is essential for transient complementation. Once

the minimum set of cosmid clones sufficient to direct thereplication of onLyt was established, we sought to identifyessential regions within each required cosmid. BecausepCM1052 does not contain a candidate homolog of anyHSV-1 replication gene, we studied it first. To determinewhich gene(s) within pCM1052 was required in our assay, wemade a series of subclones, using previously identified ORFs(13, 62) as a guide, and tested whether they could substituteindividually for pCM1052 in the cotransfection assay. Threeof the constructs, pZP1, pZP2, and pZP3, efficiently re-placed pCM1052 (Fig. 4C, lanes 2 to 7) in the transient assay.Transfections omitting pCM1052 without substitution pro-duced no detected signal (Fig. 4C, lane 8), as shown above(Fig. 3, lane 7). None of the other tested fragments derivedfrom pCM1052, including HindIII-W and -V, could substi-tute for pCM1052 (47). pZP3 contains a 3.6-kb SmaI frag-ment that spans only the ORFs IRS1 and US1 (Fig. 4A andB). Because US1 has been shown to be dispensable for virusgrowth in culture (36), we suspected that IRS1 was theessential ORF. TRS1, the terminal repeat counterpart ofIRS1, shares extensive sequence identity with IRS1 (12), andboth can cooperate with IEl and IE2 to transactivate de-layed-early gene expression (55). In light of the finding thatthe IRS1 region of pCM1052 was sufficient for complemen-tation, it was surprising to us that cosmid pCM1035, whichcontains TRS1, failed to substitute for pCM1052 (Fig. 3, lane7). However, we subsequently found that a plasmid con-

A

GpCM1 052

X W L V

pZP1pZP2LPZP

B7 6 4 Jil IRS1

UL -- A-~ - ~ US132 RL IRS

14 3 13211O 98 5 3 2 1 USi

C

+ pZP1 pZP2 pZP3 - TRS1 TRS1F

* ~S***0 1 2 3 4 5 6 7 8 9 10

FIG. 4. Evidence that IRS1/TRS1 is essential for origin-depen-dent DNA replication. (A) Physical map depicting HindIll frag-ments within pCM1052 and fragments that were subcloned asdescribed in Materials and Methods and then tested in cotransfec-tion experiments. pZP1 extends from nt 176883 to 195837. pZP2extends from nt 185495 to 195231. pZP3 extends from nt 189500 to193108. (B) ORFs predicted by Chee et al. (12) situated within the IRregion between the UL and the Us of HCMV. (C) Cotransfectionexperiments showing that IRS1/TRS1 is essential for complementa-tion of DNA synthesis. Cotransfections contained pSP50, pSVH,and cosmids pCM1017, pCM1029, pCM1039, pCM1058, andpCM1052 (lanes 0 and 1), the same cocktail with the indicatedsubstitution for pCM1052 (lanes 2 to 7, 9, and 10), or the sametransfection mixture minus pCM1052 without substitution (lane 8).The dish transfected for the sample run in lane 0 was infected asdescribed in Materials and Methods 24 h after transfection. In lanes9 and lane 10, pON2334 (TRS1) and pON2336 (TRS1F), respec-tively, were substituted for pCM1052 (55) as explained in Results.

struct spanning HCMV strain AD169 TRS1 (pXEXX-6.1;provided by T. Jones) could substitute for cosmid pCM1052following cotransfection (47); thus, the TRS1 copy present inpCM1035 is probably defective. Further supporting thiscontention was the finding that a plasmid containing onlyTRS1 (from HCMV strain Towne; pON2334; kindly pro-vided by E. Mocarski) efficiently substituted for pCM1052(Fig. 4C, lane 9) upon cotransfection, whereas a frame-shifted mutant of TRS1 (pON2336) lacked the essentialactivity (Fig. 4C, lane 10). We concluded that IRS1 is theonly ORF within cosmid pCM1052 needed for transientcomplementation and that TRS1 can substitute for IRS1.Thus, except for IRS1/TRS1, the genomic segments contain-ing Us, IRL, and IRS were dispensable for lytic-phase DNAsynthesis. Replication signals in these substituted transfec-tions were higher than with pCM1052 (Fig. 4C; compare lane1 with lanes 3 to 7), presumably because of the highereffective concentration of an essential gene that resultedfrom replacing pCM1052 with an equal mass of smallerplasmids. A related observation is that extramolar IRS1relieved the observed requirement for pSVH, although theMIE region, supplied by pCM1058, was still essential (47).Cosmid pCM1017 contains two regions essential for oriLyt-

dependent DNA replication. IRS1/TRS1 recently was shown

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pCM1017

pZP8 pAZP7

-J pZP5pZP6

C 3934 35 A

* .----- 36 -37 38 40 41 42 44 45

43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718

N. N N Nls X pZP81\1\W K \I JpZP6IN ;I \ pCMlOl

!-\ pZP8. .. < pZP6

. \s ZP.;, 4 pZP5

:pIppZP4

'% '\ pCM1017

FIG. 5. Presence in cosmid pCM1017 of two regions that are required for origin-dependent DNA replication. (A) Physical map depictingHindIll fragments within cosmid pCM1017. (B) Fragments that were subcloned from cosmid pCM1017 and used in the describedcotransfection experiments. Subclone pZP8 contains a fragment that extends from nt 42045 to 53104. pZP6 contains a fragment that extendsfrom nt 47366 to 56170. pZP5 contains a fragment that extends from nt 52260 to 59219. pAZP7 contains a fragment that extends from nt 54118to 56701 with a HincII deletion from nt 54231 to 54813. (C) Predicted ORFs (12) within the essential region of pCM1017. (D) Cotransfectionexperiments defining essential regions within cosmid pCM1017. All transfections contained pSP50, pSVH, pZP3, pCM1029, pCM1039, andpCM1058, plus pCM1017 (lanes 11 to 14) or the substitution for pCM1017 indicated below each lane with a checkmark (lanes 1 to 10 and 15to 18).

to trans activate HCMV UL44 expression in cooperationwith the MIE region (55). Elsewhere, UL44 was proposed toencode a polymerase accessory protein on the basis ofbiochemical studies of the expressed protein (18), suggestingthat it is a component of the DNA synthesis apparatus.Those findings predicted that UL44 should be essential fortransient complementation of DNA synthesis. Therefore, todetermine whether ULA4 was required, we tested a subclonethat left intact only the predicted transcription unit for UL44(pAZP7) and found that it failed to replace pCM1017 in thereplication assay (Fig. 5, lane 18). Therefore, additionalpCM1017 subclones were made and tested both individuallyand together with UL44-containing constructs (Fig. 5) (47).None of the individual constructs that were tested couldreplace pCM1017 (e.g., Fig. SD, lanes 7 to 10). However, thecombination of pAZP7 (or pZP5) with either pZP6 or pZP8(Fig. SD, lanes 1 to 4, 16, and 17) substituted for pCM1017.The 582-nt HincII deletion in pAZP7 removed all but theamino-terminal 41 nt of the 420-nt UL42 ORF as well as thecarboxyl-terminal 209 nt of the 561-nt UL43 ORF, leavingonly ULA4 intact. Furthermore, a frameshifted mutant ofUL44 in which the BglII site (nt 56170) was filled in withKlenow enzyme failed to complement replication (47), pro-viding additional evidence that UL44 is the essential ORF inpAZP7. The finding that either pZP6 or pZP8 could cooper-ate with pAZP7 to replace pCM1017 defined a 5.7-kbp regionextending from the BglII site at nt 47366 to the KpnI site atnt 53104 and spanning only ORFs UL36-38. Moreover, aconstruct containing this 5.7-kbp fragment also substituted

for pCM1017 (47). Therefore, from this series of experi-ments, we concluded that both ULA4 and the region span-ning ORFs UL36-38 were needed to replace pCM1017 in thereplication assay (Fig. SD, lane 17).

DISCUSSION

We have developed a transient complementation assay ofHCMV lytic-phase DNA replication that is based on thestrategy devised by Challberg (9) and uses available cosmidlibraries. Five cosmids, pCM1017, pCM1029, pCM1039,pCM1052, and pCM1058, plus pSVH, which comprises theMIE region, were required for complementation of DNAreplication in permissive HFF cells. In this report, wedemonstrate the validity of this assay and show resultslocating the genes essential for complementation in two ofthe required cosmids, pCM1017 and pCM1052.

Several lines of experimental evidence indicate that DNAsynthesis resulting from transient complementation accu-rately reproduces events of HCMV lytic-phase DNA repli-cation and thus is a valid approach to identifying HCMVreplication genes. First, complemented replication was on-Lyt dependent. For example, neither pSVH nor any of thecosmids not containing oriLyt were replicated, althoughreplication of pCM1029, which includes oriLyt, was readilyseen. Second, replication was PFA sensitive, as is viralDNA replication (4), arguing that the HCMV-contributedDNA polymerase is essential for complementation of lytic-phase DNA synthesis, as would be predicted. Consistent

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with this view are preliminary results indicating that HCMVUL54, which encodes the DNA polymerase, is essential forcomplementation (47). Third, complemented replication wasautonomous because recombination with cosmid DNA wasnot detected, and replicated products were tandem arrays ofinput plasmid DNA. Finally, as detailed below, complemen-tation depends on the expression of a limited subset of virusgenes, including one of the proposed homologs of HSV-1replication genes.Only one pCM1052 ORF, IRS1, was essential for transient

complementation. The promoter and the amino-terminal 684residues of IRS1 are encoded within the internal copy of theIRS region and thus are duplicated in the terminal invertedrepeat as TRS1; the unique carboxyl-terminal portion ofIRS1 is encoded in the Us segment (12). The smallest testedfragment that provided IRS1 function also included US1, buttwo lines of experimental evidence demonstrate that IRS1encodes the essential trans-acting function. First, Jones andMuzithras (36) showed that the unique ORF US1 is dispens-able for viral replication in permissive fibroblasts by con-structing viable virus deletions of this region. They alsodemonstrated that IRS1 was dispensable. Second, we foundthat a clone containing only the terminal repeat counterpartof IRS1, TRS1, complemented DNA replication as effi-ciently as did the IRS1-containing clone. Moreover, a frame-shift mutation in the common segment of TRS1 abrogated itsability to replace IRS1 (Fig. 4C, lanes 9 and 10). Thus, eitherIRS1 or TRS1 was essential to complement DNA replica-tion, but the rest of the Us and inverted repeat regions weredispensable. Our results are therefore consistent with thefindings of Jones and Muzithras (36) and further suggest thatit may not be possible to delete simultaneously both IRS1and TRS1 because at least one copy may be essential forgrowth in fibroblasts. IRS1 and TRS1 have amino-terminalsequence similarity to several other predicted HCMV ORFs,which are grouped as the US22 family (12, 62), and we notethat none of the genomic clones containing other members ofthis family that were tested, including those spanning UL36-38, could substitute for IRS1/TRS1 in our assay.Two distinct regions within pCM1017 were needed to

complement HCMV DNA replication. The two definedsegments span ORFs UL44 and UL36-38, respectively. Noother ORFs remained intact in the fragment used to provideUL44 (p/ZP7), suggesting that UL44 encodes the requiredprotein. Moreover, a reading frame mutation near the aminoterminus of UL44 abolished the ability of this fragment tocomplement replication, supporting this contention (47).UL44 encodes an abundant, phosphorylated DNA-bindingprotein of about 53 kDa (23, 45) that is expressed beginningin the early phase of infection and accumulates through thelate phase (23). Ertl and Powell expressed both HCMV DNApolymerase and UL44 in insect cells by using recombinantbaculoviruses and showed that the protein product of UL44complexes with coexpressed polymerase (18). Moreover,UL44 selectively stimulated HCMV DNA polymerase activ-ity in a template-dependent manner. On the basis of theseresults, they suggested that UL44 functions as a polymeraseaccessory protein, equivalent to HSV-1 UL42 (25, 33),although sequence similarity to HSV-1 UL42 is not readilyapparent. Thus, our results, considered together with avail-able biochemical evidence, are consistent with a model inwhich HCMV UL44 participates directly in DNA synthesisvia physical interaction with other components of the repli-cation machinery. Using a similar transient assay, Fixman etal. (20) recently demonstrated that EBV BMRF1, whichassociates with the EBV-coded polymerase (37, 38), is

essential for EBV DNA replication. Like HCMV UL44,BMRF1 shows minimal sequence similarity to HSV-1 UL42;however, it has a genomic location similar to that of theHSV-1 UL42 gene (5). In contrast, sequence similarity wasobserved between HCMV UL44 and a human herpesvirus 6phosphoprotein, p41 (18).The second essential region within pCM1017 defined by

these studies spans ORFs UL36-38 (12, 17, 57, 58). TheseORFs are expressed by a family of at least four overlappingand spliced transcripts, utilizing three different start sites.Three of the transcripts are expressed under IE conditions,and the fourth is expressed initially at early times (17, 57,58). These transcripts can encode at least four distinctproteins (17, 57, 58). Thus, like the MIE region, genomicconstructs spanning UL36-38 have the potential to expressdifferentially a variety of protein species. Prbteins encodedwithin this region regulate the expression of target genes in apromoter- and cell-specific manner, in some cases cooperat-ing with IE1 or IE2 (17). We have not yet determined whichamong these transcripts are needed for complementation ofDNA replication.

In contrast to the HSV-1 system, in which virus-encodedtransactivators are not essential for transient complementa-tion of DNA replication, we found that at least three distinctgenomic regions encoding IE transcriptional regulators, theMIE region, IRS1/TRS1, and UL36-38, were needed forcomplementation of onLyt-mediated DNA synthesis. Theessential roles played by each of these three transactivatorsin transient complementation of DNA synthesis have notbeen established, but there are several nonexclusive possi-bilities. First, it is likely that these transactivators areneeded to promote the expression of early genes required forHCMV DNA replication, because it has been shown thatIRS1/TRS1 and the MIE regions encoding IE1 and IE2 cancooperate to activate expression of essential early genes intransient transfection assays (55). The MIE proteins havebeen studied extensively with respect to their role in regu-lating HCMV and heterologous promoters (6, 15, 22, 27, 32,41, 49, 55, 56, 59). They upregulate expression from severaldelayed-early promoters, including those of the DNA poly-merase and UL44 (54, 55, 56). Like the MIE region, TRS1 isexpressed at IE times after infection and in the presence ofcycloheximide (55). We found that IRS1/TRS1 was essentialfor transient complementation of DNA synthesis, whereasStasiak and Mocarski (55) observed only a severalfold su-perinduction of UL44 by TRS1 plus the MIE region over theMIE region-activated UL44 promoter. However, we notethat this effect would be multiplied if IRS1/TRS1 similarlyinduces expression of other genes essential for DNA synthe-sis, which could readily account for its key role in-comple-mentation. UL36-38 gene products also activate transcrip-tion of selected cellular genes as well as HCMV early genesin a cell-type-specific manner in cooperation with TEl andIE2 (17). However, replication genes have not yet beenshown to be targets for transcriptional activation by UL36-38. A second possibility is that these IE regions inducecellular factors that influence HCMV viral DNA replication.The corresponding MIE proteins of mouse cytomegalovirusand HCMV have been implicated in the induction of cellularDNA replication and transcription (27, 48, 53) and thus couldplay another crucial role by increasing the availability ofneeded host factors such as topoisomerase (7). Finally, it isalso possible that one or more of the transactivators directlyaffects or regulates initiation of replication, as has recentlybeen suggested for simian virus 40 and for the papillomavi-ruses (16, 26, 46). In this regard, onLyt-dependent EBV

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replication also requires three IE proteins, Zta, Rta, andMta, although the requirement for Mta is not absolute (20).Zta binds directly to elements in two distinct domains of theDSL promoter and enhancer regions and activates divergenttranscription (30, 39, 40). This region lies within EBV oriLyt(28), and Zta may play a role in promoting EBV DNAreplication.Experiments are under way to identify specific genes

within the remaining cosmids that contribute genes essentialfor DNA replication. Results obtained thus far are consistentwith a requirement for candidate HCMV homologs of HSV-1replication genes (47). Once all of the genes needed forcomplementation are identified, it will be possible to addressexperimentally the role of viral transactivators.

ACKNOWLEDGMENTSWe thank Rich Stenberg, Tom Jones, Ed Mocarski, and Annie

Colberg-Poley for generous gifts of recombinant plasmids; J. Nelsonfor providing antibodies; and Mary Beth Coy for dedicated technicalassistance.

This work was aided by Public Health Service NIH grantsAI31249 and A133416. G.S.P. is a Wadsworth postdoctoral fellow.

REFERENCES1. Anders, D. G., A. Irmiere, and W. Gibson. 1986. Identification

and characterization of a major early cytomegalovirus DNA-binding protein. J. Virol. 58:253-262.

2. Anders, D. G., M. A. Kacica, G. S. Pari, and S. M. Punturieri.1992. Boundaries and structure of human cytomegalovirus ori-Lyt, a complex origin for lytic-phase DNA replication. J. Virol.66:3373-3384.

3. Anders, D. G., J. R. Kidd, and W. Gibson. 1987. Immunologicalcharacterization of an early cytomegalovirus single-strandDNA-binding protein with similarities to the HSV major DNA-binding protein. Virology 161:579-588.

4. Anders, D. G., and S. M. Punturieri. 1991. Multicomponentorigin of cytomegalovirus lytic-phase DNA replication. J. Virol.65:931-937.

5. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J.Farrell, T. J. Gibson, and G. Hatfull. 1984. DNA sequence andexpression of the B95-8 Epstein-Barr virus genome. Nature(London) 310:207-211.

6. Baracchini, E., E. Glezer, K. Fish, R. M. Stenberg, J. A. Nelson,and J. A. Ghazal. 1992. An isoform variant of the cytomegalo-virus immediate-early auto repressor functions as a transcrip-tional activator. Virology 188:518-530.

7. Benson, J. D., and E. S. Huang. 1988. Two specific topoi-somerase inhibitors prevent replication of human cytomegalo-virus DNA: an implied role in replication of the viral genome. J.Virol. 62:4797-4800.

8. Carmichael, E. P., M. J. Kosovsky, and S. K. Weller. 1988.Isolation and characterization of herpes simplex mutants defec-tive in viral DNA synthesis. J. Virol. 62:91-99.

9. Challberg, M. D. 1986. A method for identifying the viral genesrequired for herpes DNA replication. Proc. Natl. Acad. Sci.USA 83:9094-9098.

10. Challberg, M. D. 1991. Herpes simplex virus DNA replication.Semin. Virol. 2:247-256.

11. Challberg, M. D., and T. J. Kelly. 1989. Animal virus DNAreplication. Annu. Rev. Biochem. 58:671-717.

12. Chee, M. S., A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, R.Cerny, and T. Horsnell. 1990. Analysis of the coding content ofthe sequence of human cytomegalovirus strain AD169. Curr.Top. Microbiol. Immunol. 154:125-169.

13. Chee, M. S., S. C. Satchwell, E. Preddie, K. M. Weston, andB. G. Barrell. 1990. Human cytomegalovirus encodes three Gprotein-coupled receptor homologues. Nature (London) 344:774-777.

14. Chen, C., and H. Okayama. 1987. High-efficiency transforma-tion of mammalian cells by plasmid DNA. Mol. Cell. Biol.7:2745-2752.

15. Cherrington, J. M., E. L. Khoury, and E. S. Mocarski. 1991.Human cytomegalovirus ie2 negatively regulates a gene expres-sion via a short target sequence near the transcription start site.J. Virol. 65:887-896.

16. Chiang, C. M., G. Dong, T. R. Broker, and L. T. Chow. 1992.Control of human papillomavirus type 11 origin of replication bythe E2 family of transcription regulatory proteins. J. Virol.66:5224-5231.

17. Colberg-Poley, A. M., L. D. Santomenna, P. P. Harlow, P. A.Benfield, and D. J. Tenney. 1992. Human cytomegalovirus US3and UL36-38 immediate-early proteins regulate gene expres-sion. J. Virol. 66:95-105.

18. Ertl, P. F., and K. L. Powell. 1992. Physical and functionalinteraction of human cytomegalovirus DNA polymerase and itsaccessory protein (ICP36) expressed in insect cells. J. Virol.66:4126-4133.

19. Ertl, P. F., M. F. Thomas, and K. L. Powell. 1991. High levelexpression of DNA polymerases from herpesviruses. J. Gen.Virol. 72:1729-1734.

20. Fixman, E. D., G. S. Hayward, and S. D. Hayward. 1992.trans-acting requirements for replication of Epstein-Barr virusori-Lyt. J. Virol. 66:5030-5039.

21. Fleckenstein, B., I. Muller, and J. Collins. 1982. Cloning of thecomplete human cytomegalovirus genome in cosmids. Gene18:39-46.

22. Ghazal, P., J. Young, E. Giulietti, C. DeMattei, J. Garcia, R.Gaynor, and R. M. Stenberg. 1991. A discrete cis element in thehuman immunodeficiency virus long terminal repeat mediatessynergistic trans activation by cytomegalovirus immediate-earlyproteins. J. Virol. 65:6735-6742.

23. Gibson, W. 1984. Synthesis, structure and function of cytomeg-alovirus major nonvirion nuclear protein, p. 423-440. In F. Rapp(ed.), Herpesvirus. Alan R. Liss, New York.

24. Goldstein, D. J., and S. K. Weller. 1988. An ICP6::lacZ inser-tional mutagen is used to demonstrate that the UL52 gene ofherpes simplex virus type 1 is required for virus growth andDNA synthesis. J. Virol. 62:2970-2977.

25. Gottleib, J., A. I. Marcy, D. M. Coen, and M. D. Challberg.1990. The herpes simplex virus type 1 UL42 gene product: asubunit of DNA polymerase that functions to increase proces-sivity. J. Virol. 64:5976-5987.

26. Guo, Z.-S., and M. L. DePamphilis. 1992. Specific transcriptionfactors stimulate simian virus 40 and polyomavirus origins ofDNA replication. Mol. Cell. Biol. 12:2514-2524.

27. Hagemeier, C., S. M. Walker, P. J. G. Sissons, and J. H.Sinclair. 1992. The 72K IE1 and 80K IE2 proteins of humancytomegalovirus independently trans-activate the c-fos, c-mycand hsp70 promoters via basal promoter elements. J. Gen.Virol. 73:2385-2393.

28. Hammerschmidt, W., and B. Sugden. 1988. Identification andcharacterization of oriLyt, a lytic origin of DNA replication ofEpstein-Barr virus. Cell 55:427-433.

29. Hamzeh, F. M., P. S. Lietman, W. Gibson, and G. S. Hayward.1990. Identification of the lytic origin of DNA replication inhuman cytomegalovirus by a novel approach utilizing ganciclo-vir-induced chain termination. J. Virol. 64:6184-6195.

30. Hardwick, J. M., P. M. Lieberman, and S. D. Hayward. 1988. Anew Epstein-Barr virus transactivator, R, induces expression ofa cytoplasmic early antigen. J. Virol. 62:2274-2284.

31. Heilbronn, R., G. Jahn, A. Burkle, U.-K. Freese, B. Flecken-stein, and H. zur Hausen. 1987. Genomic localization, sequenceanalysis, and transcription of the putative human cytomegalo-virus DNA polymerase gene. J. Virol. 61:119-124.

32. Hermiston, T. W., C. L. Malone, and M. F. Stinski. 1990.Human cytomegalovirus immediate-early two protein regioninvolved in negative regulation of the major immediate-earlypromoter. J. Virol. 64:3532-3536.

33. Hernandez, T. R., and I. R. Lehman. 1990. Functional interac-tion between the herpes simplex-1 DNA polymerase and UL42protein. J. Biol. Chem. 265:11227-11232.

34. Huang, E. 1975. Human cytomegalovirus. IV. Specific inhibi-tion of virus-induced DNA polymerase activity and viral DNAreplication by phosphonoacetic acid. J. Virol. 16:1560-1565.

VOL. 67, 1993

on January 23, 2019 by guesthttp://jvi.asm

.org/D

ownloaded from

2582 PARI ET AL.

35. Huang, E.-S. 1975. Human cytomegalovirus. III. Virus-inducedDNA polymerase. J. Virol. 16:298-310.

36. Jones, T. R., and V. P. Muzithras. 1992. A cluster of dispensablegenes within the human cytomegalovirus genome short compo-nent: IRS1, US1 through US5, and the US6 family. J. Virol.66:2541-2546.

37. Kiehl, A., and D. I. Dorsky. 1991. Cooperation of EBV DNApolymerase and EA-D (BMRF1) in vitro and colocalization innuclei of infected cells. Virology 184:330-340.

38. Li, J., B.-S. Zhou, G. E. Dutschman, S. P. Grill, R.-S. Tan, andY.-C. Cheng. 1987. Association of Epstein-Barr virus earlyantigen diffuse component and virus-specified DNA polymeraseactivity. J. Virol. 61:2947-2949.

39. Lieberman, P. M., J. M. Hardwick, J. Sample, G. S. Hayward,and S. D. Hayward. 1990. The Zta transactivator involved ininduction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in targetpromoter and enhancer regions. J. Virol. 64:1143-1155.

40. Lieberman, P. M., M. Hardwick, and S. D. Hayward. 1989.Responsiveness of Epstein-Barr virus NotI repeat promoter tothe Z transactivator is mediated in a cell-type-specific mannerby independent signal regions. J. Virol. 63:3040-3050.

41. Malone, C. L., D. H. Vesole, and M. F. Stinski. 1990. Transac-tivation of a human cytomegalovirus early promoter by geneproducts from the immediate-early gene IE2 and augmentationby IEl: mutational analysis of the viral proteins. J. Virol.64:1498-1506.

42. Marchetti, M. E., C. A. Smith, and P. A. Schaffer. 1988. Atemperature-sensitive mutation in a herpes simplex virus type 1gene required for viral DNA synthesis maps to coordinates0.609 through 0.614 in UL. J. Virol. 62:715-721.

43. Masse, M. J. O., S. Karlin, G. A. Schachtel, and E. S. Mocarski.1992. Human cytomegalovirus origin of DNA replication (ori-Lyt) resides within a highly complex repetitive region. Proc.Natl. Acad. Sci. USA 89:5246-5250.

44. Matz, B., J. H. Subak-Sharpe, and V. G. Preston. 1983. Physicalmapping of temperature-sensitive mutations of herpes simplexvirus type 1 using cloned restriction endonuclease fragments. J.Gen. Virol. 64:2261-2270.

45. Mocarski, E. S., L. Pereira, and N. Michael. 1985. Preciselocalization of genes on large animal virus genomes: use of Xgtlland monoclonal antibodies to map the gene for a cytomegalo-virus protein family. Proc. Natl. Acad. Sci. USA 82:1266-1270.

46. Mohr, I. J., R. Clark, S. Sun, E. J. Androphy, P. MacPherson,and M. R. Botchan. 1990. Targeting the El replication protein tothe papillomavirus origin of replication by complex formationwith the E2 transactivator. Science 250:1694-1699.

47. Pari, G. S., and D. G. Anders. 1992. Unpublished results.48. Pari, G. S., and S. C. St. Jeor. 1990. Human cytomegalovirus

major immediate early gene product can induce SV40 DNAreplication in human embryonic lung cells. Virology 179:785-794.

49. Pizzorno, M. C., M. A. Mullen, Y. N. Chang, and G. S.Hayward. 1991. The functionally active IE2 immediate-earlyregulatory protein of human cytomegalovirus is an 80-kilodalton

polypeptide that contains two distinct activator domains and aduplicated nuclear localization signal. J. Virol. 65:3839-3852.

50. Purifoy, D. J. M., R. B. Lewis, and K. L. Powell. 1977.Identification of the herpes simplex virus DNA polymerasegene. Nature (London) 269:621-623.

51. Purifoy, D. J. M., and K. L. Powell. 1981. Temperature-sensitive mutants in two distinct complementation groups ofherpes simplex virus type 1 specify thermolabile DNA poly-merase. J. Gen. Virol. 54:219-222.

52. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed., vol. 3. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

53. Schickendanz, J., L. Philipson, W. Ansorge, R. Pepperkok, R.Klein, and U. H. Koszinowski. 1988. The 89,000-Mr murinecytomegalovirus immediate-early protein stimulates c-fos ex-pression and cellular DNA synthesis. J. Virol. 62:3341-3347.

54. Staprans, S. I., D. K. Rabert, and D. H. Spector. 1988. Identi-fication of sequence requirements and trans-acting functionsnecessary for regulated expression of a human cytomegalovirusearly gene. J. Virol. 62:3463-3473.

55. Stasiak, P. C., and E. S. Mocarski. 1992. Transactivation of thecytomegalovirus ICP36 gene promoter requires the ax geneproduct TRS1 in addition to IEl and IE2. J. Virol. 66:1050-1058.

56. Stenberg, R. M., J. Fortney, S. W. Barlow, B. P. Magrane, J. A.Nelson, and P. Ghazal. 1990. Promoter-specific trans activationand repression by human cytomegalovirus immediate-early pro-teins involves common and unique protein domains. J. Virol.64:1556-1565.

57. Tenney, D. J., and A. M. Colberg-Poley. 1991. Expression of thehuman cytomegalovirus UL36-38 immediate early region duringpermissive infection. Virology 182:199-210.

58. Tenney, D. J., and A. M. Colberg-Poley. 1991. Human cytomeg-alovirus UL36-38 and US3 immediate-early genes: temporallyregulated expression of nuclear, cytoplasmic, and polysome-associated transcripts during infection. J. Virol. 65:6724-6734.

59. Wade, M., T. F. Kowalik, M. Mudryj, E. Huang, and J. C.Azizkhan. 1992. E2F mediates dihydrofolate reductase pro-moter activation and multiprotein complex formation in humancytomegalovirus infection. Mol. Cell. Biol. 12:4364-4374.

60. Weller, S. K. 1990. Genetic analysis of HSV genes required forgenome replication, p. 105-135. In E. Wagner (ed.), Herpesvi-rus transcription and its regulation. CRC Press, Boca Raton,Fla.

61. Weller, S. K., K. J. Lee, D. J. Sabourin, and P. A. Schaffer.1983. Genetic analysis of temperature-sensitive mutants whichdefine the gene for the major herpes simplex virus type 1DNA-binding protein. J. Virol. 45:354-366.

62. Weston, K., and B. G. Barrell. 1986. Sequence of the shortunique region, short repeats, and part of the long repeats ofhuman cytomegalovirus. J. Mol. Biol. 192:177-208.

63. Wu, C. A., N. J. Nelson, D. J. McGeoch, and M. D. Challberg.1988. Identification of herpes simplex virus type 1 genes re-quired for origin-dependent synthesis. J. Virol. 62:435-443.

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.org/D

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