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 Diseases
MMIM022 - Antimicrobials
HCSM06 - Microbial Sciences, Immunology & Haematology
Infectious Diseases Group Leader
Find me on campus Room: 04 AW 01
Contact Janet Sheldrick for an appointment.
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.
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 glycoproteinM(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. © 2012, American Society for Microbiology.
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 (ΔgEbind). 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. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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