117 results found
Bodda C, Reinert LS, Fruhwürth S, et al., 2020, HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection, Journal of Experimental Medicine, Vol: 217, ISSN: 0022-1007
Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.
Serwa RA, Sekine E, Brown J, et al., 2019, Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry, PLoS Pathogens, Vol: 15, ISSN: 1553-7366
We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together w
Ahmad L, Mashbat B, Leung C, et al., 2019, Human TANK-binding kinase 1 is required for early autophagy induction upon herpes simplex virus 1 infection, Journal of Allergy and Clinical Immunology, Vol: 143, Pages: 765-769.e7, ISSN: 0091-6749
Teo CSH, O'Hare P, 2018, A bimodal switch in global protein translation coupled to eIF4H relocalisation during advancing cell-cell transmission of herpes simplex virus, PLoS Pathogens, Vol: 14, ISSN: 1553-7366
We used the bioorthogonal protein precursor, homopropargylglycine (HPG) and chemical ligation to fluorescent capture agents, to define spatiotemporal regulation of global translation during herpes simplex virus (HSV) cell-to-cell spread at single cell resolution. Translational activity was spatially stratified during advancing infection, with distal uninfected cells showing normal levels of translation, surrounding zones at the earliest stages of infection with profound global shutoff. These cells further surround previously infected cells with restored translation close to levels in uninfected cells, reflecting a very early biphasic switch in translational control. While this process was dependent on the virion host shutoff (vhs) function, in certain cell types we also observed temporally altered efficiency of shutoff whereby during early transmission, naïve cells initially exhibited resistance to shutoff but as infection advanced, naïve target cells succumbed to more extensive translational suppression. This may reflect spatiotemporal variation in the balance of oscillating suppression-recovery phases. Our results also strongly indicate that a single particle of HSV-2, can promote pronounced global shutoff. We also demonstrate that the vhs interacting factor, eIF4H, an RNA helicase accessory factor, switches from cytoplasmic to nuclear localisation precisely correlating with the initial shutdown of translation. However translational recovery occurs despite sustained eIF4H nuclear accumulation, indicating a qualitative change in the translational apparatus before and after suppression. Modelling simulations of high multiplicity infection reveal limitations in assessing translational activity due to sampling frequency in population studies and how analysis at the single cell level overcomes such limitations. The work reveals new insight and a revised model of translational manipulation during advancing infection which has important implications both mechan
Sekine E, Schmidt N, Gaboriau D, et al., 2017, Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy., PLoS Pathogens, Vol: 13, Pages: 1-36, ISSN: 1553-7366
We investigated the spatiotemporal dynamics of HSV genome transport during the initiation of infection using viruses containing bioorthogonal traceable precursors incorporated into their genomes (HSVEdC). In vitro assays revealed a structural alteration in the capsid induced upon HSVEdC binding to solid supports that allowed coupling to external capture agents and demonstrated that the vast majority of individual virions contained bioorthogonally-tagged genomes. Using HSVEdC in vivo we reveal novel aspects of the kinetics, localisation, mechanistic entry requirements and morphological transitions of infecting genomes. Uncoating and nuclear import was observed within 30 min, with genomes in a defined compaction state (ca. 3-fold volume increase from capsids). Free cytosolic uncoated genomes were infrequent (7-10% of the total uncoated genomes), likely a consequence of subpopulations of cells receiving high particle numbers. Uncoated nuclear genomes underwent temporal transitions in condensation state and while ICP4 efficiently associated with condensed foci of initial infecting genomes, this relationship switched away from residual longer lived condensed foci to increasingly decondensed genomes as infection progressed. Inhibition of transcription had no effect on nuclear entry but in the absence of transcription, genomes persisted as tightly condensed foci. Ongoing transcription, in the absence of protein synthesis, revealed a distinct spatial clustering of genomes, which we have termed genome congregation, not seen with non-transcribing genomes. Genomes expanded to more decondensed forms in the absence of DNA replication indicating additional transitional steps. During full progression of infection, genomes decondensed further, with a diffuse low intensity signal dissipated within replication compartments, but frequently with tight foci remaining peripherally, representing unreplicated genomes or condensed parental strands of replicated DNA. Uncoating and nuclear en
Barbosa S, Carreira S, O'Hare P, 2017, GSK-3-mediated phosphorylation couples ER-Golgi transport and nuclear stabilization of the CREB-H transcription factor to mediate apolipoprotein secretion, MOLECULAR BIOLOGY OF THE CELL, Vol: 28, Pages: 1565-1579, ISSN: 1059-1524
CREB-H, an ER-anchored transcription factor, plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes, including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases, releasing the N-terminal product, which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif termed the P-motif, located in the N-terminus between serines 73 and 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation, promoting ER retention, while mutation of target serines and drug inhibition of GSK-3 activity coordinately induce both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations, which regulate stability by targeting the protein to the SCFFbw1a E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilization of the active form in the nucleus.
Su Hui Teo C, Serwa RA, O'Hare P, 2016, Spatial and temporal resolution of global protein synthesis during HSV infection using bioorthogonal precursors and click chemistry, PLOS Pathogens, Vol: 12, Pages: 1-37, ISSN: 1553-7366
We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal ne
Schmidt N, Hennig T, Serwa RA, et al., 2015, Remote activation of host cell DNA synthesis in uninfected cells signalled by infected cells in advance of virus transmission., Journal of Virology, Vol: 89, Pages: 11107-1115, ISSN: 1098-5514
Viruses modulate cellular processes and metabolism in diverse ways but these are almost universally studied in the infected cell itself. Here we study spatial organisation of DNA synthesis during multi-round transmission of herpes simplex virus (HSV) using pulse-labelling with ethynyl-nucleotides and cycloaddition of azide-fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with current understanding during single step growth cycle, HSV suppresses host DNA synthesis and promotes virus DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover using pulse chase analysis we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis was then shut off and replaced with virus DNA synthesis. Using non-propagating viruses or conditioned medium we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as for mechanisms governing host DNA synthesis. IMPORTANCE: We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus induced paracrine effector. The field ha
Barbosa S, Carreira S, Bailey D, et al., 2015, Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets., Molecular Biology of the Cell, Vol: 26, Pages: 2939-2954, ISSN: 1939-4586
CREB‑H, an endoplasmic reticulum-anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87-S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCF(Fbw1a) E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV.
Serwa R, Krause E, Abaitua F, et al., 2015, Systems analysis of protein fatty acylation in herpes simplex virus infected cells using chemical proteomics., Chemistry & Biology, Vol: 22, Pages: 1008-1017, ISSN: 1074-5521
Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.
Hennig T, O'Hare P, 2015, Viruses and the nuclear envelope, CURRENT OPINION IN CELL BIOLOGY, Vol: 34, Pages: 113-121, ISSN: 0955-0674
Sun C, Schattgen SA, Pisitkun P, et al., 2015, Evasion of Innate Cytosolic DNA Sensing by a Gammaherpesvirus Facilitates Establishment of Latent Infection, JOURNAL OF IMMUNOLOGY, Vol: 194, Pages: 1819-1831, ISSN: 0022-1767
Zaborowska J, Baumli S, Laitem C, et al., 2014, Herpes Simplex Virus 1 (HSV-1) ICP22 Protein Directly Interacts with Cyclin-Dependent Kinase (CDK) 9 to Inhibit RNA Polymerase II Transcription Elongation, PLOS ONE, Vol: 9, ISSN: 1932-6203
Hennig T, Abaitua F, O'Hare P, 2014, Functional Analysis of Nuclear Localization Signals in VP1-2 Homologues from All Herpesvirus Subfamilies, JOURNAL OF VIROLOGY, Vol: 88, Pages: 5391-5405, ISSN: 0022-538X
Barbosa S, Fasanella G, Carreira S, et al., 2013, An Orchestrated Program Regulating Secretory Pathway Genes and Cargos by the Transmembrane Transcription Factor CREB-H, TRAFFIC, Vol: 14, Pages: 382-398, ISSN: 1398-9219
Abaitua F, Zia R, Hollinshead M, et al., 2013, Polarised cell migration during cell-to-cell transmission of herpes simplex virus in human skin keratinocytes., Journal of Virology
In addition to transmission involving extracellular free particles, a generally accepted model of virus propagation is one wherein virus replicates in one cell producing infectious particles that transmit to the next cell via cell junctions or induced polarised contacts. This mechanism of spread is especially important in the presence of neutralising antibody and the concept underpins analysis of virus spread, plaque size, viral and host functions and general mechanisms of virus propagation. Here we demonstrate a novel process involved in cell-to-cell transmission of herpes simplex virus (HSV) in human skin cells that has not previously been appreciated. Using time lapse microscopy of fluorescent viruses we show that HSV infection induces the polarised migration of skin cells into the site of infection. In the presence of neutralising antibody, uninfected skin cells migrate to the initial site of infection and spread over infected cells, to become infected in a spatially confined cluster containing hundreds of cells. The cells in this cluster do not undergo cytocidal cell lysis but harbour abundant enveloped particles within cells and cell-free virus within interstitial regions below the cluster surface. Cells at the base and outside the cluster were generally negative for virus immediate-early expression. We further show using spatially separated monolayer assays, that at least one component of this induced migration is the paracrine stimulation of a cytotactic response from infected cells to uninfected cells. The existence of this process changes our concept of virus transmission and the potential functions, virus and host factors involved.
Abaitua F, Hollinshead M, Bolstad M, et al., 2012, A Nuclear Localization Signal in Herpesvirus Protein VP1-2 Is Essential for Infection via Capsid Routing to the Nuclear Pore, JOURNAL OF VIROLOGY, Vol: 86, Pages: 8998-9014, ISSN: 0022-538X
Abaitua F, Daikoku T, Crump CM, et al., 2011, A single mutation responsible for temperature sensitive entry and assembly defects in the VP1-2 protein of HSV, J Virol, Vol: 85, Pages: 2024-2036
Evidence for an essential role of the HSV-1 tegument protein VP1-2 originated from the analysis of the temperature-sensitive mutant tsB7. At the non-permissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore with defective genome release and substantially reduced virus gene expression. We compare the UL36 gene of tsB7 with the parental strain HFEM or strain 17 and identify 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 genes 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 4 substitutions. A virus containing the w/t residue at position 1453 with the other three residues being from tsB7 VP1-2, exhibited w/t plaquing efficiency. Conversely a virus containing the three w/t residues but the single residue Y>H at position 1453 from tsB7, exhibited a 4-5 log drop in plaquing efficiency and was defective in at both early and late stages of infection.
Bolstad M, Abaitua F, Crump CM, et al., 2011, Autocatalytic activity of the Ubiquitin Specific Protease domain of HSV-1 VP1-2, J Virol, Vol: 85, Pages: 8738-8751
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 stabilised 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 stabilised 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.
Llarena M, Bailey D, Curtis H, et al., 2010, Different Mechanisms of Recognition and ER Retention by Transmembrane Transcription Factors CREB-H and ATF6, TRAFFIC, Vol: 11, Pages: 48-69, ISSN: 1398-9219
Llarena M, Bailey D, Curtis H, et al., 2010, Different mechanisms of recognition and ER retention by transmembrane transcription factors CREB-H and ATF6, Traffic, Vol: 11, Pages: 48-69
CREB-H and activating transcription factor 6 (ATF6) are transmembrane transcription factors that, in response to endoplasmic reticulum (ER) stress, traffic to the Golgi where they are cleaved by specific proteases, producing the N-terminal domains that effect appropriate transcriptional responses. We show that unlike in ATF6 whose lumenal tail binds BiP and contains determinants for stress sensing and Golgi transport, in CREB-H the lumenal tail is not involved in ER retention, not required for Golgi transport and does not bind BiP. The main determinant for CREB-H ER retention resides in a membrane-proximal cytoplasmic determinant that is conserved in related members of the CREB-H family, but lacking in ATF6. We refine requirements within the ER-retention motif (ERM) and show that ERM-ve variants exhibited constitutive Golgi localization and constitutive cleavage by the Golgi protease, S1P. The ERM also conferred ER retention on a heterologous protein. Furthermore, deletion of the lumenal tail of CREB-H had no effect on ER retention of parental CREB-H or Golgi localization of ERM-ve variants. Importantly, when the lumenal tail of ATF6 was transferred into an ERM-ve variant, the chimera was now retained in the ER. Together, these data demonstrate novel and qualitatively distinct mechanisms of trafficking and stress signalling in CREB-H compared to ATF6.
Roberts AP, Abaitua F, O'Hare P, et al., 2009, Differing Roles of Inner Tegument Proteins pUL36 and pUL37 During Entry of Herpes Simplex Virus Type 1 (HSV-1), J. Virol., Vol: 83, Pages: 105-116
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 genes 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 role of these genes in initial stages of infection could not be investigated. To circumvent this, cells infected at 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. Visualisation 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 HSV-1 WT, 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, whilst pUL37 is needed for formation of virions but is not required during the initial stages of infection. Analysis of non-infectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.
Abaitua F, Souto RN, Browne H, et al., 2009, Characterization of the herpes simplex virus (HSV)-1 tegument protein VP1-2 during infection with the HSV temperature-sensitive mutant tsB7, J. Gen. Virol., Vol: 90, Pages: 2353-2363
VP1-2, encoded by the UL36 gene of herpes simplex virus (HSV), is a large structural protein, conserved across the family Herpesviridae, that is assembled into the tegument and is essential for virus replication. Current evidence indicates that VP1-2 is a central component in the tegumentation and envelopment processes and that it also possesses important roles in capsid transport and entry. However, any detailed mechanistic understanding of VP1-2 function(s) remains limited. This study characterized the replication of HSV-1 tsB7, a temperature-sensitive mutant restricted at the non-permissive temperature due to a defect in VP1-2 function. A tsB7 virus expressing green fluorescent protein-fused VP16 protein was used to track the accumulation and location of a major tegument protein. After infection at the permissive temperature and shift to the non-permissive temperature, the production of infectious virus ceased. VP1-2 accumulated in altered cytosolic clusters, together with VP16 and other virion proteins. Furthermore, correlating with the results of immunofluorescence, electron microscopy demonstrated abnormal cytosolic capsid clustering and a block in envelopment. As VP1-2 encompasses a ubiquitin-specific protease domain, the occurrence of ubiquitin-conjugated proteins during tsB7 infection was also examined at the non-permissive temperature. A striking overaccumulation was observed of ubiquitin-specific conjugates in cytoplasmic clusters, overlapping and adjacent to the VP1-2 clusters. These results are discussed in relation to the possible functions of VP1-2 in the assembly pathway and the nature of the defect in tsB7.
Hofemeister H, O'Hare P, 2008, Nuclear pore composition and gating in herpes simplex virus-infected cells, J Virol, Vol: 82, Pages: 8392-8399
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.
Abaitua F, O'Hare P, 2008, Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein, J. Virol., Vol: 82, Pages: 5234-5244
VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.
Bailey D, O'Hare P, 2007, Transmembrane bZIP transcription factors in ER stress signaling and the unfolded protein response, Antioxid. Redox Signal., Vol: 9, Pages: 2305-2321
Regulated intramembrane proteolysis (RIP) of the transmembrane transcription factor ATF6 represents a key step in effecting adaptive response to the presence of unfolded or malfolded protein in the endoplasmic reticulum. Recent studies have highlighted new ATF6-related transmembrane transcription factors. It is likely that current models for ER stress signaling are incomplete and that the expansion of the bZIP transmembrane family reflects selectivity in many aspects of these responses, including the type and duration of any particular stress, the cell type in which it occurs, and the integration with other aspects of cell-type-specific organization or additional intrinsic pathways, and the integration and communication between these pathways, not only in a cell-type-specific manner, but also between different tissues and organs. This review summarizes current information on the bZIP-transmembrane proteins and discusses outstanding questions on the elucidation of the stress signals, the repertoire of components involved in regulating different aspects of the forward transport, cleavage, nuclear import, transcriptional activity, and turnover of each of these factors, and dissection of the integration of the various outputs into broad coordinated responses.
Morris JB, Hofemeister H, O'Hare P, 2007, Herpes simplex virus infection induces phosphorylation and delocalization of emerin, a key inner nuclear membrane protein, J. Virol., Vol: 81, Pages: 4429-4437
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.
Bailey D, Barreca C, O'Hare P, 2007, Trafficking of the bZIP transmembrane transcription factor CREB-H into alternate pathways of ERAD and stress-regulated intramembrane proteolysis, Traffic, Vol: 8, Pages: 1796-1814
CREB-H is an ATF6-related, transmembrane transcription factor that, in response to endoplasmic reticulum (ER)-associated stress, is cleaved by Golgi proteases and transported to the nucleus to effect appropriate adaptive responses. We characterize the ER processing and turnover of CREB-H with results which have important implications for ER stress regulation and signalling. We show that CREB-H is glycosylated and demonstrate that both the ER and nuclear forms of CREB-H have short half-lives. We also show that CREB-H is subject to cycles of retrotranslocation, deglycosylation and degradation through the ER-associated degradation (ERAD) pathway. Proteasome inhibition resulted in accumulation of a cytosolic intermediate but additionally, in contrast to inhibition of glycosylation, promoted specific cleavage of CREB-H and nuclear transport of the N-terminal-truncated product. Our data indicate that under normal conditions CREB-H is transported back from the ER to the cytosol, where it is subject to ERAD, but under conditions that repress proteasome function or promote load CREB-H is diverted from this pathway instead undergoing cleavage and nuclear transport. Finally, we identify a cytoplasmic determinant involved in CREB-H ER retention, deletion of which results in constitutive Golgi transport and corresponding cleavage. We present a model where cellular stresses may be sensed at different levels by different members of the basic and leucine zipper domain transmembrane proteins.
Barreca C, O'Hare P, 2006, Characterization of a potent refractory state and persistence of herpes simplex virus 1 in cell culture, J Virol, Vol: 80, Pages: 9171-9180
Herpes simplex virus (HSV) normally undergoes productive cytocidal infection in culture and is thought of as relatively resistant to innate immune responses such as interferon. We previously described an unusual pattern of infection in culture in MDBK cells, which after initial productive infection, surprisingly resulted in progressive suppression of replication and cell recovery. The dominance of the refractory state was due to the inability to suppress interferon production and subsequent paracrine signaling. Here, using a wild-type HSV-1 strain expressing green fluorescent protein (GFP)-VP16, we analyze aspects of long-term HSV persistence resulting from this oscillating refractory state. We show that the gradual suppression of GFP-VP16 expression correlated with a biphasic pattern of accumulation of viral DNA and extracellular virus titers. We quantify virus maintenance in a minor subpopulation of cells during subculture, show the reemergence of virus by infectious center assay, and demonstrate that this required intracellular events over a 24- to 48-h time course. We also demonstrate that conditioned medium (cMed) from infected cells induced a profound shutoff of HSV gene expression at the transcriptional level. Finally, we demonstrate that this suppression was extremely rapid, requiring only 1 h of treatment to essentially abolish HSV immediate-early expression, and surprisingly persisted for almost 2 days after removal of the cMed. These combined effects underpin the oscillating effect both in plaque progression, where infection spreads but is overwhelmed by the accumulation of inhibitory components, enabling cell recovery, and virus maintenance in a subpopulation of cells. These results may be relevant to consider in studies of HSV latency in different animal models.
Howat TJ, Barreca C, O'Hare P, et al., 2006, Modelling dynamics of the type I interferon response to in vitro viral infection, J R Soc Interface, Vol: 3, Pages: 699-709
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-beta production are investigated. Implications for in vivo infections, in particular the priming of susceptible cells by IFN-beta during infection, are considered.
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