Herpes Simplex Virus Type 1 Immediate-early Gene Research Articles

Single-cell RNA-sequencing analysis identifies host long noncoding RNA MAMDC2-AS1 as a co-factor for HSV-1 nuclear transport.

Herpes simplex virus (HSV) type 1 (HSV-1) infection exhibited high heterogeneity at individual cells level, including the different gene expression patterns and varying amounts of progeny virus. However, the underlying mechanism of such variability remains obscure. The importance of host long noncoding RNAs (lncRNAs) in virus infection had been recognized, while the contribution of lncRNAs to the heterogeneous infection remains unknown. Herein, a prior single-cell RNA sequencing data using HSV-1 reporter strain expressing ICP4-YFP was re-analyzed to obtain the differentially expressed lncRNA between the successfully initiated viral gene expression (ICP4-YFP+) cells and the aborted infection cells (ICP4-YFP-). The ICP4-YFP+ population show a higher abundance of MAMDC2 antisense 1 (MAMDC2-AS1) lncRNA than ICP4-YFP- population. MAMDC2-AS1 silencing reduces the expression of HSV-1 immediate early (IE) genes and limit HSV-1 infection in human host cells. Consistently, ectopic expression of MAMDC2-AS1 enhances HSV-1 IE genes transcription and facilitates the formation of HSV-1-induced plaques. Mechanically, both RNA-pull down and RNA immunoprecipitation assays show that MAMDC2-AS1 interacts with the RNA binding protein heat shock protein 90α (Hsp90α), a molecular chaperone involving in the nuclear import of HSV-1. The MAMDC2-AS1-Hsp90α interaction facilitates the nuclear transport of viral tegument protein VP16, the core factor initiating the expression of HSV-1 IE genes. The transcription factor YY1 mediates the induction of MAMDC2-AS1 upon HSV-1 infection. Our study elucidates the contribution of lncRNA to HSV-1 infection susceptibility in human cells and the role of Hsp90α RNA binding activity in HSV-1 infection.

Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons.

Trichosanthin affects HSV-1 replication in Hep-2 cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inhibits the replication of both human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1). The mechanism of inhibition is not clear. This investigation explored the effects of TCS on the stages of HSV-1 infection in Hep-2 cells, from attachment to release. We demonstrated that TCS reduced HSV-1 antigen and DNA content and interfered with viral replication as early as 3–15h after infection. TCS had no effect on HSV-1 attachment, penetration or immediate-early gene expression. However, the expression of early and late genes and virion release were diminished. In summary, this study demonstrates that TCS primarily affects HSV-1 replication in Hep-2 cells during the early to late infection period.

PI3K/Akt signaling mediated apoptosis blockage and viral gene expression in oral epithelial cells during herpes simplex virus infection

Phosphatidylinositol 3-kinases (PI3Ks) function in the anti-apoptotic pathway, and are commonly exploited by various viruses to accomplish the viral life cycle. This study examined the role of the PI3K pathway in human oral epithelial cells following herpes simplex virus type 1 (HSV-1) infection. The results showed that HSV-1 induced the phosphorylation of Akt and glycogen synthase kinase 3 (GSK-3). Phosphorylation of Akt, but not GSK-3, induced by HSV-1 was PI3K-dependent. The expression of HSV-1 immediate-early genes may be involved in the initial phosphorylation of Akt and GSK-3. Inhibition of HSV-1-induced PI3K activity increased DNA fragmentation and cleavage of poly ADP-ribose polymerase (PARP), caspase 3 and caspase 7 compared with infected alone. Inhibition of PI3K attenuated the expression of HSV-1-infected cell protein 0 (ICP0), but not thymidine kinase (TK) and viral replication. Collectively, these data suggested that, in oral epithelial cells, the HSV-1-induced PI3K/Akt activation was involved in the regulation of apoptosis blockage and viral gene expression.

The Presence of Lytic HSV-1 Transcripts and Clonally Expanded T Cells with a Memory Effector Phenotype in Human Sensory Ganglia

Herpes simplex virus type 1 (HSV-1) latent persistence in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T-cell infiltration. Thus far, during HSV-1 latency only a single transcript, namely the latency-associated transcript (LAT), has been identified to be synthesized but not translated into a protein. In contrast, the chronic CD8 T-cell infiltration suggests that an antigen trigger must be present. The focus of the current work was to look for HSV-1 transcription activity as a potential trigger of the immune response and to demonstrate whether the immune cells are clonally expanded and have a phenotype that suggests that they have been triggered by viral antigen. By combining in situ hybridization, laser cutting microscopy, and single-cell real time RT-PCR, we demonstrated expression of the HSV-1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T cells are clonally expanded, indicating an antigen-driven immune response. Moreover, the persisting CD8(+) T cells had prominent features of the memory effector phenotype. Chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells and the corresponding chemokine receptors CCR5 and CXCR3 were co-expressed on virtually all T cells bearing the CD8 phenotype. Thus, HSV-1 IE genes are expressed in human TG, and the infiltrating T cells bear several characteristics that suggest viral antigenic stimulation.

Transcriptional Coactivators Are Not Required for Herpes Simplex Virus Type 1 Immediate-Early Gene Expression In Vitro

Virion protein 16 (VP16) of herpes simplex virus type 1 (HSV-1) is a potent transcriptional activator of viral immediate-early (IE) genes. The VP16 activation domain can recruit various transcriptional coactivators to target gene promoters. However, the role of transcriptional coactivators in HSV-1 IE gene expression during lytic infection had not been fully defined. We showed previously that transcriptional coactivators such as the p300 and CBP histone acetyltransferases and the BRM and Brg-1 chromatin remodeling complexes are recruited to viral IE gene promoters in a manner dependent mostly on the presence of the activation domain of VP16. In this study, we tested the hypothesis that these transcriptional coactivators are required for viral IE gene expression during infection of cultured cells. The disrupted expression of the histone acetyltransferases p300, CBP, PCAF, and GCN5 or the BRM and Brg-1 chromatin remodeling complexes did not diminish IE gene expression. Furthermore, IE gene expression was not impaired in cell lines that lack functional p300, or BRM and Brg-1. We also tested whether these coactivators are required for the VP16-dependent induction of IE gene expression from transcriptionally inactive viral genomes associated with high levels of histones in cultured cells. We found that the disruption of coactivators also did not affect IE gene expression in this context. Thus, we conclude that the transcriptional coactivators that can be recruited by VP16 do not contribute significantly to IE gene expression during lytic infection or the induction of IE gene expression from nucleosomal templates in vitro.

Curcumin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity

Curcumin, a phenolic compound from the curry spice turmeric, exhibits a wide range of activities in eukaryotic cells, including antiviral effects that are at present incompletely characterized. Curcumin is known to inhibit the histone acetyltransferase activity of the transcriptional coactivator proteins p300 and CBP, which are recruited to the immediate early (IE) gene promoters of herpes simplex virus type 1 (HSV-1) by the viral transactivator protein VP16. We tested the hypothesis that curcumin, by inhibiting these coactivators, would block viral infection and gene expression. In cell culture assays, curcumin significantly decreased HSV-1 infectivity and IE gene expression. Entry of viral DNA to the host cell nucleus and binding of VP16 to IE gene promoters was not affected by curcumin, but recruitment of RNA polymerase II to those promoters was significantly diminished. However, these effects were observed using lower curcumin concentrations than those required to substantially inhibit global H3 acetylation. No changes were observed in histone H3 occupancy or acetylation at viral IE gene promoters. Furthermore, p300 and CBP recruitment to IE gene promoters was not affected by the presence of curcumin. Finally, disruption of p300 expression using a short hairpin RNA did not affect viral IE gene expression. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity.

Presence of HSV‐1 Immediate Early Genes and Clonally Expanded T‐cells with a Memory Effector Phenotype in Human Trigeminal Ganglia

The latent persistence of herpes simplex virus type 1 (HSV-1) in human trigeminal ganglia (TG) is accompanied by a chronic CD8 T-cell infiltrate. The focus of the current work was to look for HSV-1 transcription activity as a potential trigger of the immune response and to characterize the immune cell infiltrates by this feature. We combined in situ hybridization, laser cutting microscopy, and single cell RT-PCR to demonstrate the expression of the HSV-1 immediate early (IE) genes ICP0 and ICP4 in human trigeminal neurons. Using CDR3 spectratyping, we showed that the infiltrating T-cells are clonally expanded, indicating an antigen-driven immune response. Moreover, the persisting CD8+ T-cells had features of the memory effector phenotype. The voltage-gated potassium channel Kv1.3, a marker of chronic activated memory effector cells, and the chemokines CCL5 and CXCL10 were expressed by a subpopulation of infiltrating cells. The corresponding chemokine receptors CCR5 and CXCR3 were co-expressed on virtually all CD8 T-cells. In addition, T-cells expressed granzymes and perforin. In contrast to animal models of HSV-1 latency, hardly any FoxP3-positive regulatory T-cells were detected in human TG. Thus, HSV-1 IE genes are expressed in human TG and the infiltrating T-cells bear several characteristics that suggest viral antigenic stimulation.

Early Growth Response gene 1 (Egr-1) regulates HSV-1 ICP4 and ICP22 gene expression

The molecular mechanisms mediating herpes simplex virus type 1 (HSV-1) gene silencing during latent infection are not clear. Five copies of early growth response gene 1 (Egr-1) binding elements were identified in the intron of HSV-1 ICP22 (infected cell protein No. 22) gene, leading to the hypothesis that Egr-1 binds to the viral genome and regulates the viral gene expression. Transient co-transfection assays indicated that Egr-1 negatively regulated the transcription of both full-length and intron-removed ICP22 promoters. The same assays also revealed that Egr-1 repressed ICP4 (infected cell protein No. 4) promoter activity in a dose-dependent manner but showed less inhibition when the intron was removed. Histone deacetylation was not involved in this regulation since histone deacetylase inhibitor trichostatin A did not exhibit any effect on Egr-1-mediated repression. Chromatin immunoprecipitation assays showed that Egr-1 reduced the binding of Sp1 to the promoters and that the co-repressor Nab2 (NGFI-A/EGR1-binding protein) was recruited to the proximity of ICP4 in the presence of Egr-1. These results suggested that the multifunctional transcription factor Egr-1 can repress HSV-1 immediate-early gene expression through the recruitment of co-repressor Nab2 and reduction of Sp1 occupancy, and thus may play a critical role in HSV-1 gene silencing during latency.

Understanding the Relationship Between ApoE and HSV-1 and its Possible Significance in Alzheimer’s Disease

Evaluation of: Miller RM, Federoff HJ: Isoform-specific effects of ApoE on HSV immediate early gene expression and establishment of latency. Neurobiol. Aging (2006) (Epub ahead of print). It is 100 years since Alzheimer's disease was first described and there is still no consensus of opinion regarding the etiology of its sporadic form. Herpes simplex virus type 1 (HSV-1), in conjunction with apolipoprotein E (ApoE), may well play a role in this form of the disease. In the evaluated article, Miller and Federoff try to determine whether the ApoE profile affects the expression of the HSV-1 immediate-early genes and the production of the virus’ latency-associated transcript. Their results show that overexpression of the intermediate-early genes and the establishment of latency are directly correlated with the ApoE genotype.

The herpes simplex virus type 1 ICP0 promoter is activated by viral reactivation stimuli in trigeminal ganglia neurons of transgenic mice.

Herpes simplex virus type 1 (HSV-1) causes a latent infection in sensory ganglia neurons in humans and in the mouse model. The ability of the virus to latently infect neurons and reactivate is central to the ability of HSV-1 to remain in the human population and spread to new hosts. It is possible that neuronal transcriptional proteins control latency and reactivation by modulating activation of the HSV-1 immediate-early (IE) gene ICP0. We have previously shown that factors in trigeminal ganglia neurons can differentially activate the IE ICP0 promoter and the IE ICP4 promoter in developing trigeminal ganglia neurons of transgenic mice. Ultraviolet (UV) irradiation and hyperthermic stress have been shown to result in HSV-1 reactivation from sensory neurons in the mouse model. Reporter transgenic mice were exposed to UV irradiation or hyperthermia to test whether stimuli that are known to reactivate HSV-1 could activate viral IE promoters in the absence of viral proteins. Measurement of beta-galactosidase activity in trigeminal ganglia from these transgenic mice indicated that the ICP0 promoter activity was significantly increased by both UV irradiation and hyperthermia. The IE genes ICP4 and ICP27 and the late gene gC reporter transgenes failed to be activated in parallel experiments. These results suggest that the ICP0 promoter is a target for activation by host transcription factors in sensory neurons that have undergone damage. It further suggests the possibility that activation of ICP0 gene expression by neuronal transcription factors may be important in reactivation of HSV-1 in neurons.

Herpes Simplex Virus Type 1 Suppresses the Interferon Signaling Pathway by Inhibiting Phosphorylation of STATs and Janus Kinases during an Early Infection Stage

We examined the influence on the interferon (IFN) signaling pathway of infection with herpes simplex virus type 1 (HSV-1) strain VR3. Data from reporter gene assays showed that expression of both type I and type II IFN-inducible genes was dramatically suppressed during the early stage of HSV-1 infection (2 to 3 h postinfection). During these periods, phosphorylation levels of janus kinases (JAKs) and STATs did not increase after treatment of HSV-1-infected FL cells with IFN-α or IFN-γ, although cellular protein levels of the JAKs and the STATs were not significantly changed. In contrast, the inhibitory effect of HSV-1 on phosphorylation of STAT1 was not observed in U937 cells, which show resistance to steady-state accumulation of RNA for HSV-1 immediate-early genes. The phosphorylation of STAT1 in FL cells was not inhibited by infection with a UV-inactivated virus. These results indicate that viral gene expression or viral protein production is necessary for the inhibition of phosphorylation by HSV-1.

Herpes Simplex Virus Type 1 Infection Imposes a G1/S Block in Asynchronously Growing Cells and Prevents G1 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, G0-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 G1 from proceeding through the restriction point and for cells in S from completing a round of DNA replication.

Herpes Simplex Virus Type 1 Latency-Associated Transcripts Suppress Viral Replication and Reduce Immediate-Early Gene mRNA Levels in a Neuronal Cell Line

During herpes simplex virus type 1 (HSV-1) latent infection in human dorsal root ganglia, limited viral transcription, which has been linked to HSV-1 reactivation ability, takes place. To study the involvement of this transcription in HSV-1 replication in neuronal cells and consequently in viral latency, we constructed stably transfected neuronal cell lines containing (i) the entire HSV-1 latency transcriptionally active DNA fragment, (ii) the same DNA sequence with deletions of the latency-associated transcript (LAT) promoters, or (iii) the DNA coding sequence of the LAT domain. Replication of HSV-1 or a LAT-negative mutant was markedly repressed in the LAT-expressing cells, a phenomenon mediated by the LATs. To study the mechanism responsible for this effect, we examined LAT influence upon expression of HSV-1 immediate-early (IE) genes ICP0, ICP4, and ICP27, by Northern blot analysis. Following infection of a LAT-expressing neuronal cell line with a LAT-negative mutant, the steady-state levels of all three IE mRNAs were reduced compared to those for control cells. Transient transfections into a neuronal cell line indicated that the LAT suppressive effect upon ICP0 mRNA was mediated directly and was not due to the LAT effect upon the ICP0 promoter. We therefore propose that the LATs may repress viral replication in neuronal cells by reducing IE gene mRNA levels and thus facilitate the establishment of HSV-1 latency in nervous tissue.

Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis.

Herpes simplex virus type 1 (HSV-1) transcription can be arrested at the immediate early (IE) stage by continuous treatment of cells with inhibitors of protein synthesis, usually cycloheximide, from the time of infection. We have analysed the effect of cycloheximide on IE gene expression with HSV-1 mutants deficient in the production of functional levels of the three major transactivators, the virion protein (VP16) and two IE proteins (ICP0 and ICP4). Expression from the HSV-1 IE promoters that control synthesis of ICP0 and ICP27 was, unexpectedly, stimulated by inhibition of protein synthesis. The effect was observed for the ICP0 promoter in its normal genome location and also when cloned upstream of the Escherichia coli lacZ coding sequences and inserted into the viral thymidine kinase locus. Expression from the human cytomegalovirus major IE promoter, when cloned into the genome of HSV-1 mutants, was also increased by inhibition of protein synthesis. Cycloheximide did not affect the intracellular stability of lacZ-specific RNA, suggesting that the response represented an increase in mRNA production. Activation of the ICP0 promoter was observed when protein synthesis was blocked by alternative agents. Since inhibitors of protein synthesis are known to activate cellular signal transduction pathways, our findings demonstrate new mechanisms for the regulation of HSV-1 IE gene expression which may be important during latency and reactivation. The results also highlight previously unrecognized difficulties in analysing the intrinsic activities of promoters when cloned into the HSV-1 genome.

Cooperation between herpes simplex virus type 1-encoded ICP0 and Tat to support transcription of human immunodeficiency virus type 1 long terminal repeat in vivo can occur in the absence of the TAR binding site.

Expression of human immunodeficiency virus type 1 (HIV-1) provirus can be stimulated by herpes simplex virus type 1 (HSV-1) infection; the stimulation occurs at the level of transcriptional activation of the HIV long terminal repeat (LTR) and is mediated by both cellular and HSV-1-encoded transactivators. We have shown in this study that HSV-1 immediate-early gene ICP0 cooperates effectively with the HIV-1-encoded transactivator, Tat, in the stimulation of HIV-1 LTR-directed transcription. The cooperation between ICP0 and Tat is specific for the HIV-1 LTR and was not observed with other promoters (e.g., ICP0) that can be transactivated by ICP0 but not by Tat. Analyses of HIV-1 LTR deletion mutants have shown that ICP0 not only transactivates an HIV-1 LTR mutant that is unresponsive to NF-kappaB and Tat-mediated transactivation, such as the HIV-1 LTR with the enhancer deleted (-83 LTR) and TAR deleted (+20 to +81), but also restores responsiveness to Tat. ICP0 also showed cooperation with Gal4-Tat fusion protein-mediated transactivation of Gal4-HIV-1 LTR with TAR deleted. Enhancement of the transcriptional activation of ICP0 by Tat requires both the cysteine-rich and core domains of Tat and is inhibited by RO5-3335. ICP0 stimulates transcription of not only the HIV-1 LTR but also the TAR-defective HIV-1 provirus. We suggest that ICP0 can (i) recruit Tat to the vicinity of the HIV-1 promoter, thereby providing an alternative binding site for Tat, and (ii) substitute for the enhancer-binding proteins that are required for efficient Tat transactivation in T cells.

Herpes simplex virus type 1 replication and IL-1β gene expression in mouse peritoneal macrophages activated in vivo by an attenuated Salmonella typhimurium vaccine or Corynebacterium parvum

Activated macrophages (Mφ) from mice given Salmonella typhimurium or Corynebacterium parvum were compared with resident peritoneal macrophages at the molecular level for permissiveness for herpes simplex virus type 1 (HSV-1) replication and for expression of interleukin-1 beta (IL-1β). Peritoneal macrophages were harvested from mice injected 7 days previously with live, avirulent S. typhimurium (Sal-PMφ) or heat-killed C. parvum (CP-PMφ), and infected with HSV-1 in vitro. Both Sal-PMφ and CP-PMφ were activated as evidenced by characteristic changes in an ectoenzyme, by increased permissiveness for infectious virus production and viral cytopathic effect, and by induction of IL-1β mRNA. Analysis at the molecular level revealed that both types of activated Mφ demonstrated increased patterns of HSV-1 immediate-early gene expression and viral DNA replication as compared with resident cells. A novel finding was that viral infection reduced IL-1β mRNA in both types of activated Mβ. This observation has implications for the efficacy of Salmonella vaccines given in proximity to HSV-1 infection and for potential deleterious effects of HSV-1 infection in immunosuppressed patients receiving immunotherapy.

Varicella-zoster virus open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters

The varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein is the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. These are two virion tegument proteins that have extensive amino acid sequence identity in their amino-terminal and middle domains. ORF10, however, lacks the acidic carboxy terminus which is critical for transactivation by VP16. Earlier studies showed that VZV ORF10 does not form a tertiary complex with the TAATGARAT regulatory element (where R is a purine) with which HSV-1 VP16 interacts, suggesting that ORF10 may not have transactivating ability. Using transient-expression assays, we show that VZV ORF10 is able to transactivate VZV immediate-early (IE) gene (ORF62) and HSV-1 IE gene (ICP4 and ICP0) promoters. Furthermore, cell lines stably expressing ORF10 complement the HSV-1 mutant in1814, which lacks the transactivating function of VP16, and enhance the de novo synthesis of infectious virus following transfection of HSV-1 virion DNA. These results indicate that ORF10, like its HSV-1 homolog VP16, is a transactivating protein despite the absence of sequences similar to the VP16 carboxy-terminal domain. The transactivating function of the VZV ORF10 tegument protein may be critical for efficient initiation of viral infection.

Herpes simplex virus immediate early gene expression in the absence of transinduction by Vmw65 varies during the cell cycle

The requirement for the herpes simplex virus type 1 (HSV-1) protein Vmw65 (VP16) for activation of immediate early (IE) gene expression was examined in synchronized HeLa cells. Analyses of IE RNA levels were conducted during infection with a viral Vmw65 mutant, in1814. The results revealed an increased requirement for Vmw65 when cultures reached G2 phase of the cell cycle. The levels of IE RNAs 1, 2, and 4 were reduced 5–10 times more in G2 than G1/S for in1814-infected cells when compared to cells infected with wild-type virus or 1814R (a rescued virus), and similar but smaller effects were observed on IE RNA 3 levels. The relative decrease at G2 was reversed by resynchronization of cells to G1 /S. Mutant in1814 formed plaques less efficiently on cells at G2 than on cells synchronized at G1/S. The results show that, in the absence of functional Vmw65, HSV-1 IE gene expression and replication vary during the cell cycle.

Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor.

The purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).