Enhancement of HSV-1 cell-free virion release by the envelope protein gC.

Glycoprotein C (gC), one of ∼12 HSV-1 envelope glycoproteins, carries out several important functions during infection, including the enhancement of virion attachment by binding to host cell heparan sulfate proteoglycans (HSPG). Here we report that gC can also enhance the release of cell-free progeny virions at the end of the infectious cycle. This activity was observed in multiple cellular contexts including Vero cells and immortalized human keratinocytes. In the absence of gC, progeny virions bound more tightly to infected cells, suggesting that gC promotes the detachment of virions from the infected cell surface. Given this finding, we analyzed the biochemical interactions that tether progeny virions to cells and report evidence for two distinct modes of binding. One is consistent with a direct interaction between gC and HSPG, whereas the other is gC-independent and likely does not involve HSPG. Together, our results i) identify a novel function for a long-studied HSV-1 glycoprotein, and ii) demonstrate that the extracellular release of HSV-1 virions is a dynamic process involving multiple viral and host components.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.

Special Issue "Replication and Spread of Alphaherpesviruses".

Alphaherpesviruses, one of three sub-families of the Herpesviridae, are of keen interest to biomedical scientists for several reasons [...].

Release of HSV-1 Cell-Free Virions: Mechanisms, Regulation, and Likely Role in Human-Human Transmission.

Herpes simplex virus type 1, or HSV-1, is a widespread human pathogen that replicates in epithelial cells of the body surface and then establishes latent infection in peripheral neurons. When HSV-1 replicates, viral progeny must be efficiently released to spread infection to new target cells. Viral spread occurs via two major routes. In cell-cell spread, progeny virions are delivered directly to cellular junctions, where they infect adjacent cells. In cell-free release, progeny virions are released into the extracellular milieu, potentially allowing the infection of distant cells. Cell-cell spread of HSV-1 has been well studied and is known to be important for in vivo infection and pathogenesis. In contrast, HSV-1 cell-free release has received less attention, and its significance to viral biology is unclear. Here, I review the mechanisms and regulation of HSV-1 cell-free virion release. Based on knowledge accrued in other herpesviral systems, I argue that HSV-1 cell-free release is likely to be tightly regulated in vivo. Specifically, I hypothesize that this process is generally suppressed as the virus replicates within the body, but activated to high levels at sites of viral reactivation, such as the oral mucosa and skin, in order to promote efficient transmission of HSV-1 to new human hosts.

Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export.

N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

APOBECs and Herpesviruses.

The APOBEC family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes - APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new host-pathogen conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits.

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Functional comparison of herpes simplex virus 1 (HSV-1) and HSV-2 ICP27 homologs reveals a role for ICP27 in virion release.

UNLABELLED: Numerous studies have focused on the regulatory functions of ICP27, an immediate-early (IE) protein of herpes simplex virus 1 (HSV-1). However, its homolog in HSV-2, termed ICP27t2, has been little studied. Here, we used two different approaches to functionally compare ICP27t2 and ICP27. In transfection-based assays, ICP27t2 closely resembled ICP27 in its capacity to enhance HSV-1 late gene expression, suppress the splicing of a viral intron, and complement the growth of an HSV-1 ICP27 null mutant. To study ICP27t2 in the context of viral infection, we engineered K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27. In Vero cells, K2F1 replicated with wild-type (WT) kinetics and yields, expressed delayed-early and late proteins normally, and was fully capable of activating several cellular signal transduction pathways that are ICP27 dependent. Thus, we conclude that ICP27t2 and ICP27 are functionally very similar and that ICP27t2 can mediate all ICP27 activities that are required for HSV-1 replication in cell culture. Surprisingly, however, we found that K2F1 forms plaques that are morphologically different from those of WT HSV-1. Investigation of this trait demonstrated that it results from the decreased release of progeny virions into the culture medium. This appears to be due to a reduction in the detachment of K2F1 progeny from the extracellular surface of the infected cell. We identified two HSV-1 ICP27 amino-terminal deletion mutants with a similar release defect. Together, these results demonstrate that ICP27 plays a heretofore-unappreciated role in modulating the efficiency of progeny virion release. IMPORTANCE: ICP27 is an essential, multifunctional regulatory protein that has a number of critical roles in the HSV-1 life cycle. Although ICP27 homologs are encoded by all known members of the Herpesviridae, previous work with several of these homologs has shown that they cannot substitute for ICP27 in the context of HSV-1-infected cells. Here, we identify ICP27t2 as the first homolog that can efficiently replace ICP27 in HSV-1 infection. Unexpectedly, our results also reveal that the sequence of the ICP27 gene can affect the release of HSV-1 progeny virions from the infected cell. Thus, our comparative study has revealed a novel function for ICP27 in the regulation of virus release.

Role of immediate early protein ICP27 in the differential sensitivity of herpes simplex viruses 1 and 2 to leptomycin B.

Leptomycin B (LMB) is a highly specific inhibitor of CRM1, a cellular karyopherin-ß that transports nuclear export signal-containing proteins from the nucleus to the cytoplasm. Previous work has shown that LMB blocks herpes simplex virus 1 (HSV-1) replication in Vero cells and that certain mutations in viral immediate early protein ICP27 can confer LMB resistance. However, little is known of the molecular mechanisms involved. Here we report that HSV-2, a close relative of HSV-1, is naturally resistant to LMB. To see whether the ICP27 gene determines this phenotypic difference, we generated an HSV-1 mutant that expresses the HSV-2 ICP27 instead of the HSV-1 protein. This recombinant was fully sensitive to LMB, indicating that one or more other viral genes must be important in determining HSV-2's LMB-resistant phenotype. In additional work, we report several findings that shed light on how HSV-1 ICP27 mutations can confer LMB resistance. First, we show that LMB treatment of HSV-1-infected cells leads to suppression of late viral protein synthesis and a block to progeny virion release. Second, we identify a novel type of ICP27 mutation that can confer LMB resistance, that being the addition of a 100-residue amino-terminal affinity purification tag. Third, by studying infections where both LMB-sensitive and LMB-resistant forms of ICP27 are present, we show that HSV-1's sensitivity to LMB is dominant to its resistance. Together, our results suggest a model in which the N-terminal portion of ICP27 mediates a nonessential activity that interferes with HSV-1 replication when CRM1 is inactive. We suggest that LMB resistance mutations weaken or abrogate this activity.

HSV-1 ICP22: hijacking host nuclear functions to enhance viral infection.

During its productive infection, HSV-1 dramatically remodels the architecture and physiology of the host cell nucleus. The immediate-early proteins, the first viral proteins to be expressed during infection, are key players in this process. Here, we review the known properties and functions of immediate-early protein ICP22. Although this polypeptide has received less attention than other immediate-early proteins, the published evidence indicates that it mediates several striking changes to important host nuclear systems, including those involved in RNA polymerase II transcription, cell cycle regulation and protein quality control. Recent genetic analyses suggest that these alterations can promote HSV-1 productive infection. Thus, future work on ICP22 is likely to reveal novel mechanisms by which herpesviruses, and possibly other DNA viruses, manipulate the host cell nucleus to enhance their replication.

Herpes simplex virus-induced epithelial damage and susceptibility to human immunodeficiency virus type 1 infection in human cervical organ culture.

Normal human premenopausal cervical tissue has been used to derive primary cell populations and to establish ex vivo organ culture systems to study infections with herpes simplex virus (HSV-1 or HSV-2) and human immunodeficiency virus type 1 (HIV-1). Infection with either HSV-1 or HSV-2 rapidly induced multinuclear giant cell formation and widespread damage in mucosal epithelial cells. Subsequent exposure of the damaged mucosal surfaces to HIV-1 revealed frequent co-localization of HSV and HIV-1 antigens. The short-term organ culture system provides direct experimental support for the epidemiological findings that pre-existing sexually transmitted infections, including primary and recurrent herpes virus infections at mucosal surfaces, represent major risk factors for acquisition of primary HIV-1 infection. Epithelial damage in combination with pre-existing inflammation, as described here for overtly normal human premenopausal cervix, creates a highly susceptible environment for the initiation and establishment of primary HIV-1 infection in the sub-mucosa of the cervical transformation zone.

Phenotypic suppression of a herpes simplex virus 1 ICP27 mutation by enhanced transcription of the mutant gene.

ICP27 is an essential herpes simplex virus 1 (HSV-1) regulatory protein that enhances viral gene expression. Although it is predominantly nuclear, it shuttles to the cytoplasm during infection using an N-terminal nuclear export signal (NES). We previously engineered an NES-negative ICP27 mutant, dLeu, that replicates poorly in cultured cells. In this study, we isolated dLeuR, a growth-competent revertant of dLeu. We show that dLeuR possesses one or more extragenic mutations that enhance ICP27 transcription, leading to overexpression of the mutant protein and restoration of viral growth. This work provides evidence of a novel pathway regulating transcription of the ICP27 gene.

Identification of an ICP27-responsive element in the coding region of a herpes simplex virus type 1 late gene.

During productive herpes simplex virus type 1 (HSV-1) infection, a subset of viral delayed-early (DE) and late (L) genes require the immediate-early (IE) protein ICP27 for their expression. However, the cis-acting regulatory sequences in DE and L genes that mediate their specific induction by ICP27 are unknown. One viral L gene that is highly dependent on ICP27 is that encoding glycoprotein C (gC). We previously demonstrated that this gene is posttranscriptionally transactivated by ICP27 in a plasmid cotransfection assay. Based on our past results, we hypothesized that the gC gene possesses a cis-acting inhibitory sequence and that ICP27 overcomes the effects of this sequence to enable efficient gC expression. To test this model, we systematically deleted sequences from the body of the gC gene and tested the resulting constructs for expression. In so doing, we identified a 258-bp "silencing element" (SE) in the 5' portion of the gC coding region. When present, the SE inhibits gC mRNA accumulation from a transiently transfected gC gene, unless ICP27 is present. Moreover, the SE can be transferred to another HSV-1 gene, where it inhibits mRNA accumulation in the absence of ICP27 and confers high-level expression in the presence of ICP27. Thus, for the first time, an ICP27-responsive sequence has been identified in a physiologically relevant ICP27 target gene. To see if the SE functions during viral infection, we engineered HSV-1 recombinants that lack the SE, either in a wild-type (WT) or ICP27-null genetic background. In an ICP27-null background, deletion of the SE led to ICP27-independent expression of the gC gene, demonstrating that the SE functions during viral infection. Surprisingly, the ICP27-independent gC expression seen with the mutant occurred even in the absence of viral DNA synthesis, indicating that the SE helps to regulate the tight DNA replication-dependent expression of gC.

Herpes simplex virus type 1 immediate-early protein ICP22 is required for VICE domain formation during productive viral infection.

During productive infection, herpes simplex virus type 1 (HSV-1) induces the formation of discrete nuclear foci containing cellular chaperone proteins, proteasomal components, and ubiquitinated proteins. These structures are known as VICE domains and are hypothesized to play an important role in protein turnover and nuclear remodeling in HSV-1-infected cells. Here we show that VICE domain formation in Vero and other cells requires the HSV-1 immediate-early protein ICP22. Since ICP22 null mutants replicate efficiently in Vero cells despite being unable to induce VICE domain formation, it can be concluded that VICE domain formation is not essential for HSV-1 productive infection. However, our findings do not exclude the possibility that VICE domain formation is required for viral replication in cells that are nonpermissive for ICP22 mutants. Our studies also show that ICP22 itself localizes to VICE domains, suggesting that it could play a role in forming these structures. Consistent with this, we found that ICP22 expression in transfected cells is sufficient to reorganize the VICE domain component Hsc70 into nuclear inclusion bodies that resemble VICE domains. An N-terminal segment of ICP22, corresponding to residues 1 to 146, is critical for VICE domain formation in infected cells and Hsc70 reorganization in transfected cells. We previously found that this portion of the protein is dispensable for ICP22's effects on RNA polymerase II phosphorylation. Thus, ICP22 mediates two distinct regulatory activities that both modify important components of the host cell nucleus.

Herpes simplex virus type 1 ICP27 induces p38 mitogen-activated protein kinase signaling and apoptosis in HeLa cells.

The herpes simplex virus type 1 (HSV-1) protein ICP27 has been implicated in a variety of functions important for viral replication including host shutoff, viral gene expression, activation of mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK), and apoptosis inhibition. In the present study we sought to examine the functions of ICP27 in the absence of viral infection by creating stable HeLa cell lines that inducibly express ICP27. Here, we characterize two such cell lines and show that ICP27 expression is associated with a cellular growth defect. The observed defect is caused at least in part by the induction of apoptosis as indicated by caspase-3 activation, annexin V staining, and characteristic changes in cellular morphology. In an effort to identify the function of ICP27 responsible for inducing apoptosis, we show that ICP27 expression is sufficient to activate p38 signaling to a level that is similar to that observed during wild-type HSV-1 infection. However, ICP27 expression alone is unable to lead to a strong activation of JNK signaling. Using chemical inhibitors, we show that the ICP27-mediated activation of p38 signaling is responsible for the observed induction of apoptosis in the induced cell lines. Our findings suggest that during viral infection, ICP27 activates p38 and JNK signaling pathways via two distinct mechanisms. ICP27 directly activates p38 signaling, leading to stimulation of the host cell apoptotic pathways. In contrast, robust activation of JNK signaling by ICP27 requires one or more delayed early or late viral gene products and may be associated with the inhibition of apoptosis.

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

Herpes simplex virus type 1 ICP27 regulates expression of a variant, secreted form of glycoprotein C by an intron retention mechanism.

We previously showed that herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP27 can posttranscriptionally stimulate mRNA accumulation from a transfected viral late gene encoding glycoprotein C (gC) (K. D. Perkins, J. Gregonis, S. Borge, and S. A. Rice, J. Virol. 77:9872-9884, 2003). We began this study by asking whether ICP27 homologs from other herpesviruses can also mediate this activity. Although the homologs from varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) were inactive, the homolog from bovine herpesvirus 4 (BHV-4), termed HORF1/2, was a very efficient transactivator. Surprisingly, most of the mRNA produced via HORF1/2 transactivation was 225 nucleotides shorter than expected due to the removal of a previously undescribed intron from the gC transcript. We found that the gC mRNA produced in the absence of transactivation was also mostly spliced. In contrast, gC mRNA produced by ICP27 transactivation was predominantly unspliced. Based on these results, we conclude that ICP27 has two distinct effects on the transfected gC gene: it (i) stimulates mRNA accumulation and (ii) promotes the retention of an intron. Interestingly, the spliced transcript encodes a variant of gC that lacks its transmembrane domain and is secreted from transfected cells. As the gC splicing signals are conserved among several HSV-1 strains, we investigated whether the variant gC is expressed during viral infection. We report here that both the spliced transcript and its encoded protein are readily detected in Vero cells infected with three different laboratory strains of wild-type HSV-1. Moreover, the variant gC is efficiently secreted from infected cells. We have designated this alternate form of the protein as gCsec. As the extracellular domain of gC is known to bind heparan sulfate-containing proteoglycans and to inhibit the complement cascade via an interaction with complement component C3b, we speculate that gCsec could function as a secreted virulence factor.

Herpes simplex virus type 1 immediate-early protein ICP27 is required for efficient incorporation of ICP0 and ICP4 into virions.

Early in infection, herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins ICP0 and ICP4 localize to the nucleus, where they stimulate viral transcription. Later in infection, ICP0 and to a lesser extent ICP4 accumulate in the cytoplasm, but their biological role there is unknown. Previously, it was shown that the cytoplasmic localization of ICP0/4 requires the multifunctional IE protein ICP27, which is itself an activator of viral gene expression. Here, we identify a viral ICP27 mutant, d3-4, which is unable to efficiently localize ICP0 and ICP4 to the cytoplasm but which otherwise resembles wild-type HSV-1 in its growth and viral gene expression phenotypes. These results genetically separate the function of ICP27 that affects ICP0/4 localization from its other functions, which affect viral growth and gene expression. As both ICP0 and ICP4 are known to be minor virion components, we used d3-4 to test the hypothesis that the cytoplasmic localization of these proteins is required for their incorporation into viral particles. Consistent with this conjecture, d3-4 virions were found to lack ICP0 in their tegument and to have greatly reduced levels of ICP4. Thus, the cytoplasmic localization of ICP0 and ICP4 appears to be a prerequisite for the assembly of these important transcriptional regulatory proteins into viral particles. Furthermore, our results show that ICP27 plays a previously unrecognized role in determining the composition of HSV-1 virions.

Herpes simplex virus immediate-early protein ICP22 triggers loss of serine 2-phosphorylated RNA polymerase II.

During eukaryotic mRNA transcription, the synthetic activity and mRNA processing factor interactions of RNA polymerase II (RNAP II) are regulated by phosphorylation of its carboxyl-terminal domain (CTD), with modification occurring primarily on serines 2 and 5 of the CTD. We previously showed that herpes simplex virus type 1 (HSV-1) infection rapidly triggers the loss of RNAP II forms bearing serine 2 phosphorylation (Ser-2P RNAP II). Here we show that the HSV-1 immediate-early (IE) protein ICP22 is responsible for this effect during the IE phase of infection. This activity does not require the viral UL13 protein kinase, which is required for several other regulatory functions of ICP22. Additionally, we show that transient expression of ICP22 can trigger the loss of Ser-2P RNAP II in transfected cells. Thus, the ability of ICP22 to cause the loss of Ser-2 RNAP II does not require other viral factors or the context of the infected cell. Expression of the HSV-1 ICP22-related protein US1.5, which corresponds to residues 147 to 420 of ICP22, also triggers a loss of Ser-2P RNAP II in transfected cells, whereas expression of the varicella-zoster virus ICP22 homolog, ORF63, does not. Our study also provides evidence for a second, viral late gene-dependent pathway that triggers loss of Ser-2P RNAP II in infected cells, consistent with the recent work of Dai-Ju et al. (J. Q. Dai-Ju, L. Li, L. A. Johnson, and R. M. Sandri-Goldin, J. Virol. 80:3567-3581, 2006). Therefore, it appears that HSV-1 has evolved redundant mechanisms for triggering the loss of a specific phosphorylated form of RNAP II.