Peter O'Hare

Differing Roles of Inner Tegument Proteins pUL36 and pUL37 during Entry of Herpes Simplex Virus Type 1

ABSTRACT Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17+ did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.

Herpes Simplex Virus Infection Induces Phosphorylation and Delocalization of Emerin, a Key Inner Nuclear Membrane Protein

ABSTRACT The inner nuclear membrane (INM) contains specialized membrane proteins that selectively interact with nuclear components including the lamina, chromatin, and DNA. Alterations in the organization of and interactions with INM and lamina components are likely to play important roles in herpesvirus replication and, in particular, exit from the nucleus. Emerin, a member of the LEM domain class of INM proteins, binds a number of nuclear components including lamins, the DNA-bridging protein BAF, and F-actin and is thought to be involved in maintaining nuclear integrity. Here we report that emerin is quantitatively modified during herpes simplex virus (HSV) infection. Modification begins early in infection, involves multiple steps, and is reversed by phosphatase treatment. Emerin phosphorylation during infection involves one or more cellular kinases but can also be influenced by the US3 viral kinase, a protein whose function is known to be involved in HSV nuclear egress. The results from biochemical extraction analyses and from immunofluorescence of the detergent-resistant population demonstrate that emerin association with the INM significantly reduced during infection. We propose that the induction of emerin phosphorylation in infected cells may be involved in nuclear egress and uncoupling interactions with targets such as the lamina, chromatin, or cytoskeletal components.

A Nuclear Localization Signal in Herpesvirus Protein VP1-2 Is Essential for Infection via Capsid Routing to the Nuclear Pore

ABSTRACT To initiate infection, herpesviruses must navigate to and transport their genomes across the nuclear pore. VP1-2 is a large structural protein of the virion that is conserved in all herpesviruses and plays multiple essential roles in virus replication, including roles in early entry. VP1-2 contains an N-terminal basic motif which functions as an efficient nuclear localization signal (NLS). In this study, we constructed a mutant HSV strain, K.VP1-2ΔNLS, which contains a 7-residue deletion of the core NLS at position 475. This mutant fails to spread in normal cells but can be propagated in complementing cell lines. Electron microscopy (EM) analysis of infection in noncomplementing cells demonstrated capsid assembly, cytoplasmic envelopment, and the formation of extracellular enveloped virions. Furthermore, extracellular virions isolated from noncomplementing cells had similar profiles and abundances of structural proteins. Virions containing VP1-2ΔNLS were able to enter and be transported within cells. However, further progress of infection was prevented, with at least a 500- to 1,000-fold reduction in the efficiency of initiating gene expression compared to that in the revertant. Ultrastructural and immunofluorescence analyses revealed that the K.VP1-2ΔNLS mutant was blocked at the microtubule organizing center or immediately upstream of nuclear pore docking and prior to gene expression. These results indicate that the VP1-2 NLS is not required for the known assembly functions of the protein but is a key requirement for the early routing to the nuclear pore that is necessary for successful infection. Given its conservation, we propose that this motif may also be critical for entry of other classes of herpesviruses.

Evasion of Innate Cytosolic DNA Sensing by a Gammaherpesvirus Facilitates Establishment of Latent Infection

Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and β-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi’s sarcoma–associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections.

A Single Mutation Responsible for Temperature-Sensitive Entry and Assembly Defects in the VP1-2 Protein of Herpes Simplex Virus

ABSTRACT Evidence for an essential role of the herpes simplex virus type 1 (HSV-1) tegument protein VP1-2 originated from the analysis of the temperature-sensitive (ts) mutant tsB7. At the nonpermissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore, with defective genome release and substantially reduced virus gene expression. We compared the UL36 gene of tsB7 with that of the parental strain HFEM or strain 17 and identified four amino acid substitutions, 1061D → G, 1453Y → H, 2273Y → H, and 2558T → I. We transferred the UL36 gene from tsB7, HFEM, or strain 17 into a KOS background. While KOS recombinants containing the HFEM or strain 17 UL36 gene exhibited no ts defect, recombinants containing the tsB7 UL36 VP1-2 exhibited a 5-log deficiency at the NPT. Incubation at the NPT resulted in little or no virus gene expression, though limited expression could be detected in a highly delayed fashion. Using shift-down regimes, gene expression recovered and recapitulated the time course normally observed, indicating that the initial block was in a reversible pathway. Using temperature shift-up regimes, a second defect later in the replication cycle was also observed in the KOS.ts viruses. We constructed a further series of recombinants which contained subsets of the four substitutions. A virus containing the wild-type (wt) residue at position 1453 and with the other three residues being from tsB7 VP1-2 exhibited wt plaquing efficiency. Conversely, a virus containing the three wt residues but the single Y → H change at position 1453 from tsB7 exhibited a 4- to 5-log drop in plaquing efficiency and was defective at both early and late stages of infection.

Nuclear Pore Composition and Gating in Herpes Simplex Virus-Infected Cells

ABSTRACT The mechanism by which herpes simplex virus (HSV) exits the nucleus remains a matter of controversy. The generally accepted route proposes that capsids exit via primary envelopment at the inner nuclear membrane and subsequent fusion of this primary particle with the outer nuclear membrane to gain capsid entry to the cytoplasm. However, recent observations indicate that HSV may induce gross morphological alterations of nuclear pores, resulting in the loss of normal pores and the appearance of dilated gaps in the nuclear membrane of up to several 100 nm. On this basis, it was proposed that a main route of capsid exit from the nucleus is directly through these altered pores. Here, we examine the biochemical composition of some of the major nuclear pore components in uninfected and HSV-infected cells. We show that total levels of major nucleoporins and their sedimentation patterns in density gradients remain largely unchanged up to 18 h after HSV infection. Some alteration in modification of one nucleoporin, Nup358/RanBP2, was observed during enrichment with anti-nucleoporin antibody and probing for O glycosylation. In addition, we examine functional gating within the nucleus in live cells, using microinjection of labeled dextran beads and a recombinant virus expressing GFP-VP16 to track the progress of infection. The nuclear permeability barrier for molecules bigger than 70 kDa remained intact throughout infection. Thus, in a functional assay in live cells, we find no evidence for gross perturbation to the gating of nuclear pores, although this might not exclude a small population of modified pores.

Modelling dynamics of the type I interferon response to in vitro viral infection.

Innate immunity is crucial in the early stages of resistance to novel viral infection. The family of cytokines known as the interferons (IFNs) forms an essential component of this system: they are responsible for signalling that an infection is underway and for promoting an antiviral response in susceptible cells. We construct a spatial stochastic model, parameterized by experimental data and informed by analytic approximation, to capture the dynamics of virus–IFN interaction during in vitro infection of Madin–Darby bovine kidney cell monolayers by Herpes simplex virus 1. The dose dependence of infection progression, subsequent monolayer destruction and IFN-β production are investigated. Implications for in vivo infections, in particular the priming of susceptible cells by IFN-β during infection, are considered.

Comparison of the SUMO1 and ubiquitin conjugation pathways during the inhibition of proteasome activity with evidence of SUMO1 recycling.

To investigate potential interplay between the SUMO1 (small ubiquitin-related modifier-1) and ubiquitin pathways of post-translational protein modification, we examined aspects of their localization and conjugation status during proteasome inhibition. Our results indicate that these pathways converge upon the discrete sub-nuclear domains known as PML (promyelocytic leukaemia protein) NBs (nuclear bodies). Proteasome inhibition generated an increased number of PML bodies, without any obvious increase in size. Using a cell line that constitutively expresses an epitope-tagged version of SUMO1, which was incorporated into high-molecular-mass conjugates, we observed SUMO1 accumulating in clusters around a subset of the NBs. Nuclear ubiquitin was initially observed in numerous speckles and foci, which bore no relationship to PML NBs in the absence of proteasome inhibition. However, during proteasome inhibition, total ubiquitin-conjugated species increased in the cell, as judged by Western blotting. Concomitantly the number of nuclear ubiquitin clusters decreased, and were almost quantitatively associated with the PML NBs, co-localizing with the SUMO-conjugated pool. Proteasome inhibition depleted the pool of free SUMO1 in the cell. Reversal of proteasome inhibition in the presence or absence of protein synthesis demonstrated that free SUMO1 was regenerated from the conjugated pool. The results indicate that a significant fraction of the free SUMO1 pool could be accounted for by recycling from the conjugated pool and indeed it may be that, as for ubiquitin, SUMO1 needs to be removed from conjugated species prior to processing by the proteasome. Taken together with other recent reports on the proteasome and PML NBs, these results suggest that the PML NBs may play an important role in integrating these pathways.

Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2

ABSTRACT The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances.

Characterization of VP22 in Herpes Simplex Virus-Infected Cells

ABSTRACT We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.