Efficient replication by herpes simplex virus type 1 involves activation of the IkappaB kinase-IkappaB-p65 pathway.

Infection by herpes simplex virus type 1 (HSV-1) induces a persistent nuclear translocation of NFkappaB. To identify upstream effectors of NFkappaB and their effect on virus replication, we employed mouse embryo fibroblast (MEF)-derived cell lines with deletions of either IKK1 or IKK2, the catalytic subunits of the IkappaB kinase (IKK) complex. Infected MEFs were assayed for virus yield, loss of IkappaBalpha, nuclear translocation of p65, and NFkappaB DNA-binding activity. Absence of either IKK1 or IKK2 resulted in an 86 to 94% loss of virus yield compared to that of normal MEFs, little or no loss of IkappaBalpha, and greatly reduced NFkappaB nuclear translocation. Consistent with reduced virus yield, accumulation of the late proteins VP16 and gC was severely depressed. Infection of normal MEFs, Hep2, or A549 cells with an adenovirus vector expressing a dominant-negative (DN) IkappaBalpha, followed by superinfection with HSV, resulted in a 98% drop in virus yield. These results indicate that the IKK-IkappaB-p65 pathway activates NFkappaB after virus infection. Analysis of NFkappaB activation and virus replication in control and double-stranded RNA-activated protein kinase-null MEFs indicated that this kinase plays no role in the NFkappaB activation pathway. Finally, in cells where NFkappaB was blocked because of DNIkappaB expression, HSV failed to suppress two markers of apoptosis, cell surface Annexin V staining and PARP cleavage. These results support a model in which activation of NFkappaB promotes efficient replication by HSV, at least in part by suppressing a host innate response to virus infection.

Pocket protein p130/Rb2 is required for efficient herpes simplex virus type 1 gene expression and viral replication.

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.

Herpes simplex virus type 1 infection imposes a G(1)/S block in asynchronously growing cells and prevents G(1) entry in quiescent cells.

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G(0)-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein-E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G(1) from proceeding through the restriction point and for cells in S from completing a round of DNA replication.

Activation of cJUN N-terminal kinase by herpes simplex virus type 1 enhances viral replication.

Signal transduction pathways convey signals generated at the cell surface into the cell nucleus in order to initiate a program of gene expression that is characteristic for particular stimuli. Here we present evidence that infection by herpes simplex virus type 1 activated the two terminal kinases, cJUN N-terminal kinase (JNK) and p38, of stress-activated signal transduction kinase cascades. By using a solid-phase kinase assay, a phospho-specific antibody, and extracts prepared from a variety of infected cell types, we determined that activation of both kinases began 3 to 4 h postinfection (p.i.) and remained elevated out to 14 h p.i. Through the use of UV-irradiated or antibody-neutralized wild-type virus and the temperature-sensitive mutant tsB7, the high level of JNK activation was shown to be dependent on viral gene expression. Activation of JNK following infection by vi13, an ICP4 mutant virus that does not express early or late genes, suggested that only virus entry and immediate-early gene expression were necessary for JNK activation. The activation of JNK and p38 correlated with increased chloramphenicol acetyltransferase (CAT) activity in reporter assays dependent upon the activity of cJUN and ATF2 trans-activation domains. Increased CAT activity dependent on TRE and CRE promoter sites was also observed in response to herpes simplex virus infection. The activities of ERK and ERK-dependent transcription factors were unchanged or depressed following infection, showing that activation of JNK and p38 was a specific event. Finally, the activation of JNK was important for the efficiency of viral replication. The yield of virus in NIH 3T3 cells stably expressing JIP-1, an inhibitor of JNK translocation to the nucleus, was reduced 70% compared to that of control cells, in single-step growth experiments.

Herpes simplex virus induces intracellular redistribution of E2F4 and accumulation of E2F pocket protein complexes.

Accumulation of E2F-p107 and E2F-pRB DNA binding complexes occurred after herpes simplex virus infection of U2-OS cells. Accumulation of E2F-p107 also occurred by 4 h p.i. in C33 cells. This corresponded to a time when host DNA synthesis was reduced by 50%, and lagged by >/=1 h, the onset of viral DNA synthesis. To determine the basis for increased nuclear E2F complexes, we investigated the effects of virus infection on the intracellular distribution of the E2F-dependent DNA binding complexes and their protein constituents. Western blot analyses of whole cell extracts revealed that amounts of E2F4, E2F1, DP1, and p107 remained unchanged after infection of C33 cells. Analysis of cytoplasmic and nuclear fractions, however, revealed that cytoplasmic E2F4 decreased and nuclear E2F4 increased. This correlated with a loss of cytoplasmic E2F DNA-binding activity and a corresponding increase in nuclear DNA-binding activity. Concomitant with its redistribution, the apparent molecular weight of total and p107-associated E2F4 increased, at least partially as a result of protein phosphorylation. Increased nuclear E2F-pRB in U2-OS cells was accompanied by the conversion of pRB from a hyper- to a hypophosphorylated state. Infection of U2-OS cells with viral mutants indicated that viral protein IE ICP4 was necessary for the decrease in cytoplasmic E2F-p107, and that viral protein DE ICP8 was required for nuclear accumulation of p107-E2F. In contrast, ICP8 was not required for accumulation of E2F-pRB. These results indicate that the increase in E2F-p107 may be explained by the redistribution and modification of E2F4 and the increase in E2F-pRB by modification of pRB.

Herpes simplex type 1 induction of persistent NF-kappa B nuclear translocation increases the efficiency of virus replication.

The latent form of the dimeric transcription factor NF-kappa B is sequestered in the cytoplasm by proteins containing ankyrin repeats, such as 1 kappa B alpha and beta, or by the p105 precursor form of the NF-kappa B p50 subunit. Tumor necrosis factor alpha or virus infection can cause targeted destruction of 1 kappa B and nuclear translocation of NF-kappa B. Following translocation, NF-kappa B mediates immune, inflammatory, or anti-apoptotic responses. Here we present evidence that beginning at around 6 h postinfection, herpes simplex virus (HSV) induces a persistent translocation of NF-kappa B into the nucleus of C33 cells, coincident with loss of both 1 kappa B alpha and 1 kappa B beta. Translocation failed to occur when infecting virus was preincubated with neutralizing antibody to viral envelope glycoproteins gD or gH, thus preventing entry, or when cells infected with viruses expressing mutated forms of immediate-early regulatory proteins lCP4 or lCP27. Surprisingly, no increase in the trans-activation function of NF-kappa B, as assayed by transient expression of CAT, was detected following HSV infection. The significance of NF-kappa B nuclear translocation for virus replication was demonstrated by an 80-90% reduction in virus yield following infection of C33 cells expressing a constitutive repressor form of 1 kappa B alpha. Models that reconcile nuclear translocation of NF-kappa B with the inability to detect NF-kappa B-dependent gene expression are discussed.

Induction by herpes simplex virus of free and heteromeric forms of E2F transcription factor.

We have determined that HSV causes rapid and large increases in cell-cycle-regulated free E2F and S-phase p107/E2F DNA binding activities in asynchronous cultures of C33A cells. Induction occurred by 4 hr postinfection and coincided with the appearance of viral encoded immediate-early and delayed-early proteins, i.e., when viral DNA replication normally commences. No increase in E2F activities occurred when cells were infected with viruses expressing mutant regulatory proteins ICP4 or ICP27, or mutant replication proteins ICP8, pol or helicase, or when cells were infected with wild-type virus in the presence of inhibitors of DNA synthesis. In contrast, ICP8 mutant-infected cells contained elevated amounts of NF kappa B activity equivalent to WT virus, no induction of Sp1 relative to WT virus, and reduced ATF/CREB activity relative to WT virus. Results of transient expression assays with E2F-responsive reporters indicated that the net effect of induction of both active (free E2F) and repressive (p107/E2F) complexes was a decrease in AdE2 promoter activity and an increase in c-myc promoter activity. Taken together these results suggest that HSV can cause unscheduled changes in the amount and functional status of a cell-cycle-regulated transcription factor. These results are discussed in light of possible roles for viral-induced alterations in E2F, especially as related to imposing or overriding cell-cycle checkpoints.

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein-Barr virus early promoter, BMRF1.

Disruption of Epstein-Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243-9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085-4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at -59 and -106), whereas two additional Z binding sites (located at -42 and -170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251-8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.

Differential dependence of herpes simplex virus immediate-early gene expression on de novo-infected cell protein synthesis.

The time course of accumulation of herpes simplex virus immediate-early (IE) mRNA and the requirement for infected cell protein synthesis for mRNA transcription and accumulation were compared. Measurements of transcription in nuclear run-on assays, accumulation of cytoplasmic mRNA by Northern (RNA) blot hybridization, and rates of infected cell protein synthesis by pulse-labeling did not indicate differences among the five IE gene, consistent with previous studies. However, as a result of varying the amount of de novo protein synthesis after infection, at least three patterns of maximal expression of the IE genes were revealed. Addition of the protein synthesis inhibitor anisomycin to cells coincident with infection resulted in maximal rates of transcription and accumulation of functional ICP0 mRNA, while 0.5 h of infected cell protein synthesis prior to addition of the drug was required for maximal expression of ICP22/47 and ICP27 mRNAs. Maximal expression of ICP4 mRNA occurred only when 1 h of de novo protein synthesis occurred prior to the addition of the drug. These results are discussed in the context of alternative mechanisms for regulating IE gene expression.

Intracellular organization of herpes simplex virus type 1 DNA assayed by staphylococcal nuclease sensitivity.

The nucleoprotein organization of herpes simplex virus type 1 (HSV-1) DNA during productive infection was analyzed using staphylococcal nuclease. Both prior to and during the genome replication phase of infection, digestion of nuclei revealed two readily discernible forms of viral DNA, resistant and sensitive. The identity of these forms was established by the use of a variety of assays, including velocity sedimentation, nucleic acid hybridization and restriction endonuclease digestion and by employing temperature sensitive (ts) mutants impaired in either DNA replication or encapsidation of progeny DNA. Thus, nuclease resistant DNA was derived from encapsidated unit length genomes while sensitive DNA represented digestion products of replicating viral genomes. Importantly, no evidence was obtained for the arrangement of either parental or progeny viral DNA in nucleosomes. These findings are discussed with regard to the nucleoprotein structure of replicating viral DNA.

Variable requirements for herpes simplex virus immediate-early proteins in the expression of the adenovirus E2 gene.

Regulation of the adenovirus E2 gene by trans-activating proteins encoded by herpes simplex virus was investigated. Coinfection of Vero cells was performed with Ad5 dl312 (an E1 deletion mutant) and either wildtype HSV, mutant virus encoding a temperature-sensitive ICP4 protein (tsK), or mutants carrying deletions in the ICP4 (d120) or ICP0 (dl x 3.1) gene. As detected by the presence of E2 mRNA, or the product of the E2 gene, 72-kDa DNA binding protein (DBP), functional ICP4 was sufficient for expression of the E2 gene. Regulation of E2 gene expression was at the level of transcription activation as judged by nuclear run-on assay. In contrast to results when Vero cells were coinfected, expression of 72-kDa DBP in CN3 cells, carrying an integrated copy of the E2 gene, required expression of both HSV immediate-early proteins. These results suggest that the DNA-protein organization of the target gene sequence may play a significant role in the ability of viral regulatory proteins to activate expression of heterologous as well as homologous genes.

Characterization of intranuclear capsids made by ts morphogenic mutants of HSV-1.

We have characterized capsids made by seven temperature-sensitive (ts) mutants of HSV-1 previously shown to be defective in viral DNA processing and packaging at the nonpermissive temperature (NPT). The empty capsids isolated from mutant-infected cells at the NPT were devoid of DNA, cosedimented in sucrose with wt B capsids, and contained the same structural proteins found in wt B capsids (W. Gibson and B. Roizman (1972). J. Virol. 10, 1044-1052). The presence of VP22a in empty capsids suggests that the processing of this protein from higher-molecular-weight precursors and its association with capsids is required, but not sufficient, for DNA encapsidation. Mutants made no detectable A capsids at the NPT, but did so at the permissive temperature (PT), suggesting that A particles are generated during or subsequent to, rather than prior to, encapsidation. In temperature-shift experiments, it was demonstrated that capsids of one of the mutants, F18, made at the NPT did not participate in DNA encapsidation when cells were subsequently shifted to the PT. Only those capsids made after temperature shift to the PT acquired viral DNA, implying that the ts mutation in F18 may lie in a gene coding for a structural protein, or in a protein involved in the processing of viral DNA.

Synthesis and metabolism of cellular transcripts in HSV-1 infected cells.

The effects of productive herpes simplex virus infection on host gene expression were examined by measuring the rates of synthesis and subsequent fates of several Vero cell mRNAs. The rates of transcription of actin, beta-tubulin, and histone-3 and -4 RNAs were measured by pulse-labeling of intact cells as well as by run-on transcription in isolated nuclei. At both early (2 hr) and late (6 hr) times, the relative rates of transcription of these RNAs were greater than in uninfected cells. Kinetic labeling experiments performed at late times also revealed increased turnover rates of nuclear RNAs. That the rate of appearance of these RNAs in the cytoplasm was also reduced suggests that these cellular RNAs are being specifically retained and degraded in the nucleus. Levels of pre-existing cytoplasmic RNAs as measured by Northern blot analysis declined rapidly after infection though the nuclear steady-state levels of these RNAs increased up to 3 hr postinfection and then declined between 3 and 10 hr postinfection. At no time was the accumulation of processing intermediates detectable. Finally, we also determined that, consistent with the decline in levels of histone mRNA, rates of histone protein synthesis declined rapidly after infection.

DNA processing in temperature-sensitive morphogenic mutants of HSV-1.

The anatomy of DNA synthesized by five HSV-1 mutants previously shown to accumulate predominantly empty capsids at the nonpermissive temperature (NPT) was analyzed with Bg/II restriction digestion. At the NPT, all five generated DNA lacking termini, indicating that in the absence of packaging, viral DNA is not processed to unit length. One mutant, F18, was able to process DNA made at the NPT to unit length molecules during a 6-hr period after shift to the permissive temperature. The appearance of unit length molecules correlated with the appearance of staphylococcal nuclease-resistant F18 DNA.

Degradation of cellular mRNAs induced by a virion-associated factor during herpes simplex virus infection of Vero cells.

We have used Northern blot hybridization to study the accumulation of specific cellular mRNAs in Vero cells infected with herpes simplex virus (HSV) type 1 or type 2. HSV-1 infection decreased the cytoplasmic levels of beta- and gamma-actin, beta-tubulin, and histone H3 and H4 mRNAs, though not all at the same rate. HSV-2 infection resulted in a more rapid decrease in actin and histone mRNA levels compared with HSV-1 infection. The turnover rate of each type of mRNA studied was accelerated in HSV-infected cells compared with the rate in uninfected cells. Cellular mRNA degradation was induced by HSV infection under conditions of (i) inhibition of de novo protein synthesis, (ii) inhibition of de novo RNA synthesis, (iii) infection with HSV-1(17) tsK, which fails to produce early and late viral gene products at the nonpermissive temperature, and (iv) infection with purified virions in the presence of actinomycin D. We have concluded that, in Vero cells, cellular mRNA degradation is induced by a factor associated with the infecting HSV virion and thus does not require de novo RNA or protein synthesis. Despite the overall inhibition of cellular mRNA accumulation, a novel 2.2-kilobase cytoplasmic actin transcript was produced in HSV-infected cells when viral gene expression was allowed. The level of accumulation of cytoplasmic host mRNAs was compared with the rate of cellular protein synthesis under different conditions of infection. This analysis suggests that both HSV-1 and HSV-2 require an additional function(s) to completely inhibit cellular protein synthesis.

Accumulation of herpes simplex virus type 1 RNAs of different kinetic classes in the cytoplasm of infected cells.

We have analyzed the accumulation of herpes simplex virus type 1 RNA of the immediate early (IE; infected cell polypeptide types 4 and 0 [ICP-4 and ICP-0]), early (thymidine kinase), and early late (ICP-5) kinetic classes in the cytoplasm of infected cells in the presence of anisomycin, canavanine, or phosphonoacetic acid and in the course of a normal infection. IE RNAs were overproduced and were the only class of transcript detected in anisomycin-blocked cells. Phosphonoacetic acid treatment resulted in overaccumulation of early RNAs and underaccumulation of early late RNAs. Although low-stringency canavanine treatment resulted in accumulation of RNA from all kinetic classes, high-stringency conditions restricted accumulation of herpes simplex virus type 1 RNAs to the IE class. More importantly, the IE RNAs for ICP-4 and ICP-0 accumulated to a lesser extent under high-stringency canavanine conditions compared with their accumulation in anisomycin-treated cells. Therefore, the absence of newly synthesized viral proteins (anisomysin treatment) and the presence of analog proteins (stringent canavanine treatment) have different consequences with regard to the accumulation of these two IE RNAs. The kinetics of cytoplasmic accumulation for these RNAs was different for each class of RNA. The IE RNAs were detectable at 1 h postinfection and reached a maximum accumulation at ca. 3 h postinfection. The IE RNAs for both ICP-4 and ICP-0 persisted at late times of infection; however, they differed in that the RNA for ICP-4 remained at relatively low levels and the RNA for ICP-0 remained at relatively high levels as compared with their peak levels of accumulation. The 1.4-kilobase RNA for the herpes simplex virus type 1 thymidine kinase was detected by 2 h, with maximum accumulation occurring at ca. 5 h postinfection. After the peak of accumulation, the amount of thymidine kinase RNA declined rapidly from 8 to 14 h postinfection. The early late RNA for ICP-5 was detected between 2 and 3 h, after which accumulation increased to a peak between 8 and 10 h postinfection. The level of ICP-5 RNA remained at close to the peak level until 14 h postinfection. We also compared the accumulation of viral mRNAs in the cytoplasm with the rates of synthesis of their respective polypeptides. Our results suggest that translational controls may be involved in the regulation of IE genes but not early or late genes.

Structure of replicating herpes simplex virus DNA.

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication.

Characterization of adenovirus type 2 high molecular weight nuclear transcripts.

The structure of adenovirus transcripts in the nuclei of infected cells has been studied. Evidence is presented for the existence in the steady-state population of nuclear RNA of two classes of adenovirus type 2 (Ad2)-specified large transcripts. The minor class consisted of molecules containing 3' poly(A), 5' cap structures and internal methylated nucleotides. Leader sequences at the 5' end of these large poly(A)+ transcripts were identified and characterized by S1 hybridization analyses on alkaline agarose. They represented a spectrum of intermediates which ranged from completely unprocessed (5' end at 16.5) to completely condensed (5' end at 26.5) forms. S1 hybrid analyses located 3' ends of large poly(A)+ nuclear RNA at 39, 50 and 61.5 on the Ad2 map. A fraction of these nuclear molecules consisted of transcripts that had undergone processing at both 3' and 5' ends of mRNA-coding regions since S1 bands corresponding to mature mRNAs for late proteins 52,55K, III, pVI and hexon, as well as 72K early protein were detected. The major class (75 to 85%) of large nuclear transcripts was not polyadenylated, but did contain capped leader sequences. These molecules yielded discrete bands, by S1 analysis, whose 3' termini could be positioned at sites corresponding to 5' ends of cytoplasmic mRNA (e.g. 47, 50, 51.5 and 66.5). The possible significance of these poly(A)-RNAs to the transcriptional programme of Ad2 is discussed.

Replication of herpes simplex virus type I DNA in permeabilized infected cells.

Herpes simplex type 1 (HSV)-infected Vero cells can be permeabilized by a combination of hypotonic shock and a mild emulsifier, gum arabic. Permeabilized cells will incorporate triphosphate precursors into viral and host DNA in vitro in ratios similar to those seen in vivo. This reaction is ATP-dependent and is shown to be replicative by the single strand density shift of DNA synthesized in the presence of BrdUTP. The product is heterogeneous in size, and contains a significant proportion of rapidly sedimenting forms and of unit size (55S) viral DNA. The presence of polyamines and EGTA (a specific chelator of Ca2+ ions) in the labeling medium is shown to be necessary to maintain the integrity of the replicating DNA. The average size of newly synthesized single strands, however, is smaller than seen in vivo. The reaction is sensitive to phosphonoacetic acid added at the time of labeling, at concentrations which inhibit in vivo synthesis only after one hour of pre-exposure. These properties make permeabilized cell monolayers an attractive system for the study of HSV DNA replication.