James R. Smiley

Herpes Simplex Virus ICP0 Mutants Are Hypersensitive to Interferon

ABSTRACT Interferon (IFN) is an important immune system molecule capable of inducing an antiviral state within cells. Herpes simplex virus type 1 (HSV-1) replication is somewhat reduced in tissue culture in the presence of IFN, presumably due to decreased viral transcription. Here, we show mutations that inactivate immediate-early (IE) gene product ICP0 render HSV-1 exquisitely sensitive to IFN inhibition, resulting in greatly decreased levels of viral mRNA transcripts and the resulting polypeptides and a severe reduction in plaque formation ability. Mutations in other HSV-1 genes, including the genes coding for virion transactivator VP16 and the virion host shutoff protein vhs, IE gene ICP22, and the protein kinase UL13 gene, do not increase the IFN sensitivity of HSV-1. Interestingly, ICP0 mutants demonstrate the same level of sensitivity to IFN as wild-type virus on U2OS cells, an osteosarcoma cell line that is known to complement mutations in ICP0 and VP16. Thus, in some cell types, functional ICP0 is required for HSV-1 to efficiently bypass the inhibitory effects of IFN in order to ensure its replication. The significance of this link between ICP0 and IFN resistance is discussed.

Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase?

Herpes simplex virus type 1 (HSV-1) is the prototypical member of the Herpesviridae, a large family of enveloped DNA viruses that infect diverse metazoans. It is also the defining example of the Alphaherpesvirinae, the neurotropic subfamily of herpesviruses. Like all herpesviruses, HSV displays both lytic and latent modes of interaction with its natural human host. Primary infection of epithelial cells produces the lytic response—virus replication followed by cell death. Progeny virus particles then infect adjacent sensory neurons, establishing a lifelong latent interaction. The latent viral genome is maintained in an extrachromosomal state in which only a restricted portion of the genome is transcribed. Latent genomes occasionally reactivate into the lytic cycle, producing a limited amount of progeny virus that gives rise to secondary infections of the epithelial sites enervated by the latently infected neurons. n nHSV executes a complex genetic program during lytic infection (reviewed in reference 47). Expression of most cellular genes is strongly suppressed, and three temporal classes of viral genes are sequentially activated in a regulatory cascade. Five viral immediate-early (IE) genes are expressed first, and four of these (ICP0, ICP4, ICP22, and ICP27) encode regulatory proteins that stimulate expression of the viral early (E) and late (L) genes. The E genes are activated next, giving rise to proteins required for replication of the viral genome. Viral DNA replication then ensues, augmenting IE-dependent expression of the L genes that encode the structural components of the virion. n nHSV differs from many other nuclear DNA viruses in that some of its key regulatory polypeptides are delivered into the host cell by the infecting virus particle. These virion regulators are located in the viral tegument—the space between the envelope and the nucleocapsid—and as such are injected into the newly infected cell immediately upon fusion of the viral envelope with the host cell plasma membrane. These proteins are therefore strategically poised to influence the very earliest events in the viral replication cycle. In the best-known case, the abundant tegument protein VP16 activates transcription of the viral IE genes, thereby contributing to the initial launch of the lytic program of gene expression (reviewed in reference 17). The tegument also contains vhs, the virion host shutoff protein encoded by HSV gene UL41. vhs is an mRNA-specific RNase that triggers rapid shutoff of host cell protein synthesis, disruption of preexisting polyribosomes, and degradation of host mRNAs in the absence of de novo viral gene expression (reviewed in reference 55). Here I summarize our present understanding of the mechanism of vhs action and discuss recent studies that point to intriguing roles in viral pathogenesis and immune evasion. Space limitations preclude an exhaustive review of the earlier literature; I therefore seek the indulgence of my colleagues and refer the interested reader to a recent review (55) and the introductory sections of two recent articles (9, 10) for more details. Unless otherwise stated below, vhs refers to the UL41 gene product of HSV-1.

Evidence that Herpes Simplex Virus VP16 Is Required for Viral Egress Downstream of the Initial Envelopment Event

ABSTRACT During infection with herpes simplex virus type 1 (HSV-1), VP16 serves multiple functions, including transcriptional activation of viral immediate early genes and downregulation of the virion host shutoff protein vhs. Furthermore, VP16 has been shown to be involved in some aspect of virus assembly and/or maturation. Experiments with a VP16 null virus, 8MA, suggested that VP16 plays a direct role in virion assembly, since removal of VP16 from the HSV-1 genome results in reduced levels of encapsidated DNA and a failure to produce extracellular enveloped particles. However, VP16 null mutants display a severe translational arrest due to unrestrained vhs activity, thus complicating interpretation of these data. We examine here the role of VP16 in virion assembly and egress in the context of a vhs null background, using the virus 8MA/ΔSma (VP16−vhs−). Comparison of 8MA and 8MA/ΔSma with respect to viral DNA accumulation and encapsidation and accumulation of the major capsid protein, VP5, revealed that the 8MA lethal phenotype is only partially due to uncontrolled vhs activity, indicating that VP16 is required in HSV-1 virion formation. Electron microscopy confirmed these results and further showed that VP16 is required for HSV-1 egress beyond the perinuclear space. In addition, we describe the isolation and characterization of an 8MA derivative capable of propagation on Vero cells, due to second site mutations in the vhs and UL53 (gK) genes. Taken together, these results show that VP16 is required for viral egress downstream of the initial envelopment step and further underscore the importance of VP16 in controlling vhs activity within an infected cell.

Herpes Simplex Virus ICP0 and ICP34.5 Counteract Distinct Interferon-Induced Barriers to Virus Replication

ABSTRACT Interferon inhibits virus replication through multiple mechanisms. Here we show that herpes simplex virus proteins ICP0 and ICP34.5 overcome interferon-induced barriers to viral transcription and translation, respectively. These cytokine-induced antiviral mechanisms are differentially expressed in established cell lines: U2OS cells do not mount the IFN-induced mechanism targeted by ICP0, and Vero cells may be defective for the mechanism targeted by ICP34.5.

Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences

Mature Herpes Simplex Virus (HSV) genomes are cleaved from concatemeric precursors by a site-specific mechanism. These cleavage events are probably coupled to the encapsidation process. Sequences within the terminal repeat of HSV DNA are necessary for the cleavage and packaging reactions, and are also thought to be responsible for high frequency genome isomerization events. Here we present evidence to show that two viral DNA cleavage and packaging signals reside within a 250 bp subfragment of the terminal repeat, that the termini of mature viral DNA are generated by a process involving two separate DNA cleavages at sites distal to the cleavage signals, and that the sequences between these two cleavage sites are duplicated by the DNA maturation system.

Identification and characterization of the virion-induced host shutoff product of herpes simplex virus gene UL41

The virion-induced host shutoff product of the herpes simplex virus UL41 gene is required for shutoff of host translation and degradation of cellular mRNAs. We employed a rabbit antipeptide antiserum to identify a 58K UL41-related phosphoprotein in infected cells. We also provide evidence that this protein is a component of the virus particle, consistent with its role in virion-induced shutoff.

Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major component of infected cell membranes and virion envelopes. Glycoprotein B is known to be essential for entry of viruses into cells and may play important roles in virus-induced cell fusion and other alterations in cell morphology. In order to study the biochemical and immunological properties of gB in isolation from other HSV-1 polypeptides we have constructed human adenovirus vectors capable of expressing high levels of gB. The gB gene was coupled to the SV40 early promoter and inserted into the E3 region of two adenovirus vectors, one in which the E1 region was deleted (AdgB-1) and another which contained E1 sequences (AdgB-2). In AdgB-1 the orientation of the chimeric gB-SV40 gene was right to left, i.e., opposite to the direction of late and E3 mRNA transcription, whereas in AdgB-2 the orientation was left to right. Human 293 cells which express E1 functions supported replication of AdgB-1 and gB was expressed in these cells but not in mouse cells and only at very low levels in human cells other than 293. Replication of AdgB-2 was not limited to 293 cells and the virus was able to induce synthesis of gB at levels equal to or higher than those expressed in HSV-1-infected human or mouse cells. Microscopic examination of AdgB-2-infected cells revealed extensive vacuolization in a manner completely uncharacteristic of adenovirus-infected cells, and fluorescent antibody staining indicated that gB was not only present at the cell surface but also concentrated in the cytoplasmic vacuoles.

Construction of a double-jointed herpes simplex viral DNA molecule: Inverted repeats are required for segment inversion, and direct repeats promote deletions

Abstract In order to study the mechanisms of herpes simplex virus (HSV) DNA segment inversion and packaging of viral DNA a mutant “double-jointed” virus was constructed, by inserting an additional copy of the L-S junction into the viral thymidine kinase gene. This additional joint promotes the formation of all of the novel joints and termini which are predicted to arise if any region of viral DNA which is flanked by inverted repeats is capable of inverting independently. In addition to promoting the formation of new isomeric forms of complete viral DNA by recombination between sets of inverted repeats, the additional joint participates in recombination events with direct repeats, resulting in the formation of two half-genomes. These two half-genomes are both packaged into virions, but only as dimers. This result suggests that the recombination events which result in segment inversion frequently occur prior to the formation of concatemeric forms of viral DNA.

Herpes Simplex Virus vhs Protein

Publisher Summary The virion host shutoff (vhs) protein encoded by herpes simplex virus (HSV) gene UL41 is responsible for the rapid shutoff of host protein synthesis that occurs during the earliest stages of HSV infection. This chapter summarizes the current understanding of the mechanism of action and regulation of vhs activity, and provides a detailed description of a simple and convenient In Vitro assay for vhs-dependent ribonuclease activity. vhs is a structural component of the HSV virion that is synthesized late in infection and packaged into the tegument of the mature virus particle (the space between the envelope and the nucleocapsid). It is then delivered into the cytoplasm of newly infected cells after fusion of the virion envelope with the host plasma membrane, where it triggers host shutoff before the onset of de novo viral gene expression, vhs-induced shutoff is characterized by strong inhibition of host protein synthesis, disruption of preexisting polyribosomes, and accelerated turnover of host mRNAs. The vhs-dependent shutoff system exhibits little specificity, destabilizing most, if not all, cellular and viral mRNAs in the infected cell. The rapid decline in host mRNA levels presumably helps viral mRNAs gain access to the cellular translational apparatus. The vhs system provides a striking and readily dissected example of gene regulation at the level of mRNA stability in mammalian cells, and may therefore help illuminate the host mRNA turnover pathways that regulate cell growth, differentiation, and oncogenesis.

Herpes Simplex Virus ICP27 Is Required for Virus-Induced Stabilization of the ARE-Containing IEX-1 mRNA Encoded by the Human IER3 Gene

ABSTRACT Herpes simplex virus (HSV) stifles cellular gene expression during productive infection of permissive cells, thereby diminishing host responses to infection. Host shutoff is achieved largely through the complementary actions of two viral proteins, ICP27 and virion host shutoff (vhs), that inhibit cellular mRNA biogenesis and trigger global mRNA decay, respectively. Although most cellular mRNAs are thus depleted, some instead increase in abundance after infection; perhaps surprisingly, some of these contain AU-rich instability elements (AREs) in their 3′-untranslated regions. ARE-containing mRNAs normally undergo rapid decay; however, their stability can increase in response to signals such as cytokines and virus infection that activate the p38/MK2 mitogen-activated protein kinase (MAPK) pathway. We and others have shown that HSV infection stabilizes the ARE mRNA encoding the stress-inducible IEX-1 mRNA, and a previous report from another laboratory has suggested vhs is responsible for this effect. However, we now report that ICP27 is essential for IEX-1 mRNA stabilization whereas vhs plays little if any role. A recent report has documented that ICP27 activates the p38 MAPK pathway, and we detected a strong correlation between this activity and stabilization of IEX-1 mRNA by using a panel of HSV type 1 (HSV-1) isolates bearing an array of previously characterized ICP27 mutations. Furthermore, IEX-1 mRNA stabilization was abrogated by the p38 inhibitor SB203580. Taken together, these data indicate that the HSV-1 immediate-early protein ICP27 alters turnover of the ARE-containing message IEX-1 by activating p38. As many ARE mRNAs encode proinflammatory cytokines or other immediate-early response proteins, some of which may limit viral replication, it will be of great interest to determine if ICP27 mediates stabilization of many or all ARE-containing mRNAs.