Herpes Simplex Virus Protein Research Articles

Aggregation Propensities of Herpes Simplex Virus-1 Proteins and Derived Peptides: An In Silico and In Vitro Analysis.

Recurrent infections of neurotropic herpes simplex virus-1 (HSV-1) have been implicated in etiology and pathology of Alzheimer’s disease (AD). Although protein and peptide aggregation events are at the center of the AD pathophysiology, except a single study where a peptide derived from glycoprotein B of HSV-1 was reported to form β-amyloid-like aggregates, similar investigations with the entire proteome of HSV-1 have not been attempted. In the current study, 70 HSV-1 proteins were screened using bioinformatics tools to identify aggregation-prone candidates. Thereafter, the 20S proteasome cleavage sites within the sequence of the selected proteins were determined using Pcleavage and NetChop algorithms, thereby mimicking a cellular proteasomal activity providing short peptides. Here, we report the biochemical characterization of a 28-residue-long peptide (HSV-1 gK208–235) derived from glycoprotein K of HSV-1. The peptide showed high aggregation propensity and homology to the C-terminus of Aβ1–42 peptide. The aggregates of gK208–235 peptide were characterized by the Congo red and Thioflavin T assays and Fourier transform infrared (FTIR) spectroscopy, and their spheroid oligomeric structure was established by atomic force microscopy (AFM). Furthermore, the aggregates demonstrated dose-dependent cytotoxicity to primary mouse splenocytes. The current findings hypothesize a mechanism by which HSV-1 may contribute to AD, which may be pursued further in the future.

BX795 demonstrates potent antiviral benefits against herpes simplex Virus-1 infection of human cell lines

Herpes simplex virus-1 (HSV-1) infection is known to cause skin blisters, keratitis as well as deadly cases of encephalitis in some situations. Only a few therapeutic modalities are available for this globally prevalent infection. Very recently, a small molecule BX795 was identified as an inhibitor of HSV-1 protein synthesis in an ocular model of infection. In order to demonstrate its broader antiviral benefits, this study was aimed at evaluating the antiviral efficacy, mode-of-action, and toxicity of BX795 against HSV-1 infection of three human cell lines: HeLa, HEK, and HCE. Several different assays, including cell survival analysis, imaging, plaque analysis, Immunoblotting, and qRT-PCR, were performed. In all cases, BX795 demonstrated low toxicity at therapeutic concentration and showed strong antiviral benefits. Quite interestingly, cell line-dependent differences in the mechanism of antiviral action and cytokine response to infection were seen upon BX795 treatment. Taken together, our results suggest that BX795 may exert its antiviral benefits via cell-line specific mechanisms.

Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27.

Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity.IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

The ICP0 Protein of Herpes Simplex Virus 1 (HSV-1) Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection.

Herpes simplex virus 1 (HSV-1) infects mucosal epithelial cells and establishes lifelong infections in sensory neurons. Following reactivation, the virus is transferred anterograde to the initial site of infection or to sites innervated by infected neurons, causing vesicular lesions. Upon immunosuppression, frequent HSV-1 reactivation can cause severe diseases, such as blindness and encephalitis. Autophagy is a process whereby cell components are recycled, but it also serves as a defense mechanism against pathogens. HSV-1 is known to combat autophagy through the functions of the γ134.5 protein, which prevents formation of the autophagophore by binding to Beclin 1, a key factor involved in the elongation of the isolation membrane, and by redirecting the protein phosphatase 1α (PP1α) to dephosphorylate the translation initiation factor 2α (eIF2α) to prevent host translational shutoff. Other viral proteins that counteract innate immunity negatively impact autophagy. Here, we present a novel strategy of HSV-1 to evade the host through the downregulation of the autophagy adaptor protein sequestosome (p62/SQSTM1) and of the mitophagy adaptor optineurin (OPTN). This down-modulation occurs during the early steps of the infection. We also found that infected cell protein 0 (ICP0) of the virus mediates the down-modulation of the two autophagy adaptors in a mechanism independent of its E3 ubiquitin ligase activity. Cells depleted of either p62 or OPTN were able to mount greater antiviral responses, whereas cells expressing exogenous p62 displayed decreased virus yields. We conclude that downregulation of p62/SQSTM1 and OPTN is a viral strategy to counteract the host.IMPORTANCE Autophagy is a homeostatic mechanism of cells to recycle components, as well as a defense mechanism to get rid of pathogens. Strategies that HSV-1 has developed to counteract autophagy have been described and involve inhibition of autophagosome formation or indirect mechanisms. Here, we present a novel mechanism that involves downregulation of two major autophagy adaptor proteins, sequestosome 1 (p62/SQSTM1) and optineurin (OPTN). These findings generate the question of why the virus targets two major autophagy adaptors if it has mechanisms to block autophagosome formation. P62/SQSTM1 and OPTN proteins have pleiotropic functions, including regulation of innate immunity, inflammation, protein sorting, and chromatin remodeling. The decrease in virus yields in the presence of exogenous p62/SQSTM1 suggests that these adaptors have an antiviral function. Thus, HSV-1 may have developed multiple strategies to incapacitate autophagy to ensure replication. Alternatively, the virus may target another antiviral function of these proteins.

Total-Body PET and Highly Stable Chelators Together Enable Meaningful 89Zr-Antibody PET Studies up to 30 Days After Injection.

The use of 89Zr-antibody PET imaging to measure antibody biodistribution and tissue pharmacokinetics is well established, but current PET systems lack the sensitivity needed to study 89Zr-labeled antibodies beyond 2-3 isotope half-lives (7-10 d), after which a poor signal-to-noise ratio is problematic. However, studies across many weeks are desirable to better match antibody circulation half-life in human and nonhuman primates. These studies investigated the technical feasibility of using the primate mini-EXPLORER PET scanner, making use of its high sensitivity and 45-cm axial field of view, for total-body imaging of 89Zr-labeled antibodies in rhesus monkeys up to 30 d after injection. Methods: A humanized monoclonal IgG antibody against the herpes simplex viral protein glycoprotein D (gD) was radiolabeled with 89Zr via 1 of 4 chelator-linker combinations (benzyl isothiocyanate-DFO [DFO-Bz-NCS], where DFO is desferrioxamine B; DFO-squaramide; DFO*-Bz-NCS, where DFO* is desferrioxamine*; and DFO*-squaramide). The pharmacokinetics associated with these 4 chelator-linker combinations were compared in 12 healthy young male rhesus monkeys (∼1-2 y old, ∼3 ± 1 kg). Each animal was initially injected intravenously with unlabeled antibody in a peripheral vessel in the right arm (10 mg/kg, providing therapeutic-level antibody concentrations), immediately followed by approximately 40 MBq of one of the 89Zr-labeled antibodies injected intravenously in a peripheral vessel in the left arm. All animals were imaged 6 times over a period of 30 d, with an initial 60-min dynamic scan on day 0 (day of injection) followed by static scans of 30-45 min on approximately days 3, 7, 14, 21, and 30, with all acquired using a single bed position and images reconstructed using time-of-flight list-mode ordered-subsets expectation maximization. Activity concentrations in various organs were extracted from the PET images using manually defined regions of interest. Results: Excellent image quality was obtained, capturing the initial distribution phase in the whole-body scan; later time points showed residual 89Zr mainly in the liver. Even at 30 d after injection, representing approximately 9 half-lives of 89Zr and with a total residual activity of only 20-40 kBq in the animal, the image quality was sufficient to readily identify activity in the liver, kidneys, and upper and lower limb joints. Significant differences were noted in late time point liver uptake, bone uptake, and whole-body clearance between chelator-linker types, whereas little variation (±10%) was observed within each type. Conclusion: These studies demonstrate the ability to image 89Zr-radiolabeled antibodies up to 30 d after injection while maintaining satisfactory image quality, as provided by the primate mini-EXPLORER with high sensitivity and long axial field of view. Quantification demonstrated potentially important differences in the behavior of the 4 chelators. This finding supports further investigation.

Disassembly of the TRIM23-TBK1 Complex by the Us11 Protein of Herpes Simplex Virus 1 Impairs Autophagy.

The Us11 protein encoded by herpes simplex virus 1 (HSV-1) functions to impair autophagy; however, the molecular mechanisms of this inhibition remain to be fully established. Here, we report that the Us11 protein targets tripartite motif protein 23 (TRIM23), which is a key regulator of autophagy-mediated antiviral defense mediated by TANK-binding kinase 1 (TBK1). In virus-infected cells, the Us11 protein drastically reduces the formation of autophagosomes mediated by TRIM23 or TBK1. This autophagy-inhibitory effect is attributable to the binding of the Us11 protein to the ARF domain in TRIM23. Furthermore, such interaction spatially excludes TBK1 from the TRIM23 complex that also contains heat shock protein 90 (Hsp90). When stably expressed alone in host cells, the Us11 protein recapitulates the observed phenotypes seen in cells infected with the US11-expressing or wild-type virus. Consistent with this, expression of the Us11 protein promotes HSV-1 growth, while expression of TRIM23 restricts HSV-1 replication in the absence of US11. Together, these results suggest that disruption of the TRIM23-TBK1 complex by the Us11 protein inhibits autophagy-mediated restriction of HSV-1 infection.IMPORTANCE Autophagy is an evolutionarily conserved process that restricts certain intracellular pathogens, including HSV-1. Although HSV-1 is well known to inhibit autophagy, little is known about the precise molecular mechanisms of autophagy inhibition. We demonstrate that the Us11 protein of HSV-1 spatially disrupts the TRIM23-TBK1 complex, which subsequently suppresses autophagy and autophagy-mediated virus restriction. Thus, expression of the Us11 protein facilitates HSV-1 replication. These data unveil new insight into viral escape from autophagy-mediated host restriction mechanisms.

Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

Expression, Purification, and Antiserum Production of the Truncated UL31 Protein of Herpes Simplex Virus 1.

BackgroundThe UL31 protein of herpes simplex virus 1 (HSV-1) plays an important role in the HSV-1 replication, however, its pinpoint functions in the life cycle of the virus have yet to be adequately elucidated.ObjectivesAn antiserum specific for detecting HSV-1 UL31 was prepared as the foundation for future research on the role of UL31 in the course of HSV-1 infection.Materials and MethodsRecombinant protein of UL31 was expressed in Escherichia coli, which was then purified and employed to raise the level of antiserum in mice. Subsequently, western blot and immunofluorescence assay (IFA) were utilized to detect the specific antiserum.ResultsThe recombinant UL31 protein consisting of N-terminal 27 aa of UL31 was fused to EYFP and His-tag. It was expressed, purified, and applied to the preparation of the antiserum. Western blot analysis and IFA demonstrated that this antiserum could detect both the recombinant UL31 and the native UL31.ConclusionsOur results manifest that this antiserum could be conducive to further investigations concerning the roles of UL31 in the HSV-1 infection.

Structural Basis for the Interaction between p53 Transactivation Domain and the Mediator Subunit MED25.

Eukaryotic transcription initiation is mediated by interactions between transcriptional activators and the mediator coactivator complex. Molecular interaction of p53 transcription factor with mediator complex subunit 25 (MED25) is essential for its target gene transcription. In this study, we characterized the molecular interaction between p53 transactivation domain (p53TAD) and activator interaction domain (ACID) of MED25 using nuclear magnetic resonance (NMR) spectroscopy. The NMR chemical shift perturbation and isothermal titration calorimetry (ITC) data showed that p53TAD interacted with MED25 ACID mainly through the p53TAD2 sequence motif. Taken together with the mutagenesis data, the refined structural model of MED25 ACID/p53TAD2 peptide complex showed that an amphipathic α-helix of p53TAD2 peptide bound an elongated hydrophobic groove of MED25 ACID. Furthermore, our results revealed the highly conserved mechanism of MED25 interaction with intrinsically unfolded acidic TADs from the transcriptional activators p53, ERM (Ets-related molecule), and herpes simplex virus protein 16 (VP16).

A Mutation in the UL24 Gene Abolishes Expression of the Newly Identified UL24.5 Protein of Herpes Simplex Virus 1 and Leads to an Increase in Pathogenicity in Mice.

Herpes simplex virus 1 (HSV-1) infects the host via epithelia and establishes latency in sensory neurons. The UL24 gene is conserved throughout the Herpesviridae family, and the UL24 protein is important for efficient viral replication and pathogenesis. Multiple transcripts are expressed from the UL24 gene. The presence of a transcription initiation site inside the open reading frame of UL24 and an ATG start codon in the same open reading frame led us to suspect that another protein was expressed from the UL24 locus. To test our hypothesis, we constructed a recombinant virus that expresses a hemagglutinin tag at the C terminus of UL24. Western blot analysis revealed the expression of an 18-kDa protein that is not a degradation product of the full-length UL24, which we refer to as UL24.5. Ectopically expressed UL24.5 did not induce the dispersal of nucleolar proteins, as seen for UL24. In order to characterize the role of UL24.5, we constructed a mutant virus encoding a substitution of the predicted initiation methionine to a valine. This substitution eliminated the expression of the 18-kDa polypeptide. Unlike the UL24-null mutant (UL24X), which exhibits reduced viral yields, the UL24.5-null mutant exhibited the same replication phenotype in cell culture as the parental strain. However, in a murine ocular infection model, we observed an increase in the incidence of neurological disorders with the UL24.5 mutant. Alignment of amino acid sequences for various herpesviruses revealed that the initiation site of UL24.5 is conserved among HSV-1 strains and is present in many herpesviruses.IMPORTANCE We discovered a new HSV-1 protein, UL24.5, which corresponds to the C-terminal portion of UL24. In contrast to the replication defects observed with HSV-1 strains that do not express full-length UL24, the absence of UL24.5 did not affect viral replication in cell culture. Moreover, in mice, the absence of UL24.5 did not affect viral titers in epithelia or trigeminal ganglia during acute infection; however, it was associated with a prolonged persistence of signs of inflammation. Strikingly, the absence of UL24.5 also led to an increase in the incidence of severe neurological impairment compared to results for wild-type control viruses. This increase in pathogenicity is in stark contrast to the reduction in clinical signs associated with the absence of full-length UL24. Bioinformatic analyses suggest that UL24.5 is conserved among all human alphaherpesviruses and in some nonhuman alphaherpesviruses. Thus, we have identified UL24.5 as a new HSV-1 determinant of pathogenesis.

Construction of enhanced transcriptional activators for improving cellulase production in Trichoderma reesei RUT C30

Enhancing cellulase production in Trichoderma reesei is of great interest for an economical biorefinery. Artificial transcription factors are a potentially powerful molecular strategy for improving cellulase production in T. reesei. In this study, enhanced transcriptional activators XYR1VP, ACE2VP, and ACE1VP were constructed by linking the C terminus of XYR1, ACE2, or ACE1 with an activation domain of herpes simplex virus protein VP16. T. reesei transformants TXYR1VP, TACE2VP, and TACE1VP showed improved cellulase and/or xylanase production. TXYR1VP has a cellulase-free phenotype but with significantly elevated xylanase production. Xylanase I and xylanase II activities [U/(mg biomass)] increased by 51% and 80%, respectively, in TXYR1VP in comparison with parental strain RUT C30. The filter paper activity of TACE2VP in the Avicel-based medium increased by 52% compared to that of RUT C30. In the Avicel-based medium, TACE1VP manifested an 80% increase in FPase activity and a 50% increase in xylanase activity as compared to those of RUT C30. Additionally, when pretreated corn stover was hydrolyzed, crude enzymes produced from TACE1VP yielded a greater glucose release than did the enzymes produced by parental strain RUT C30.

Analysis of Recombinant VP22-SOX2 Fusion Protein Subcellular Location in HepG2 Cell Culture

Cell penetrating peptides (CPPs) are cell permeable proteins that help facilitate impermeable molecules into the cells. Herpes simplex virus protein VP22 is one of the CPP with mechanism of facilitating proteins into the cells by non-classical Golgi-independent. SRY-related HMG-box (SOX) family of transcription factors are well associated with the regulation of the development of embryonic cells. Recombinant fusion protein VP22 is hoped to translocate the protein into the cell. The research conducted is an experimental study using VP22-SOX2 as intervention and human hepatoma HepG2 cells as subject to determine the efficacy of VP22-SOX2. There are 2 main groups: HepG2 cells with VP22-SOX2 incubated for 6 hours and 1 hours, where both are accompanied with the control group in the absence of VP22-SOX2. Both undergo immunostaining using indirect immunostaining method with antibody specific against SOX2 and observation is done using confocal microscope. Counting the total number of cells and number of cells with the fluorescence is performed using ImageJ program. Based on statistic, there is no significant difference between the experiment and the control group incubated for 6 hours. In addition, there is no significant difference between experimental group incubated in 6 hours and experimental group incubated in 1 hour. VP22-SOX2 do not achieve statistically significant protein translocation and incubation period has no effect in the rates of protein translocation. Possible explanation is the requirement of media supplements such as serum replacement needed to stabilize the protein in the prevention of precipitation.

A Longitudinal Study of the Association Between Persistent Pathogens and Incident Depression Among Older U.S. Latinos.

Depression is estimated to affect more than 6.5 million Americans 65 years of age and older and compared with non-Latino whites older U.S. Latinos have a greater incidence and severity of depression, warranting further investigation of novel risk factors for depression onset among this population. We used data on 771/1,789 individuals ≥60 years of age from the Sacramento Area Latino Study on Aging (1998-2008) who were tested for cytomegalovirus (CMV), herpes simplex virus, varicella zoster, Helicobacter pylori, Toxoplasma gondii, and C-reactive protein (CRP) and interleukin-6 (IL-6) level. Among those without elevated depressive symptoms at baseline, we examined the association between each pathogen, inflammatory markers and incident depression over up to nearly 10 years of follow-up using discrete-time logistic regression. We found that only CMV seropositivity was statistically significantly associated with increased odds of incident depression (odds ratio [OR]: 1.38, 95% confidence interval [CI]: 1.00-1.90) in the total sample as well as among women only (OR: 1.70, 95% CI: 1.01-2.86). These associations were not mediated by CRP or IL-6 levels. Our findings suggest that CMV seropositivity may serve as an important risk factor for the onset of depression among older U.S. Latinos, but act outside of inflammatory pathways.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase, RIG-I, and MDA-5. However, its precise role is incompletely defined. By screening a human cDNA library, we showed that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes TBK1 from access to Hsp90 and interferon (IFN) promoter activation. Consistently, the Us11 protein, upon HSV infection, suppresses the expression of beta interferon (IFN-β), RANTES, and interferon-stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome-dependent pathway. Accordingly, Us11 expression facilitates HSV growth. In contrast, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrated that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons.

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.

Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways.IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4+ T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell.

Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting.

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells.

Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26.

Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.

The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype.

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20Syn and UL24Syn To test whether UL21 is needed only for the A855V mutant, additional gBSyn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gBSyn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci.IMPORTANCE UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.

Evasion of the STING DNA-Sensing Pathway by VP11/12 of Herpes Simplex Virus 1.

The stimulator of interferon (IFN) genes (STING) is a broad antimicrobial factor that restricts herpes simplex virus (HSV) by activating type I interferon and proinflammatory responses upon sensing of foreign DNA. UL46 is one of the most abundant tegument proteins of HSV-1, but a well-established function has yet to be found. We found that the HSV-1 UL46 protein interacts with and colocalizes with STING. A ΔUL46 virus displayed growth defects and activated innate immunity, but both effects were alleviated in STING knockdown cells. UL46 was also required for the inhibition of the 2',3'-cyclic GMP-AMP (cGAMP)-dependent immune responses during infection. In cells expressing UL46, out of the context of the infection, innate immunity to a ΔICP0 virus was largely compromised, and that permitted ICP0-deficient mutants to replicate. The UL46-expressing cell lines also rescued the defects of the ΔUL46 virus and enhanced wild-type virus infection. The UL46-expressing cell lines did not activate interferon-stimulated gene (ISG) transcription following treatment with the noncanonical cyclic dinucleotide 2',3'-cGAMP, suggesting that the STING pathway may be compromised. Indeed, we found that both proteins STING and IFI16 were eliminated in cells constitutively expressing UL46 and that the accumulation of their transcripts was blocked. Finally, we demonstrated that UL46 via its N terminus binds to STING and, via its C terminus, to TBK1. These interactions appear to modulate the functions of STING during HSV-1 infection. Taken together, our studies describe a novel function for one of the least-studied proteins of HSV, the tegument protein UL46, and that function involves the evasion of foreign DNA-sensing pathways.IMPORTANCE Herpes simplex virus 1 (HSV-1) afflicts 80% of the population worldwide, causing various diseases. After initial infection, the virus establishes latent reservoirs in sensory neurons and persists for life. Here we describe novel interactions between HSV-1 and the DNA sensor STING. We found that (i) HSV-1 tegument protein UL46 interacts with and colocalizes with STING; (ii) UL46 expressed out of the context of the infection blocks type I interferon triggered by STING stimuli, through the elimination of STING and of interferon-inducible protein 16 (IFI16); (iii) a ΔUL46 virus displayed growth defects, which were rescued in STING knockdown cells; (iv) the ΔUL46 virus failed to block innate immunity triggered by ligands of STING such as 2',3'-cGAMP and also activated IFN-β and ISG expression; and (v) UL46 binds to both STING and TBK1 through different domains. We conclude that UL46 counteracts the actions of STING during HSV-1 infection.