Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+   hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants.

Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated.

Dysregulated metabolism of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production.

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 (Δ22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 a number of virus transcripts are sequestered in the nucleus late in infection.

Herpes Simplex Virus 1 Expressing GFP-Tagged Virion Host Shutoff (vhs) Protein Uncouples the Activities of RNA Degradation and Differential Nuclear Retention of the Virus Transcriptome.

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). vhs causes several changes to the infected cell environment that favor the translation of late (L) virus proteins: cellular mRNAs are degraded, immediate early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C terminus with GFP.

Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1.

Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection which was not blocked by a nectin1 antibody and hence was not a consequence of residual nectin1 expression.

Novel Role for ESCRT-III Component CHMP4C in the Integrity of the Endocytic Network Utilized for Herpes Simplex Virus Envelopment.

Enveloped viruses exploit cellular trafficking pathways for their morphogenesis, providing potential scope for the development of new antiviral therapies. We have previously shown that herpes simplex virus 1 (HSV1) utilizes recycling endocytic membranes as the source of its envelope, in a process involving four Rab GTPases. To identify novel factors involved in HSV1 envelopment, we have screened a small interfering RNA (siRNA) library targeting over 80 human trafficking proteins, including coat proteins, adaptor proteins, fusion factors, fission factors, and Rab effectors.

Genetic and phenotypic intrastrain variation in herpes simplex virus type 1 Glasgow strain 17 syn+-derived viruses.

The Glasgow s17 syn+ strain of herpes simplex virus 1 (HSV1) is arguably the best characterized strain and has provided the reference sequence for HSV1 genetic studies. Here we show that our original s17 syn+ stock was a mixed population from which we have isolated a minor variant that, unlike other strains in the laboratory, fails to be efficiently released from infected cells and spreads predominantly by direct cell-to-cell transmission. Analysis of other s17-derived viruses that had been isolated elsewhere revealed a number with the same release phenotype.

Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins.

HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus proteins-VP22 and VP16 -are proposed to regulate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown.

Qualitative Differences in Capsidless L-Particles Released as a By-Product of Bovine Herpesvirus 1 and Herpes Simplex Virus 1 Infections.

Despite differences in the pathogenesis and host range of alphaherpesviruses, many stages of their morphogenesis are thought to be conserved. Here, an ultrastructural study of bovine herpesvirus 1 (BoHV-1) envelopment revealed profiles similar to those previously found for herpes simplex virus 1 (HSV-1), with BoHV-1 capsids associating with endocytic tubules. Consistent with the similarity of their genomes and envelopment strategies, the proteomic compositions of BoHV-1 and HSV-1 virions were also comparable.

Multiple Posttranscriptional Strategies To Regulate the Herpes Simplex Virus 1 vhs Endoribonuclease.

The herpes simplex virus 1 (HSV-1) virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in the shutoff of host protein synthesis. Hence, its unrestrained activity is considered lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex.

Shining a Light: Examining Similarities and Differences in the Work Psychology of Health Support Workers Employed in Long-Term Care and Home and Community Care Settings.

Health Support Workers (HSWs) provide up to 80% of care to residents and clients in the long-term care (LTC) and home and community care (HCC) sectors but have received little research attention compared with the regulated professions. The authors explore similarities and differences in the work psychology of HSWs employed in LTC and HCC settings. Data were collected via survey from 276 LTC and 184 HCC HSWs.

Herpes Simplex Virus 1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway That Functions at Low Temperature.

Unlabelled: Herpes simplex virus 1 (HSV-1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV-1 entry into keratinocytes has been the subject of limited investigation, but it is proposed to involve pH-dependent endocytosis, requiring the gD-binding receptor nectin-1. Here, we have utilized the nTERT human keratinocyte cell line as a new model for dissecting the mechanism of HSV-1 entry into the host.

Rab6 dependent post-Golgi trafficking of HSV1 envelope proteins to sites of virus envelopment.

Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi-to-plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis.

Mode of virus rescue determines the acquisition of VHS mutations in VP22-negative herpes simplex virus 1.

It has been proposed that herpes simplex virus 1 with VP22 deleted requires secondary mutation of VHS for viability. Here we show that a replication-competent Δ22 virus constructed by homologous recombination maintains a wild-type (Wt) VHS gene and has no other gross mutations. By contrast, Δ22 viruses recovered from a bacterial artificial chromosome contain multiple amino acid changes within a conserved region of VHS.

A network of protein interactions around the herpes simplex virus tegument protein VP22.

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM).

Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus.

Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)-infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface.

Recruitment of herpes simplex virus type 1 immediate-early protein ICP0 to the virus particle.

Although the herpes simplex virus type 1 (HSV-1) tegument is comprised of a large number of viral and cellular proteins, how and where in the cell these proteins are recruited into the virus structure is poorly understood. We have shown previously that the immediate-early gene product ICP0 is packaged by a mechanism dependent on the major tegument protein VP22, while others have shown a requirement for ICP27. We now extend our studies to show that ICP0 packaging correlates directly with the ability of ICP0 to complex with VP22 in infected cells.

Virion incorporation of the herpes simplex virus type 1 tegument protein VP22 occurs via glycoprotein E-specific recruitment to the late secretory pathway.

The mechanism by which herpesviruses acquire their tegument is not yet clear. One model is that outer tegument proteins are recruited by the cytoplasmic tails of viral glycoproteins. In the case of herpes simplex virus tegument protein VP22, interactions with the glycoproteins gE and gD have been shown.

Characterization of a CRM1-dependent nuclear export signal in the C terminus of herpes simplex virus type 1 tegument protein UL47.

The herpes simplex virus type 1 tegument protein known as VP13/14, or hUL47, localizes to the nucleus and binds RNA. Using fluorescence loss in photobleaching analysis, we show that hUL47 undergoes nucleocytoplasmic shuttling during infection. We identify the hUL47 nuclear export signal (NES) as a C-terminal 10-residue hydrophobic peptide and measure its efficiency relative to that of the classical human immunodeficiency virus type 1 Rev NES.

RNA binding by the herpes simplex virus type 1 nucleocytoplasmic shuttling protein UL47 is mediated by an N-terminal arginine-rich domain that also functions as its nuclear localization signal.

The function of the alphaherpesvirus UL47 tegument protein has not yet been defined. Nonetheless, previous studies with transfected cells have shown that both the herpes simplex virus type 1 homologue (hUL47, or VP13/14) and the bovine herpesvirus type 1 (BHV-1) homologue (bUL47, or VP8) have the capacity to shuttle between the nucleus and the cytoplasm. Furthermore, hUL47 packaged into the virion has also been shown to bind several individual virus-specific RNA transcripts.

Characterization of a novel transferable CRM-1-independent nuclear export signal in a herpesvirus tegument protein that shuttles between the nucleus and cytoplasm.

A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before.

Nucleocytoplasmic shuttling of bovine herpesvirus 1 UL47 protein in infected cells.

Previous studies with transfected cells have shown that the herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BHV-1) UL47 proteins shuttle between the nucleus and the cytoplasm. HSV-1 UL47 has also been shown to bind RNA. Here we examine the BHV-1 UL47 protein in infected cells using a green fluorescent protein-UL47-expressing virus.

Phosphorylation of the herpes simplex virus tegument protein VP22 has no effect on incorporation of VP22 into the virus but is involved in optimal expression and virion packaging of ICP0.

Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Delta22 virus replicated poorly in epithelial MDBK cells.

Herpes simplex virus tegument protein VP22 contains an internal VP16 interaction domain and a C-terminal domain that are both required for VP22 assembly into the virus particle.

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear.

Deletion of the herpes simplex virus VP22-encoding gene (UL49) alters the expression, localization, and virion incorporation of ICP0.

The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Delta22) displays a cell-specific replication defect in epithelial MDBK cells.