After a degree in Microbiology at Queen's University Belfast, I obtained my PhD working with Bert Rima on the molecular biology of the RNA virus mumps virus. I subsequently worked as a postdoc with Sue and Alan Kingsman in Oxford where I studied molecular aspects of HIV transcription. My herpesvirus research began when I obtained a Wellcome Trust Junior Fellowship to work at the University of Leeds. A further move to Marie Curie Research Institute in Surrey, allowed me to pursue my interests in herpesviruses, initially in the lab of Peter O'Hare, and subsequently in my own group. In 2007 I moved to the Section of Virology, Imperial College London where I obtained an MRC Senior Nonclinical Fellowship working on the cell biology of herpes simplex virus morphogenesis. I took up my current position of Professor of Virology in the Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, in June 2013.
My research focuses on the cell biology of herpes simplex virus (HSV) infection, and we aim to identify viral and cellular molecules that are crucial for virus production in the infected cell. Unlike the majority of human viruses, HSV establishes lifelong latent infection (in sensory neurons), and is reactivated periodically to produce new disease and infectivity. This reactivated HSV has a major impact on human health throughout the world. Apart from oral cold sores and genital herpes, reactivated HSV is also the leading cause of infectious blindness in the developed world, and the major viral cause of encephalitis that can often be fatal. Serious complications of the disease are particularly problematic in immunosuppressed patients such as transplant and chemotherapy patients. Additionally, HSV is a major cofactor for infection with HIV.Like all viruses, HSV exploits pre-existing cellular activities in its replication. We aim to determine how HSV hijacks cellular machinery to coordinate the assembly of its large, complex particles. We are particularly interested in how HSV utilises the cellular secretory pathway to direct the process of assembly; how the individual virus structural molecules interact as the new particle is built; where in the cell these interactions occur; and which cellular molecules are crucial to this process. We use a combination of live cell studies of cells infected with fluorescently tagged viruses, virus genetics, siRNA depletion, and biochemical assays to address these steps in virus morphogenesis. In this way we aim to pinpoint critical molecules - both viral and cellular - as potential targets for antiviral intervention.
Prof Geoffrey Smith, University of Cambridge.
Prof Judith Breuer, University College London.
Prof Iain McNeish, University of Glasgow.
BMS3079 - Human Microbial DiseasesMMIM022 - AntimicrobialsHCSM06 - Microbial Sciences, Immunology & Haematology
University roles and responsibilities
- Research Director, School of Biosciences and Medicine
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. Strikingly at later times, the population of cells originally resistant to HSV1 infection had also become infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a Delta UL34 virus defective in capsid export to the cytoplasm. Moreover, newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. Neutralizing human serum also failed to block virus transmission in nectin1 KO cells, which was dependent on the receptor binding protein glycoprotein D and the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission.
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. The depletion of 11 factors reduced virus yields by 20- to 100-fold, including three early secretory pathway proteins, four late secretory pathway proteins, and four endocytic pathway proteins, three of which are membrane fission factors. Five of the 11 targets were chosen for further analysis in virus infection, where it was found that the absence of only 1, the fission factor CHMP4C, but not the CHMP4A or CHMP4B paralogues, reduced virus production at the final stage of morphogenesis. Ultrastructural and confocal microscopy of CHMP4C-depleted, HSV1-infected cells showed an accumulation of endocytic membranes; extensive tubulation of recycling, transferrin receptor-positive endosomes indicative of aberrant fission; and a failure in virus envelopment. No effect on the late endocytic pathway was detected, while exogenous CHMP4C was shown to localize to recycling endosomes. Taken together, these data reveal a novel role for the CHMP4C fission factor in the integrity of the recycling endosomal network, which has been unveiled through the dependence of HSV1 on these membranes for the acquisition of their envelopes. IMPORTANCE Cellular transport pathways play a fundamental role in secretion and membrane biogenesis. Enveloped viruses exploit these pathways to direct their membrane proteins to sites of envelopment and, as such, are powerful tools for unraveling subtle activities of trafficking factors, potentially pinpointing therapeutic targets. Using the sensitive biological readout of virus production, over 80 trafficking factors involved in diverse and poorly defined cellular processes have been screened for involvement in the complex process of HSV1 envelopment. Out of 11 potential targets, CHMP4C, a key component in the cell cycle abscission checkpoint, stood out as being required for the process of virus wrapping in endocytic tubules, where it localized. In the absence of CHMP4C, recycling endocytic membranes failed to undergo scission in infected cells, causing transient tubulation and accumulation of membranes and unwrapped virus. These data reveal a new role for this important cellular factor in the biogenesis of recycling endocytic membranes.
Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). Vhs causes a number of changes to the infected cell environment that favour 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. When first expressed, vhs-GFP localised to juxtanuclear clusters, and later it colocalised and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Δvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. Moreover, a representative cellular transcript which is ordinarily highly susceptible to vhs degradation, was also retained in the nucleus. These results reveal that the vhs-induced nuclear retention of the infected cell transcriptome is dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs.
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. When first expressed, vhs-GFP localized to juxtanuclear clusters, and later it colocalized and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, which is in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. These results revealed that the vhs-induced nuclear retention of IE and E transcripts was dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. Like many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here, we describe a virus expressing vhs tagged at its C terminus with a green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the early virus transcriptome in the nucleus to favor late protein translation, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus, therefore, has uncoupled the nuclear retention and degradation activities of vhs, providing a new understanding of vhs during infection.
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 similar profiles to those previously found for HSV-1, with BoHV-1 capsids associating with endocytic tubules. Consistent with the similarity of their genomes and envelopment strategies, the proteomic composition of BoHV-1 and HSV-1 virions was also comparable. However, BoHV-1 morphogenesis exhibited a diversity in envelopment events. First, heterogeneous primary envelopment profiles were readily detectable at the inner nuclear membrane of BoHV-1 infected cells. Second, the BoHV-1 progeny comprised not just full virions, but also an abundance of capsidless, non-infectious light (L)-particles that were released from the infected cell in similar numbers to virions, and in the absence of DNA replication. Proteomic analysis of BoHV-1 L- particles and the much less abundant HSV-1 L-particles revealed that they contained the same complement of envelope proteins as virions but showed variations in tegument content. In the case of HSV-1, the UL46 tegument protein was reproducibly found to be more than 6-fold enriched in HSV-1 L-particles. More strikingly, the tegument proteins UL36, UL37, UL21 and UL16 were depleted in BoHV-1 but not HSV-1 L-particles. We propose that these combined differences reflect the presence of a truly segregated “inner” and “outer” tegument in BoHV-1, making it a critical system for studying the structure and process of tegumentation and envelopment.
Herpes simplex virus 1 deleted for VP22 is proposed to require secondary mutation of VHS for viability. Here we show that a replication-competent Δ22 virus constructed by homologous recombination maintains a Wt VHS gene and has no other gross mutations. By contrast, Δ22 viruses recovered from a bacterial artificial chromosome contain multiple codon changes within a conserved region of VHS. Hence, the mode of virus rescue influences the acquisition of secondary mutations.
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). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22's major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.
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. We have previously shown that the C-terminal half of VP22 contains the necessary signal for assembly into the virus. Here, we show that during infection VP22 interacts with gE and gM, as well as its tegument partner VP16. However, by using a range of techniques we were unable to demonstrate VP22 binding to gD. By using pulldown assays, we show that while the cytoplasmic tails of both gE and gM interact with VP22, only gE interacts efficiently with the C-terminal packaging domain of VP22. Furthermore, gE but not gM can recruit VP22 to the Golgi/trans-Golgi network region of the cell in the absence of other virus proteins. To examine the role of the gE-VP22 interaction in infection, we constructed a recombinant virus expressing a mutant VP22 protein with a 14-residue deletion that is unable to bind gE ( gEbind). Coimmunoprecipitation assays confirmed that this variant of VP22 was unable to complex with gE. Moreover, VP22 was no longer recruited to its characteristic cytoplasmic trafficking complexes but exhibited a diffuse localization. Importantly, packaging of this variant into virions was abrogated. The mutant virus exhibited poor growth in epithelial cells, similar to the defect we have observed for a VP22 knockout virus. These results suggest that deletion of just 14 residues from the VP22 protein is sufficient to inhibit binding to gE and hence recruitment to the viral envelope and assembly into the virus, resulting in a growth phenotype equivalent to that produced by deleting the entire reading frame.
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. Consistent with this, capsids did not colocalise with either the trans-Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to
Herpes simplex virus 1 (HSV1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV1 entry into keratinocytes has been the subject of limited investigation, but 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 HSV1 entry in to the host. Although immortalised, these cells nonetheless retain normal growth and differentiation properties of primary cells. Using siRNA depletion studies, we confirm that, despite nTERT cells expressing high levels of the alternative gD receptor HVEM, HSV1 requires nectin-1, not HVEM, to enter these cells. Strikingly, virus entry into nTERT cells occured with unusual rapidity, such that maximum penetration was achieved within 5 minutes. Moreover, HSV1 was able to enter keratinocytes but not other cell types at temperatures as low as 7°C, conditions where endocytosis was shown to be completely inhibited. Transmission electron microscopy of early entry events at both 37°C and 7°C identified numerous examples of naked virus capsids located immediately beneath the plasma membrane, with no evidence of virions in cytoplasmic vesicles. Taken together, these results imply that HSV1 uses the nectin-1 receptor to enter human keratinocyte cells via a previously uncharacterised rapid plasma membrane fusion pathway that functions at low temperature. These studies have important implications for current understanding of the relationship between HSV1 and its relevant in vivo target cell.
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. ICP27 is not, however, present in this VP22-ICP0 complex but is packaged into the virion in a VP22- and ICP0-independent manner. Biochemical fractionation of virions indicated that ICP0 associates tightly with the virus capsid, but intranuclear capsids contained no detectable ICP0. The RING finger domain of ICP0 and the N terminus of VP22 were both shown to be essential but not sufficient for ICP0 packaging and complex formation. Strikingly, however, the N-terminal region of VP22, while unable to form a complex with ICP0, inhibited its translocation from the nucleus to the cytoplasm. PML degradation by ICP0 was efficient in cells infected with this VP22 mutant virus, confirming that ICP0 retains activity. Hence, we would suggest that VP22 is an important molecular partner of ICP0 that controls at least one of its activities: its assembly into the virion. Moreover, we propose that the pathway by which VP22 recruits ICP0 to the virion may begin in the nucleus prior to ICP0 translocation to its final site of assembly in the cytoplasm. Copyright © 2010, American Society for Microbiology.
The Glasgow s17 syn+ strain of herpes simplex virus 1 (HSV1) is arguably the best characterised 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. Second generation sequencing of eight plaque-purified s17-derived viruses revealed sequences that vary by 50 SNPs including approximately 10 coding SNPs. This compared to interstrain variations of around 800 SNPs in strain Sc16, of which a quarter were coding changes. Amongst the variations found within s17, we identified thirteen variants of glycoprotein C within the original stock of virus which were predominantly a consequence of altered homopolymeric runs of C residues. Characterisation of seven isolates coding for different forms of gC indicated that all were expressed, despite six of them lacking a transmembrane domain. While the release phenotype did not correlate directly with any of these identified gC variations, further demonstration that nine clinical isolates of HSV1 also fail to spread through extracellular release raises the possibility that propagation in tissue culture had altered the HSV1 s17 transmission phenotype. Hence, this s17 intrastrain variation identified here offers an excellent model for understanding both HSV1 transmission and tissue culture adaptation.
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. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in Δ22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention later in Wt infection but late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also late transcripts were retained in the nucleus by a vhs-dependent mechanism, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient late protein production.
The HSV1 virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in shut-off of host protein synthesis. Hence its unrestrained activity is considered to be 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. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from co-transfected plasmids were also retained in the same nuclei where vhs mRNA was located, while polyA binding protein (PABP) was relocalised to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Co-expression of VP16 and VP22 rescued cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5’ region of vhs that blocked its translation and, when transferred to a heterologous GFP transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and auto-induced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.
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. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 Δ22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on Δ22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of both cellular and viral mRNA suggesting that vhs mutation in the absence of VP22 is necessary to overcome a more complex disturbance in mRNA metabolism than mRNA degradation alone. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production.
The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.