Wild-type Herpes Simplex Virus Research Articles

Herpes simplex virus 1 counteracts viperin via its virion host shutoff protein UL41.

The interferon (IFN)-inducible viperin protein restricts a broad range of viruses. However, whether viperin plays a role during herpes simplex virus 1 (HSV-1) infection is poorly understood. In the present study, it was shown for the first time that wild-type (WT) HSV-1 infection couldn't induce viperin production, and ectopically expressed viperin inhibited the replication of UL41-null HSV-1 but not WT viruses. The underlying molecular mechanism is that UL41 counteracts viperin's antiviral activity by reducing its mRNA accumulation.

Herpes simplex virus gE/gI extracellular domains promote axonal transport and spread from neurons to epithelial cells.

Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: (i) anterograde axonal transport of virus particles from neuron bodies to axon tips and (ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. The HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport, and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (∼400-residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants, gE-277 and gE-348, contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers, and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport; there were markedly reduced numbers of viral capsids and glycoproteins compared with wild-type HSV. The defects in axonal transport were manifest in neuronal cell bodies, involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild-type HSV. In contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite numbers of virus particles similar to those for HSV gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains function in both processes. An essential phase of the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is the capacity to reactivate from latency and then spread from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to axon tips followed by (ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important for identifying novel targets to reduce alphaherpesvirus disease.

A mutation in the DNA polymerase accessory factor of herpes simplex virus 1 restores viral DNA replication in the presence of raltegravir.

Previous reports showed that raltegravir, a recently approved antiviral compound that targets HIV integrase, can inhibit the nuclease function of human cytomegalovirus (HCMV terminase) in vitro. In this study, subtoxic levels of raltegravir were shown to inhibit the replication of four different herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, HCMV, and mouse cytomegalovirus, by 30- to 700-fold, depending on the dose and the virus tested. Southern blotting and quantitative PCR revealed that raltegravir inhibits DNA replication of HSV-1 rather than cleavage of viral DNA. A raltegravir-resistant HSV-1 mutant was generated by repeated passage in the presence of 200 μM raltegravir. The genomic sequence of the resistant virus, designated clone 7, contained mutations in 16 open reading frames. Of these, the mutations F198S in unique long region 15 (UL15; encoding the large terminase subunit), A374V in UL32 (required for DNA cleavage and packaging), V296I in UL42 (encoding the DNA polymerase accessory factor), and A224S in UL54 (encoding ICP27, an important transcriptional regulator) were introduced independently into the wild-type HSV-1(F) genome, and the recombinant viruses were tested for raltegravir resistance. Viruses bearing both the UL15 and UL32 mutations inserted within the genome of the UL42 mutant were also tested. While the UL15, UL32, and UL54 mutant viruses were fully susceptible to raltegravir, any virus bearing the UL42 mutation was as resistant to raltegravir as clone 7. Overall, these results suggest that raltegravir may be a valuable therapeutic agent against herpesviruses and the antiviral activity targets the DNA polymerase accessory factor rather than the nuclease activity of the terminase. This paper shows that raltegravir, the antiretrovirus drug targeting integrase, is effective against various herpesviruses. Drug resistance mapped to the herpesvirus DNA polymerase accessory factor, which was an unexpected finding.

Herpes simplex virus: isolation, cytopathological characterization and antiviral sensitivity.

BACKGROUND Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection.OBJECTIVE This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine.METHODS Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay.RESULTS From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%.CONCLUSIONSThe methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood.

Phosphorylation of herpes simplex virus 1 dUTPase upregulated viral dUTPase activity to compensate for low cellular dUTPase activity for efficient viral replication.

We recently reported that herpes simplex virus 1 (HSV-1) protein kinase Us3 phosphorylated viral dUTPase (vdUTPase) at serine 187 (Ser-187) to upregulate its enzymatic activity, which promoted HSV-1 replication in human neuroblastoma SK-N-SH cells but not in human carcinoma HEp-2 cells. In the present study, we showed that endogenous cellular dUTPase activity in SK-N-SH cells was significantly lower than that in HEp-2 cells and that overexpression of cellular dUTPase in SK-N-SH cells increased the replication of an HSV-1 mutant with an alanine substitution for Ser-187 (S187A) in vdUTPase to the wild-type level. In addition, we showed that knockdown of cellular dUTPase in HEp-2 cells significantly reduced replication of the mutant vdUTPase (S187A) virus but not that of wild-type HSV-1. Furthermore, the replacement of Ser-187 in vdUTPase with aspartic acid, which mimics constitutive phosphorylation, and overexpression of cellular dUTPase restored viral replication to the wild-type level in cellular dUTPase knockdown HEp-2 cells. These results indicated that sufficient dUTPase activity was required for efficient HSV-1 replication and supported the hypothesis that Us3 phosphorylation of vdUTPase Ser-187 upregulated vdUTPase activity in host cells with low cellular dUTPase activity to produce efficient viral replication.virus. Importance: It has long been assumed that dUTPase activity is important for replication of viruses encoding a dUTPase and that the viral dUTPase (vdUTPase) activity was needed if host cell dUTPase activity was not sufficient for efficient viral replication. In the present study, we showed that the S187A mutation in HSV-1 vdUTPase, which impaired its enzymatic activity, reduced viral replication in SK-N-SH cells, which have low endogenous cellular dUTPase activity, and that overexpression of cellular dUTPase restored viral replication to the wild-type level. We also showed that knockdown of cellular dUTPase in HEp-2 cells, which have higher dUTPase activity than do SK-N-SH cells, reduced replication of HSV-1 with the vdUTPase mutation but had no effect on wild-type virus replication. This is the first report, to our knowledge, directly showing that dUTPase activity is critical for efficient viral replication and that vdUTPase compensates for low host cell dUTPase activity to produce efficient viral replication.

Herpes Simplex Virus 1 Serine/Threonine Kinase US3 Hyperphosphorylates IRF3 and Inhibits Beta Interferon Production

Viral infection initiates a series of signaling cascades that lead to the transcription of interferons (IFNs), finally inducing interferon-stimulated genes (ISGs) to eliminate viruses. Viruses have evolved a variety of strategies to modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) US3, a Ser/Thr kinase conserved in alphaherpesviruses, was previously reported to counteract host innate immunity; however, the molecular mechanism is elusive. In this study, we report that US3 blocks IFN-β production by hyperphosphorylating IFN regulatory factor 3 (IRF3). Ectopic expression of US3 protein significantly inhibited Sendai virus (SeV)-mediated activation of IFN-β and IFN-stimulated response element (ISRE) promoters and the transcription of IFN-β, ISG54, and ISG56. US3 was also shown to block SeV-induced dimerization and nuclear translocation of IRF3. The kinase activity was indispensable for its inhibitory function, as kinase-dead (KD) US3 mutants K220M and D305A could not inhibit IFN-β production. Furthermore, US3 interacted with and hyperphosphorylated IRF3 at Ser175 to prevent IRF3 activation. Finally, the US3 KD mutant viruses were constructed and denoted K220M or D305A HSV-1, respectively. Cells and mice infected with both mutant viruses produced remarkably larger amounts of IFN-β than those infected with wild-type HSV-1. For the first time, these findings provide convincing evidence that US3 hyperphosphorylates IRF3, blocks the production of IFN-β, and subverts host innate immunity.

Role of Interleukin-2 and Herpes Simplex Virus 1 in Central Nervous System Demyelination in Mice

We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.

Roles of p53 in Herpes Simplex Virus 1 Replication

p53 is a critical factor in the cellular response to a broad range of stress factors through its ability to regulate various cellular pathways. In this study, tandem affinity purification of transiently expressed herpes simplex virus 1 (HSV-1) regulatory protein ICP22 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP22 interacted with p53 in HSV-1-infected cells. In p53(-/-) cells, replication of wild-type HSV-1 was reduced compared to that in parental p53(+/+) cells, indicating that p53 had a positive effect on HSV-1 replication. In contrast, the levels of viral replication of an ICP22-null mutant virus were similar in both p53(-/-) and p53(+/+) cells. At 2 h postinfection, the level of expression of ICP27, an essential viral regulatory protein, in p53(-/-) cells infected with wild-type HSV-1 or the ICP22-null mutant virus was lower than in p53(+/+) cells. In contrast, at 18 h postinfection, the level of expression of ICP0, a critical viral regulatory protein, in p53(-/-) cells infected with the ICP22-null mutant virus was higher than in p53(+/+) cells, although the levels of ICP0 expression in p53(-/-) and p53(+/+) cells infected with wild-type HSV-1 were almost identical. These results suggested that p53 overall promoted HSV-1 replication and that p53 played both positive and negative roles in HSV-1 replication: upregulating ICP27 expression very early in infection and downregulating ICP0 expression later in infection, which was antagonized by ICP22.

Pan-HSV-2 IgG Antibody in Vaccinated Mice and Guinea Pigs Correlates with Protection against Herpes Simplex Virus 2

We lack a correlate of immunity to herpes simplex virus 2 (HSV-2) that may be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes. This gap in knowledge is often attributed to a failure to measure the correct component of the adaptive immune response to HSV-2. However, efforts to identify a correlate of immunity have focused on subunit vaccines that contain less than 3% of HSV-2's 40,000-amino-acid proteome. We were interested to determine if a correlate of immunity might be more readily identified if 1. animals were immunized with a polyvalent immunogen such as a live virus and/or 2. the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2's antigens (pan-HSV-2 IgG). Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0 − viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.

A herpes simplex virus scaffold peptide that binds the portal vertex inhibits early steps in viral replication.

Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.

Methyl-2-arylidene hydrazinecarbodithioates: synthesis and biological activity.

Methyl-2-arylidene hydrazine-carbodithioate has not been of particular interest to researchers even though its metal complexes are extensively reported on due to their biological activity. This study examined the cytostatic and antiviral activity of twelve methyl-2-arylidene hydrazinecarbodithioates reported by many researchers as intermediates for the synthesis of thiosemicarbazides and the preparation of their metal complexes. Compounds IIc, IIi, and IIl with tridentate ligand features were found to have the lowest IC50 value (6.5 μM, ≈ 1 μM, and 0.8 μM, respectively) against HL60 human promyelocytic leukemia cells. They were also most inhibitory to human embryonic lung (HEL) fibroblast proliferation (5.3 μM, 17 μM, and 2.6 μM). Compound IIc and IIl show antiviral activity against wild-type herpes simplex virus (HSV), varicella zoster virus (VZV), and acyclovirresistant HSV; however, these activities were observed at concentrations at which the compounds also markedly inhibit HL60 and HEL cell proliferation.

A Herpes Simplex Virus 2 Glycoprotein D Mutant Generated by Bacterial Artificial Chromosome Mutagenesis Is Severely Impaired for Infecting Neuronal Cells and Infects Only Vero Cells Expressing Exogenous HVEM

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

Characterization of virus strains resistant to the herpes virus helicase–primase inhibitor ASP2151 (Amenamevir)

ASP2151 is an antiherpes agent targeting the helicase–primase complex of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV). We characterized the ASP2151-resistant HSV-1 and HSV-2 variants or mutants based on findings from sequencing analysis, growth, pathogenicity, and susceptibility testing, identifying several single base-pair substitutions resulting in amino acid changes in the helicase and primase subunit of ASP2151-resistant mutants. Amino acid alterations in the helicase subunit were clustered near helicase motif IV in the UL5 helicase gene of both HSV-1 and HSV-2, while the primase subunit substitution associated with reduced susceptibility, R367H, was found in ASP2151-resistant HSV-1 mutants. However, while susceptibility in the ASP2151-resistant HSV mutants to existing antiherpes agents was equivalent to that in wild-type HSV strains, ASP2151-resistant HSV mutants showed attenuated in vitro growth capability and in vivo pathogenicity compared with the parent strains. Taken together, our present findings demonstrated that important amino acid substitutions associated with reduced susceptibilities of HSV-1 and HSV-2 to ASP2151 exist in both the helicase and primase subunits of the helicase–primase complex, and that mutations in this complex against ASP2151 might confer defects in viral replication and pathogenicity.

Replication-competent Herpes Simplex Virus Retargeted to HER2 as Therapy for High-grade Glioma

Oncolytic herpes simplex viruses (HSVs) represent a novel frontier against tumors resistant to standard therapies, like glioblastoma (GBM). The oncolytic HSVs that entered clinical trials so far showed encouraging results; however, they are marred by the fact that they are highly attenuated. We engineered HSVs that maintain unimpaired lytic efficacy and specifically target cells that express tumor-specific receptors, thus limiting the cytotoxicity only to cancer cells, and leaving unharmed the neighboring tissues. We report on the safety and efficacy in a high-grade glioma (HGG) model of R-LM113, an HSV recombinant retargeted to human epidermal growth factor receptor 2 (HER2), frequently expressed in GBMs. We demonstrated that R-LM113 is safe in vivo as it does not cause encephalitis when intracranially injected in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, extremely sensitive to wild-type HSV. The efficacy of R-LM113 was assessed in a platelet-derived growth factor (PDGF)-induced infiltrative glioma model engineered to express HER2 and transplanted intracranially in adult NOD/SCID mice. Mice injected with HER2-engineered glioma cells infected with R-LM113 showed a doubled survival time compared with mice injected with uninfected cells. A doubling in survival time from the beginning of treatment was obtained also when R-LM113 was administered into already established tumors. These data demonstrate the efficacy of R-LM113 in thwarting tumor growth.

The Herpes Simplex Virus Type 1 Latency-Associated Transcript Inhibits Phenotypic and Functional Maturation of Dendritic Cells

We recently found that the herpes simplex virus-1 (HSV-1) latency-associated transcript (LAT) results in exhaustion of virus-specific CD8⁺ T cells in latently-infected trigeminal ganglia (TG). In this study we sought to determine if this impairment may involve LAT directly and/or indirectly interfering with DC maturation. We found that a small number of HSV-1 antigen-positive DCs are present in the TG of latently-infected CD11c/eYFP mice; however, this does not imply that these DCs are acutely or latently infected. Some CD8⁺ T cells are adjacent to DCs, suggesting possible interactions. It has previously been shown that wild-type HSV-1 interferes with DC maturation. Here we show for the first time that this is associated with LAT expression, since compared to LAT⁻ virus: (1) LAT⁺ virus interfered with expression of MHC class I and the co-stimulatory molecules CD80 and CD86 on the surface of DCs; (2) LAT⁺ virus impaired DC production of the proinflammatory cytokines IL-6, IL-12, and TNF-α; and (3) DCs infected in vitro with LAT⁺ virus had significantly reduced the ability to stimulate HSV-specific CD8⁺ T cells. While a similar number of DCs was found in LAT⁺ and LAT⁻ latently-infected TG of CD11c/eYFP transgenic mice, more HSV-1 Ag-positive DCs and more exhausted CD8 T cells were seen with LAT⁺ virus. Consistent with these findings, HSV-specific cytotoxic CD8⁺ T cells in the TG of mice latently-infected with LAT⁺ virus produced less IFN-γ and TNF-α than those from TG of LAT⁻-infected mice. Together, these results suggest a novel immune-evasion mechanism whereby the HSV-1 LAT increases the number of HSV-1 Ag-positive DCs in latently-infected TG, and interferes with DC phenotypic and functional maturation. The effect of LAT on TG-resident DCs may contribute to the reduced function of HSV-specific CD8⁺ T cells in the TG of mice latently infected with LAT⁺ virus.

Abstract 4374: Combination of Bevacizumab and oncolytic virus hRr3 in an experimentalmodel of pancreatic carcinoma

Abstract Aim: Proangiogenic growth factors stimulate endothelial cells in nearby vessels to proliferate and migrate into the tumor. Vascular endothelial growth factor (VEGF) is an endothelial cell specific growth factor that is known to facilitate angiogenesis. Excess levels of VEGF are linked with poor prognosis. The effect oncolytic herpes viruses (OHSV) on VEGF A are not accurately determined. Nevertheless; limited number of studies on wild type herpes simplex type 1 (HSV1) have been shown to enhance VEGF A. The aim of the present study was to evaluate the effect of combination of OHSV hrR3 with a novel monoclonal VEGF A antibody Bevacizumab in a xenograft experimental model. Materials and Methods: Several cell were tested for expression of VEGFA. BxPC3 cell line had the highest and MIA PaCa-2 had the lowest expression of VEGFA among the other cell lines. 20 BALB-C nu/nu mice were divided into 4 experimental groups. All animals were implanted 5 mm diameter of BxPC-3 tumor xenograft on each side of their flank. After 2 week of observation; Control group (n=5) received no therapy; oncolytic viral therapy group (OVT) (n=5) received 5x106 pfu hrR3 injected intratumorally per week for two weeks; Bevacizumab treated group (BVT) (n=5) received 5mg/kg Bevacizumab intraperitoneally two times a week for 3 weeks; Combination therapy group (CT) (n=5) received both Bevacizumab and hRr3 virus injected intratumorally the same periods as other groups. The same was performed for MIA PaCa-2 cell lines as In order to confirm our results in a low expression cell line. Tumor diameters were evaluated every 3 days by caliper for 8 weeks. Tumor specimen was collected for conventional hematoxylin eosin stain, immunohistochemistochistry for VEGFA, Thrombospondin1, CD 31, HSV type 1 antigen and TUNEL. In MIA PaCa-2 cell line; VEGFA antigen distribution was especially evaluated on day 1, 3, 5, 7, 14 following hRr3 treatment. Results: The BxPC3 tumor volumes of the animals in the combination therapy were significantly lower than the other groups (p<0.05). Nevertheless; we were unable to show a statistically significant difference among OVT and BVT groups in terms of tumor volume. Histopathologically the CT and the OVT group had degenerated cells and low cellular density and expressed HSV antigen. Interestingly we found highest VEGFA expression in OVT group when compared to other groups (p<0.001). Tumor response was highest in combination group in the MIA PaCa-2 tumors as well (p<0.05). In Mia PACA2 tumors VEGFA expression was increasingly expressed starting from day 5 (p<0.05). Conclusion: Oncolytic virus seems to induce the VEGFA and this may be the proangiogenic stimulus in OHSV to induce angiogenesis. If a combination therapy towards blocking the VEGF A can be administered with OHSV; it may enhance the effect of the virus. Combination of OHSV with an antiangiogenic agent such as Bevacizumab seems to be a safe and effective treatment modality. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4374. doi:1538-7445.AM2012-4374

Herpes Simplex Virus 1 Tegument Protein US11 Downmodulates the RLR Signaling Pathway via Direct Interaction with RIG-I and MDA-5

The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.

Activation of NF-κB in CD8 + Dendritic Cells Ex Vivo by the γ 1 34.5 Null Mutant Correlates with Immunity against Herpes Simplex Virus 1

The γ(1)34.5 protein of herpes simplex viruses (HSV) is essential for virulence. Accordingly, an HSV mutant lacking γ(1)34.5 is attenuated in vivo. Despite its vaccine potential, the mechanism by which the γ(1)34.5 null mutant triggers protective immunity is unknown. In this report we show that vaccination with the γ(1)34.5 null mutant protects against lethal challenge from wild-type virus via IκB kinase in dendritic cells (DCs), which sense virus-associated molecular patterns. Unlike mock-treated DCs, DCs primed with the γ(1)34.5 null mutant ex vivo mediate resistance to wild-type HSV after adoptive transfer into naïve mice. Furthermore, the γ(1)34.5 null mutant activates IκB kinase, which facilitates p65/RelA phosphorylation and nuclear translocation, resulting in DC maturation. While unable to produce infectious virus in DCs, this mutant virus expresses early and late genes. In its abortive infection, the γ(1)34.5 null mutant induces protective immunity more effectively in CD8(+) DCs than in CD8(-) DCs. This is mirrored by a higher level of interleukin-6 (IL-6) and IL-12 secretion by CD8(+) DCs than CD8(-) DCs. Remarkably, inhibition of p65/RelA phosphorylation or nuclear translocation in CD8(+) DCs disrupts protective immunity. These results suggest that engagement of the γ(1)34.5 null mutant with CD8(+) DCs elicits innate immunity to activate NF-κB, which translates into protective immunity.

Δγ 1 34.5 Herpes Simplex Viruses Encoding Human Cytomegalovirus IRS1 or TRS1 Induce Interferon Regulatory Factor 3 Phosphorylation and an Interferon-Stimulated Gene Response

The chimeric herpes simplex viruses (HSV) are Δγ₁34.5 vectors encoding the human cytomegalovirus (HCMV) IRS1 or TRS1 genes. They are capable of late viral protein synthesis and are superior to Δγ₁34.5 HSVs in oncolytic activity. The interferon (IFN) response limits efficient HSV gene expression and replication. HCMV TRS1 and IRS1 restore one γ₁34.5 gene function: evasion of IFN-inducible protein kinase R, allowing late viral protein synthesis. Here we show that, unlike wild-type HSV, the chimeric HSV do not restore another γ₁34.5 function, the suppression of early IFN signaling mediated by IFN regulatory factor 3 (IRF3).

Herpes Simplex Virus 1 ICP0 Phosphorylation Site Mutants Are Attenuated for Viral Replication and Impaired for Explant-Induced Reactivation

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.