Herpes simplex virus type 2 tegument protein UL56 relocalizes ubiquitin ligase Nedd4 and has a role in transport and/or release of virions

<p>Abstract</p> <p>Background</p> <p>The ubiquitin system functions in a variety of cellular processes including protein turnover, protein sorting and trafficking. Many viruses exploit the cellular ubiquitin system to facilitate viral replication. In fact, herpes simplex virus (HSV) encodes a ubiquitin ligase (E3) and a de-ubiquitinating enzyme to modify the host's ubiquitin system. We have previously reported HSV type 2 (HSV-2) tegument protein UL56 as a putative adaptor protein of neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) E3 ligase, which has been shown to be involved in protein sorting and trafficking.</p> <p>Results</p> <p>In this study, we visualized and characterized the dynamic intracellular localization of UL56 and Nedd4 using live-cell imaging and immunofluorescence analysis. UL56 was distributed to cytoplasmic vesicles, primarily to the trans-Golgi network (TGN), and trafficked actively throughout the cytoplasm. Moreover, UL56 relocalized Nedd4 to the vesicles in cells transiently expressing UL56 and in cells infected with HSV-2. We also investigated whether UL56 influenced the efficiency of viral replication, and found that extracellular infectious viruses were reduced in the absence of UL56.</p> <p>Conclusion</p> <p>These data suggest that UL56 regulates Nedd4 and functions to facilitate the cytoplasmic transport of virions from TGN to the plasma membrane and/or release of virions from the cell surface.</p

Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

<p>Abstract</p> <p>Background</p> <p>Herpes simplex viruses (HSVs) rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1), a host cell protein markedly up-regulated by HSV infection.</p> <p>Results</p> <p>INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP) assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA.</p> <p>Conclusions</p> <p>The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.</p

Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch

<p>Abstract</p> <p>Background</p> <p>Herpes simplex virus type 2 (HSV-2) is one of many viruses that exploits and modifies the cellular ubiquitin system. HSV-2 expresses the tegument protein UL56 that has been implicated in cytoplasmic transport and/or release of virions, and is a putative regulatory protein of Nedd4 ubiquitin ligase. In order to elucidate the biological function of UL56, this study examined the interaction of UL56 with the Nedd4-family ubiquitin ligase Itch and its role in the regulation of Itch. Additionally, we assessed the similarity between UL56 and regulatory proteins of Itch and Nedd4, Nedd4-family-interactins proteins (Ndfip).</p> <p>Results</p> <p>UL56 interacted with Itch, independent of additional viral proteins, and mediated more striking degradation of Itch, compared to Nedd4. Moreover, it was suggested that the lysosome pathway as well as the proteasome pathway was involved in the degradation of Itch. Other HSV-2 proteins with PY motifs, such as VP5 and VP16, did not mediate the degradation of endogenous Itch. Ndfip1 and Ndfip2 were similar in subcellular distribution patterns to UL56 and colocalized with UL56 in co-transfected cells.</p> <p>Conclusions</p> <p>We believe that this is the first report demonstrating the interaction of a HSV-specific protein and Itch. Thus, UL56 could function as a regulatory protein of Itch. The mechanism, function and significance of regulating Itch in HSV-2 infection remain unclear and warrant further investigation.</p

Immunization with a highly attenuated replication-competent herpes simplex virus type 1 mutant, HF10, protects mice from genital disease caused by herpes simplex virus type 2

Genital herpes is an intractable disease caused mainly by herpes simplex virus (HSV) type 2 (HSV-2), and is a major concern in public health. A previous infection with HSV type 1 (HSV-1) enhances protection against primary HSV-2 infection to some extent. In this study, we evaluated the ability of HF10, a naturally occurring replication-competent HSV-1 mutant, to protect against genital infection in mice caused by HSV-2. Subcutaneous inoculation of HF10-immunized mice against lethal infection by HSV-2, and attenuated the development of genital ulcer diseases. Immunization with HF10 inhibited HSV-2 replication in the mouse vagina, reduced local inflammation, controlled emergence of neurological dysfunctions of HSV-2 infection, and increased survival. In HF10-immunized mice, we observed rapid and increased production of interferon-γ in the vagina in response to HSV-2 infection, and numerous CD4+ and a few CD8+ T cells localized to the infective focus. CD4+ T cells invaded the mucosal subepithelial lamina propria. Thus, the protective effect of HF10 was related to induction of cellular immunity, mediated primarily by Th1 CD4+ cells. These data indicate that the live attenuated HSV-1 mutant strain HF10 is a promising candidate antigen for a vaccine against genital herpes caused by HSV-2

Replication of Epstein-Barr Virus Primary Infection in Human Tonsil Tissue Explants

Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95–8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12–15 days through 24 days post-infection in tissue models infected with B95–8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP+ cells were CD3− CD56− CD19+ HLA-DR+, and represented both naïve (immunoglobulin D+) and memory (CD27+) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents

Determination and analysis of the DNA sequence of highly attenuated herpes simplex virus type 1 mutant HF10, a potential oncolytic virus

A spontaneously occurring herpes simplex virus type 1 (HSV-1) mutant, designated HF10, replicates very efficiently and induces extensive cell fusion in most transformed cells as well as Vero cells, but is highly attenuated in mice when inoculated by peripheral routes of infection. Recent studies have shown that HF10 is a promising agent for use in oncolytic virotherapy. In this study, we sequenced the genome of HF10 and compared it with that of HSV-1 strain 17, a reference strain with the syn+ phenotype. The sequencing covered whole regions corresponding to all open reading frames of strain 17, and the overall putative amino acid identity between HF10 and strain 17 was 99.1% except for proteins encoded by three genes with frame-shift mutations. HF10 had a number of deletions and insertions in the genome, resulting in the lack of the functional expression of UL43, UL49.5, UL55, UL56 and latency-associated transcripts. Additionally, HF10 had amino acid changes in genes involved in the regulation of syncytium formation, including UL1, UL20, UL22, UL24, UL27 and UL53. The proteins encoded by UL1, UL2, UL11, UL44, US1, US7, US8.5, US10 and US12 exhibited a relatively high divergence. These data provide the genetic background of HF10 and insight into the molecular mechanism of HSV-1 replication and pathogenicity.</p