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Public health interventions such as social distancing and mask wearing decrease the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is unclear whether they decrease the viral load of infected patients and whether changes in viral load impact mortality from coronavirus disease 2019 (COVID-19). We evaluated 6923 patients with COVID-19 at six New York City hospitals from March 15-May 14, 2020, corresponding with the implementation of public health interventions in March. We assessed changes in cycle threshold (CT) values from reverse transcription-polymerase chain reaction tests and in-hospital mortality and modeled the impact of viral load on mortality. Mean CT values increased between March and May, with the proportion of patients with high viral load decreasing from 47.7% to 7.8%. In-hospital mortality increased from 14.9% in March to 28.4% in early April, and then decreased to 8.7% by May. Patients with high viral loads had increased mortality compared to those with low viral loads (adjusted odds ratio 2.34). If viral load had not declined, an estimated 69 additional deaths would have occurred (5.8% higher mortality). SARS-CoV-2 viral load steadily declined among hospitalized patients in the setting of public health interventions, and this correlated with decreases in mortality.

During latent infections with herpes simplex virus 1 (HSV-1), viral transcription is restricted and the genomes are mostly maintained in silenced chromatin, whereas in lytically infected cells all viral genes are transcribed and the genomes are dynamically chromatinized. Histones in the viral chromatin bear markers of silenced chromatin at early times in lytic infection or of active transcription at later times. The virion protein VP16 activates transcription of the immediate-early (IE) genes by recruiting transcription activators and chromatin remodelers to their promoters. Two IE proteins, ICP0 and ICP4 which modulate chromatin epigenetics, then activate transcription of early and late genes. Although chromatin is involved in the mechanism of activation of HSV- transcription, its precise role is not entirely understood. In the cellular genome, chromatin dynamics often modulate transcription competence whereas promoter-specific transcription factors determine transcription activity. Here, biophysical fractionation of serially digested HSV-1 chromatin followed by short-read deep sequencing indicates that nuclear HSV-1 DNA has different biophysical properties than protein-free or encapsidated HSV-1 DNA. The entire HSV-1 genomes in infected cells were equally accessible. The accessibility of transcribed or non-transcribed genes under any given condition did not differ, and each gene was entirely sampled in both the most and least accessible chromatin. However, HSV-1 genomes fractionated differently under conditions of generalized or restricted transcription. Approximately 1/3 of the HSV-1 DNA including fully sampled genes resolved to the most accessible chromatin when HSV-1 transcription was active, but such enrichment was reduced to only 3% under conditions of restricted HSV-1 transcription. Short sequences of restricted accessibility separated genes with different transcription levels. Chromatin dynamics thus provide a first level of regulation on HSV-1 transcription, dictating the transcriptional competency of the genomes during lytic infections, whereas the transcription of individual genes is then most likely activated by specific transcription factors. Moreover, genes transcribed to different levels are separated by short sequences with limited accessibility., Author summary Although chromatin epigenetics modulate transcription of the nuclear replicating DNA viruses, and play major roles in the process of establishment of, and reactivation from, latency, the specific mechanisms of this modulation are not totally clear. Chromatin often regulates the transcriptional competency of cellular genes, rather than the actual level of transcription of individual genes. Here, we show that chromatin dynamics regulate the transcription competency of entire herpes simplex virus 1 (HSV-1) genomes, rather than the actual transcription level of individual genes. Moreover, CTCF/ insulator containing sequences flanking the immediate-early gene loci are more inaccessible when these genes are highly transcribed in a context of little transcription from the rest of the genome than when no gene was highly transcribed or all genes were. We postulate that chromatin dynamics modulate the transcriptional competency of the HSV-1 genome. Genes in genomes rendered transcriptionally inactive by chromatin dynamics cannot be transcribed, whereas transcription of individual genes, or of group of genes, is regulated separately in the transcriptionally competent genomes.

Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells.

Entry of enveloped viruses requires fusion of viral and cellular membranes. Fusion requires the formation of an intermediate stalk structure, in which only the outer leaflets are fused. The stalk structure, in turn, requires the lipid bilayer of the envelope to bend into negative curvature. This process is inhibited by enrichment in the outer leaflet of lipids with larger polar headgroups, which favor positive curvature. Accordingly, phospholipids with such shape inhibit viral fusion. We previously identified a compound, 5-(perylen-3-yl)ethynyl-2′- deoxy -uridine (dUY11), with overall shape and amphipathicity similar to those of these phospholipids. dUY11 inhibited the formation of the negative curvature necessary for stalk formation and the fusion of a model enveloped virus, vesicular stomatitis virus (VSV). We proposed that dUY11 acted by biophysical mechanisms as a result of its shape and amphipathicity. To test this model, we have now characterized the mechanisms against influenza virus and HCV of 5-(perylen-3-yl)ethynyl- arabino -uridine (aUY11), which has shape and amphipathicity similar to those of dUY11 but contains an arabino-nucleoside. aUY11 interacted with envelope lipids to inhibit the infectivity of influenza virus, hepatitis C virus (HCV), herpes simplex virus 1 and 2 (HSV-1/2), and other enveloped viruses. It specifically inhibited the fusion of influenza virus, HCV, VSV, and even protein-free liposomes to cells. Furthermore, aUY11 inhibited the formation of negative curvature in model lipid bilayers. In summary, the arabino-derived aUY11 and the deoxy-derived dUY11 act by the same antiviral mechanisms against several enveloped but otherwise unrelated viruses. Therefore, chemically unrelated compounds of appropriate shape and amphipathicity target virion envelope lipids to inhibit formation of the negative curvature required for fusion, inhibiting infectivity by biophysical, not biochemical, mechanisms.

The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono- and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono- to polynucleosome sizes in complexes fractionating as cellular mono- to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono- to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.

Histones interact with herpes simplex virus type 1 (HSV-1) genomes and localize to replication compartments early during infections. However, HSV-1 genomes do not interact with histones in virions and are deposited in nuclear domains devoid of histones. Moreover, late viral replication compartments are also devoid of histones. The processes whereby histones come to interact with HSV-1 genomes, to be later displaced, remain unknown. However, they would involve the early movement of histones to the domains containing HSV-1 genomes and the later movement away from them. Histones unbind from chromatin, diffuse through the nucleoplasm, and rebind at different sites. Such mobility is upregulated by, for example, phosphorylation or acetylation. We evaluated whether HSV-1 infection modulates histone mobility, using fluorescence recovery after photobleaching. All somatic H1 variants were mobilized to different degrees. H1.2, the most mobilized, was mobilized at 4 h and further so at 7 h after infection, resulting in increases in its “free” pools. H1.2 was mobilized to a “basal” degree under conditions of little to no HSV-1 protein expression. This basal mobilization required nuclear native HSV-1 genomes but was independent of HSV-1 proteins and most likely due to cellular responses. Mobilization above this basal degree, and increases in H1.2 free pools, however, depended on immediate-early or early HSV-1 proteins, but not on HSV-1 genome replication or late proteins. Linker histone mobilization is a novel consequence of cell-virus interactions, which is consistent with the dynamic interactions between histones and HSV-1 genomes during lytic infection; it may also participate in the regulation of viral gene expression.

Since the 1950's, herpes simplex viruses (HSV) have played prominent roles in the development of antivirals. The first efficacious antivirals, as well as the first safe for systemic use, were developed against HSV. It was only in 1995 when the first antiviral against a target not validated first with HSV was approved for clinical use (sequinavir). The early successes of targeting HSV DNA replication were most influential in developing the concept that antiviral drugs must target viral proteins. Such concept has ensured the safety of current antiviral drugs, which all target viral proteins. Current antivirals have proven to have no major negative effects on non-infected cells or treated patients. Because of their success widespread and use, however, these drugs have been found to have some limitations. Perhaps the clinically most important one is their ability to promptly select for drug-resistant viral strains. Owing to such limitations, cellular proteins are now considered as valid targets for novel antiviral drugs. The discovery that pharmacological CDK inhibitors (PCIs) inhibit HSV replication through novel mechanisms played a major role in this new appreciation of cellular proteins as potential targets for antivirals. This appreciation is reflected in the scheduling of PCIs to enter clinical trials as antivirals (against HIV). Herein, we will review the roles of HSV as model viruses in the discovery and development of antiviral drugs. The major focus will be on the recent discovery on the activities of PCIs against HSV and other viruses.

Targeting viral proteins has lead to many successful antivirals. Yet, such antivirals rapidly select for resistance, tend to be active against only a few related viruses, and require previous characterization of the target proteins. Alternatively, antivirals may be targeted to cellular proteins. Replication of many viruses requires cellular CDKs and pharmacological CDK inhibitors (PCIs), such as the purine-based roscovitine (Rosco), are proving safe in clinical trials against cancer. Rosco inhibits replication of wild-type or (multi-)drug resistant HIV, HCMV, EBV, VZV, and HSV-1 and 2. However, the antiviral mechanisms of purine PCIs remain unknown. Our objective is to characterize these mechanisms using HSV as a model We have shown that Rosco prevents initiation of transcription from viral, but not cellular, genomes. This inhibition is promoter independent, but genome dependent, and requires no viral proteins. This is a novel antiviral mechanism and a previously unknown activity for purine PCIs.

Background: Antiviral drugs that target viral proteins are very successful but have certain limitations. Consequently, cellular proteins have begun to be considered as potential targets for novel antivirals. Cyclin-dependent kinases (CDKs) are arguably the cellular proteins best studied as potential targets. Several pharmacological CDK inhibitors (PCIs) have antiviral activity against wild-type or drug-resistant strains of HIV, human cytomegalovirus, herpes simplex virus (HSV), Epstein–Barr virus, varicella-zoster virus, Kaposi’s sarcoma herpesvirus, and human T-cell leukemia virus. Some PCIs such as roscovitine are apparently well tolerated and are scheduled to enter clinical trials as antivirals in 2005. However, the antiviral mechanisms of PCIs remain incompletely characterized, and PCIs with different molecular specificities may inhibit different viral functions. Objective: To evaluate whether PCIs with different molecular specificities target common viral functions. Methods: We evaluated the activiti...

Cyclin-dependent kinases (cdks) are required for replication of viruses that replicate only in dividing cells, such as adeno- and papillomaviruses. Recently, cdks have been shown to be required also for replication of viruses that can replicate in non-dividing cells, such as HIV-1 and herpes simplex virus types 1 and 2 (HSV-1 and -2). In these experiments, pharmacological cdk inhibitors (PCIs) were shown to have potent antiviral activity in vitro against HIV-1, HSV-1 and -2, human cytomegalovirus, varicella-zoster virus, and to inhibit specific functions of other viruses. Since two PCIs, flavopiridol and roscovitine, are proving to be non-toxic in human clinical trials against cancer, PCIs may be useful as antivirals. As significant advantages, PCIs are active in vitro against many viruses, including drug-resistant strains of HIV-1 and HSV-1, and mutant strains of HIV-1 or HSV-1 resistant to PCIs have not been identified in spite of intense efforts. Furthermore, the antiviral effects of a PCI and a conventional antiviral drug are additive. The aetiopathogenesis of several diseases, such as Kaposi's sarcoma, HPV-induced cervical carcinoma and HIV-associated nephropathy (HIVAN), among others, includes replication or expression of proteins by viruses that require cdks. Thus, PCIs could target both the aetiological agent (the virus) and the pathogenic mechanisms (cell replication). Two important questions regarding the antiviral activities of PCIs are the focus of current research efforts, (i) the identity of the specific cdks that mediate the antiviral activities of PCIs, and (ii) whether PCIs have antiviral activity in vivo at non-toxic doses.

Primary attachment to cellular glycans is a critical entry step for most human viruses. Some viruses, such as herpes simplex virus type 1 (HSV-1) and hepatitis C virus (HCV), bind to heparan sulfate, whereas others, such as influenza A virus (IAV), bind to sialic acid. Receptor mimetics that interfere with these interactions are active against viruses that bind to either heparan sulfate or to sialic acid. However, no molecule that inhibits the attachment of viruses in both groups has yet been identified. Epigallocatechin gallate (EGCG), a green tea catechin, is active against many unrelated viruses, including several that bind to heparan sulfate or to sialic acid. We sought to identify the basis for the broad-spectrum activity of EGCG. Here, we show that EGCG inhibits the infectivity of a diverse group of enveloped and nonenveloped human viruses. EGCG acts directly on the virions, without affecting the fluidity or integrity of the virion envelopes. Instead, EGCG interacts with virion surface proteins to inhibit the attachment of HSV-1, HCV, IAV, vaccinia virus, adenovirus, reovirus, and vesicular stomatitis virus (VSV) virions. We further show that EGCG competes with heparan sulfate for binding of HSV-1 and HCV virions and with sialic acid for binding of IAV virions. Therefore, EGCG inhibits unrelated viruses by a common mechanism. Most importantly, we have identified EGCG as the first broad-spectrum attachment inhibitor. Our results open the possibility for the development of small molecule broad-spectrum antivirals targeting virion attachment. IMPORTANCE This study shows that it is possible to develop a small molecule antiviral or microbicide active against the two largest groups of human viruses: those that bind to glycosaminoglycans and those that bind to sialoglycans. This group includes the vast majority of human viruses, including herpes simplex viruses, cytomegalovirus, influenza virus, poxvirus, hepatitis C virus, HIV, and many others.

We have previously shown that two inhibitors specific for cellular cyclin-dependent kinases (cdks), Roscovitine (Rosco) and Olomoucine (Olo), block the replication of herpes simplex virus (HSV). Based on these results, we demonstrated that HSV replication requires cellular cdks that are sensitive to these drugs (L. M. Schang, J. Phillips, and P. A. Schaffer. J. Virol. 72:5626–5637, 1998). We further established that at least two distinct steps in the viral replication cycle require cdks: transcription of immediate-early (IE) genes and transcription of early (E) genes (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161–2172, 1999). Since Rosco inhibits HSV replication efficiently even when added to infected cells at 6 h postinfection, we postulated that cdks may also be required for viral functions that occur after E gene expression. In the study presented herein, we tested this hypothesis directly by measuring the efficiency of viral replication, viral DNA synthesis, and expression of several viral genes during infections in which Rosco was added after E proteins had already been synthesized. Rosco inhibited HSV replication, and specifically viral DNA synthesis, when the drug was added at the time of release from a 12-h phosphonoacetic acid (PAA)-induced block in viral DNA synthesis. Inhibition of DNA synthesis was not a consequence of inhibition of expression of IE or E genes in that Rosco had no effect on steady-state levels of two E transcripts under the same conditions in which it inhibited viral DNA synthesis. Moreover, viral DNA synthesis was inhibited by Rosco even in the absence of protein synthesis. In a second series of experiments, the replication of four HSV mutants harboring temperature-sensitive mutations in genes essential for viral DNA replication was inhibited when Rosco was added at the time of shift-down from the nonpermissive to the permissive temperature. Viral DNA synthesis was inhibited by Rosco under these conditions, whereas expression of viral E genes was not affected. We conclude that cellular Rosco-sensitive cdks are required for replication of viral DNA in the presence of viral E proteins. This requirement may indicate that HSV DNA synthesis is functionally linked to transcription, which requires cdks, or that both viral transcription and DNA replication, independently, require viral or cellular factors activated by Rosco-sensitive cdks.

Initiation of productive infection by human herpes simplex virus type 1 (HSV-1) requires cell cycle-dependent protein kinase (cdk) activity. Treatment of cells with inhibitors of cdks blocks HSV-1 replication and prevents accumulation of viral transcripts, including immediate-early (IE) transcripts (26). Inhibition of IE transcript accumulation suggests that virion proteins, such as VP16, require functional cdks to activate viral transcription. In this report, we show that a cdk inhibitor, Roscovitine, blocks VP16-dependent IE gene expression. In the presence of Roscovitine, the level of virion-induced activation of a transfected reporter gene (the gene encoding chloramphenicol acetyltransferase) linked to the promoter-regulatory region of the ICP0 gene was reduced 40-fold relative to that of untreated samples. Roscovitine had little effect on the interaction of VP16 with VP16-responsive DNA sequences as measured by electrophoretic mobility shift assays. These data indicate that VP16-dependent activation of IE gene expression requires functional cdks and that this requirement is independent of the ability of VP16 to bind to DNA.

Several observations indicate that late-G 1 /S-phase-specific cellular functions may be required for herpes simplex virus (HSV) replication: (i) certain mutant HSV strains are replication impaired during infection of cells in the G 0 /G 1 but not in the G 1 /S phase of the cell cycle, (ii) several late-G 1 /S-phase-specific cellular proteins and functions are induced during infection, and (iii) the activity of a cellular protein essential for expression of viral immediate-early (IE) genes, HCF, is normally required during the late G 1 /S phase of the cell cycle. To test the hypothesis that late-G 1 /S-phase-specific cellular functions are necessary for HSV replication, HEL or Vero cells were infected in the presence of the cell cycle inhibitors roscovitine (Rosco) and olomoucine (Olo). Both drugs inhibit cyclin-dependent kinase 1 (cdk-1) and cdk-2 (required for cell cycle progression into the late G 1 /S phase) and cdk-5 (inactive in cycling cells) but not cdk-4 or cdk-6 (active at early G 1 ). We found that HSV replication was inhibited by Rosco and Olo but not by lovastatin (a cell cycle inhibitor that does not inhibit cdk activity), staurosporine (a broad-spectrum protein serine-threonine kinase inhibitor), PD98059 (an inhibitor specific for erk-1 and -2) or iso-Olo (a structural isomer of Olo that does not inhibit cdk activity). The concentrations of Rosco and Olo required to inhibit cell cycle progression and viral replication in both HEL and Vero cells were similar. Inhibition of viral replication was found not to be mediated by drug-induced cytotoxicity. Efforts to isolate Rosco- or Olo-resistant HSV mutants were unsuccessful, indicating that these drugs do not act by inhibiting a single viral target. Viral DNA replication and accumulation of IE and early viral RNAs were inhibited in the presence of cell cycle-inhibitory concentrations of Rosco or Olo. We therefore conclude that one or more cdks active from late G 1 onward or inactive in nonneuronal cells are required for accumulation of HSV transcripts, viral DNA replication, and production of infectious virus.

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly., Author Summary H3.1 is typically assembled into chromatin during DNA replication-dependent chromatin assembly. However, histones undergo exchange with those not bound in chromatin. During such exchanges, DNA replication-independent chromatin assembly incorporates histone variants, such as H3.3. The HSV-1 genomes are chromatinized, albeit in unstable nucleosomes. The viral genomes initially associate with H3.3, then associate with H3.1 only after HSV-1 DNA replication initiates. These differential interactions are consistent with the DNA replication-independent or -dependent assembly of H3.3 or H3.1, respectively, in cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, but the significance of this mobilization remained unknown. We now find that H3.3 and H3.1 are also mobilized during infection. H3.3 is mobilized to a similar extent before or after HSV-1 DNA replication, which is consistent with its DNA replication-independent assembly into HSV-1 chromatin. In contrast, H3.1 mobilization decreases during HSV-1 DNA replication, which is consistent with the assembly of previously mobilized H3.1 into HSV-1 chromatin concomitant with HSV-1 DNA replication. The mobilizations of H3.1 and H3.3 are consistent with their kinetics of association with HSV-1 genomes, providing the first indication that histone mobilization relates to the assembly of viral chromatin.

Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes., Author Summary The nuclear-replicating DNA viruses of the family herpesviridae cause a variety of diseases. Eight herpesviruses infect humans. Three of them, including herpes simplex virus 1 (HSV-1), belong to the alpha-herpesvirus sub-family. Viruses in this family have the fastest replication cycles of all herpesviruses, producing acute symptoms. During lytic infection, the genomes of HSV-1 associate with histones in more dynamic chromatin than those of the beta- and gamma- herpesviruses. The transcription activator ICP4 is conserved only among alpha-herpesviruses. Although ICP4 is essential, relatively little is known about its mechanisms of action. We have shown that histone dynamics are enhanced in HSV-1 lytically infected cells. Here we show that HSV-1 mutants in ICP4 are deficient in their ability to enhance histone dynamics. ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or DNA. The dynamics of histones were greater in the viral replication compartments, where ICP4 localizes, than in the cellular chromatin. ICP4 may thus mobilize histones away from HSV-1 genomes to activate transcription. Such a mechanism of transcription activation would result in the highly dynamic nature of the viral chromatin and the fast replication cycles, and the acute pathologies, of the alpha-herpesviruses.

Herpes simplex virus 1 (HSV-1) DNA is chromatinized during latency and consequently regularly digested by micrococcal nuclease (MCN) to nucleosome-size fragments. In contrast, MCN digests HSV-1 DNA in lytically infected cells to mostly heterogeneous sizes. Yet HSV-1 DNA coimmunoprecipitates with histones during lytic infections. We have shown that at 5 h postinfection, most nuclear HSV-1 DNA is in particularly unstable nucleoprotein complexes and consequently is more accessible to MCN than DNA in cellular chromatin. HSV-1 DNA was quantitatively recovered at this time in complexes with the biophysical properties of mono- to polynucleosomes following a modified MCN digestion developed to detect potential unstable intermediates. We proposed that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infections. Physiologically, nucleosome assembly typically associates with DNA replication, although DNA replication transiently disrupts nucleosomes. It therefore remained unclear whether the instability of the HSV-1 nucleoprotein complexes was related to the ongoing viral DNA replication. Here we tested whether HSV-1 DNA is in unstable nucleosome-like complexes before, during, or after the peak of viral DNA replication or when HSV-1 DNA replication is inhibited. HSV-1 DNA was quantitatively recovered in complexes fractionating as mono- to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h after infection, even if viral DNA replication was inhibited. Therefore, most HSV-1 DNA is in unstable nucleosome-like complexes throughout the lytic replication cycle, and the instability of these complexes is surprisingly independent of HSV-1 DNA replication. The specific accessibility of nuclear HSV-1 DNA, however, varied at different times after infection.

The infecting genomes of herpes simplex virus 1 (HSV-1) are assembled into unstable nucleosomes soon after nuclear entry. The source of the histones that bind to these genomes has yet to be addressed. However, infection inhibits histone synthesis. The histones that bind to HSV-1 genomes are therefore most likely those previously bound in cellular chromatin. In order for preexisting cellular histones to associate with HSV-1 genomes, however, they must first disassociate from cellular chromatin. Consistently, we have shown that linker histones are mobilized during HSV-1 infection. Chromatinization of HSV-1 genomes would also require the association of core histones. We therefore evaluated the mobility of the core histones H2B and H4 as measures of the mobilization of H2A-H2B dimers and the more stable H3-H4 core tetramer. H2B and H4 were mobilized during infection. Their mobilization increased the levels of H2B and H4 in the free pools and decreased the rate of H2B fast chromatin exchange. The histones in the free pools would then be available to bind to HSV-1 genomes. The mobilization of H2B occurred independently from HSV-1 protein expression or DNA replication although expression of HSV-1 immediate-early (IE) or early (E) proteins enhanced it. The mobilization of core histones H2B and H4 supports a model in which the histones that associate with HSV-1 genomes are those that were previously bound in cellular chromatin. Moreover, this mobilization is consistent with the assembly of H2A-H2B and H3-H4 dimers into unstable nucleosomes with HSV-1 genomes.

Although targeting viral proteins has lead to many successful antiviral drugs, these antivirals have certain limitations. They rapidly select for resistance, tend to be active against only a few related viruses and the proteins of a pathogen must be characterized before such drugs can be developed. Consequently, a long period is required from the identification of a new pathogen to the development of relevant antivirals, a major concern for emerging diseases. Cellular proteins are now considered as potential targets for antivirals. Drugs that target cellular proteins required for several viral functions might not easily select for drug-resistance. They may also be active against a variety of unrelated viruses, which commonly require the same cellular proteins, and against viral strains resistant to conventional antiviral drugs. These antivirals could be promptly tested against emerging viruses because even distantly related viruses commonly require the same cellular proteins. Cellular cyclin-dependent kinases (CDKs) are required for replication of many viruses and specific pharmacological CDK inhibitors (PCIs) are proving to have surprisingly few negative side effects in clinical trials (against cancer). PCIs inhibit replication of wild-type and multi-drug resistant strains of HIV, HSV-1, HSV-2, HCMV, EBV and VZV. Two PCIs, roscovitine and flavopiridol, were recently proven active in a mouse model of HIV-induced nephropathy. Because the antiviral mechanisms of PCIs require no viral proteins, mutations in viral genes may not easily overcome inhibition by these drugs. In fact, no PCI-resistant viral mutant has been reported. PCIs are scheduled to enter clinical trials as antivirals in 2005.

Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock- and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.

Although herpes simplex virus (HSV) replicates in noncycling as well as cycling cells, including terminally differentiated neurons, it has recently been shown that viral replication requires the activities of cellular cyclin-dependent kinases (cdks) (L. M. Schang, J. Phillips, and P. A. Schaffer, J. Virol. 72:5626–5637, 1998). Since we were unable to isolate HSV mutants resistant to two cdk inhibitors, Olomoucine and Roscovitine (Rosco), we hypothesized that cdks may be required for more than one viral function during HSV replication. In the experiments presented here, we tested this hypothesis by measuring the efficiency of (i) viral replication; (ii) expression of selected immediate-early (IE) (ICP0 and ICP4), early (E) (ICP8 and TK), and late (L) (gC) genes; and (iii) viral DNA synthesis in infected cultures to which Rosco was added after IE or IE and E proteins had already been synthesized. Rosco inhibited HSV replication, transcription of IE and E genes, and viral DNA synthesis when added at 1, 2, or 6 h postinfection or after release from a 6-h cycloheximide block. Transcription of a representative L gene, gC, was also inhibited by Rosco under all conditions examined. We conclude from these studies that cellular cdks are required for transcription of E as well as IE genes. In contrast, steady-state levels of at least one cellular housekeeping gene were not affected by Rosco. The requirement of viral IE and E transcription for cellular cdks may reflect either a requirement for specific cdk-activated cellular and/or viral transcription factors or a more global requirement for cdks in the transcriptional activation of the viral genome.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of infected animals. Expression of latency-related (LR) gene products is controlled by a 980-bp fragment (LR promoter). DNA sequence analysis revealed that two major open reading frames (ORFs) are in the LR gene. Antibodies directed against both ORFs were generated in rabbits by using synthetic peptides. Antibody P2, which is directed to sequences near the amino terminus of ORF 2, recognized a 41-kDa protein in lytically infected cells, suggesting that ORF 2 encodes a protein. When the LR gene was inserted into a mammalian expression vector and subsequently transfected into COS-7 cells, a 41-kDa protein was detected by use of silver-stained sodium dodecyl sulfate-polyacrylamide gels and by the P2 antibody. In contrast, this protein was not detected in mock-transfected cells. Deletion of DNA sequences containing ORF 2 blocked synthesis of the 41-kDa protein in COS-7 cells. Reverse transcriptase-mediated PCRs indicated that splicing occurs near the C terminus of ORF 2. Further studies indicated that LR RNA was alternatively spliced in latently infected cattle and that a fraction of LR RNA was poly(A)+. Taken together, these studies suggested that a spliced LR transcript has the potential to encode a 41-kDa protein.

We compared the levels of latent pseudorabies virus (PRV) DNA in trigeminal ganglia (TG) of pigs after intranasal inoculation of different PRV strains by using quantitative DNA PCR. The extent of colonization attained in each case varied significantly according to the type of strain and inoculum dose, wild-type (WT) PRV being the most efficient strain in colonizing TG. When groups of pigs representing different levels of precolonization of TG with an attenuated PRV strain were challenged with WT PRV, it became evident that there is a statistically significant inverse correlation between the extent of precolonization attained by an attenuated PRV strain in TG and the level of establishment of latency by superinfecting WT PRV. The protection against WT PRV latency did not correlate with the extent of WT PRV replication at the portal of entry.

A quantitative and differential polymerase chain reaction (PCR) was developed that measures the ability of Pseudorabies virus (PRV) to colonize tissues that are targets for latency. This PCR is based on the co-amplification of viral target sequences and that of a gene of the host species: the porcine Nuclear Factor 1 gene, which serves as an internal standard. The technique was found to be sensitive and reproducible. Using this technique, individual variabilities were detected in the level of colonization of trigeminal ganglia established by a PRV strain among different animals. At a population level, it was established that two different PRV attenuated strains establish latency with different efficiencies, when applied by the same route and at similar inoculation dose.