Oncolytic activity of naturally attenuated herpes-simplex virus HF10 against an immunocompetent model of oral carcinoma.

Prognosis for advanced oral carcinoma remains poor. Oncolytic virotherapy uses replication-competent viruses to infect and kill only the tumor cells. However, it has been difficult to investigate the oncolytic activity of viruses against oral carcinomas in mouse models. This study established a mouse model of oral cancer and investigated the in vitro and in vivo anti-tumor effects of HF10, a highly attenuated, replication-competent herpes simplex virus (HSV)-1. Mouse tongue cancer was induced by injecting 4-nitroquinoline 1-oxide into the mouse tongue. The murine oral cancer cell line isolated from this tumor, named NMOC1, formed invasive carcinoma within a week when injected into mouse tongue. HF10 successfully infected, replicated, and spread in the cancer cells in vitro. HF10 was able to kill cancer cells isolated from human or mouse tongue tumor. HF10 injection into tongue carcinomas prolonged mouse survival without any side effects or weight loss. Intertumoral injection of GFP-expressing HF10 confirmed that viral spread was confined within the tumors. Immunohistochemical staining showed that HF10 induced infiltration of CD8-positive T cells around HSV-infected cells in the tumor mass, implying increased anti-tumor immunity. We successfully established an oral cancer cell line and showed that HF10 is a promising therapeutic agent for oral cancer.

Oncolytic activity of HF10 in head and neck squamous cell carcinomas.

Recent developments in therapeutic strategies have improved the prognosis of head and neck squamous cell carcinoma (HNSCC). Nevertheless, 5-year survival rate remains only 40%, necessitating new therapeutic agents. Oncolytic virotherapy entails use of replication-competent viruses to selectively kill cancer cells. We aimed to explore the potential of HF10 as an oncolytic virus against human or mouse HNSCC cell lines, and primary-cultured HNSCC cells. HF10 replicated well in all the HNSCC cells, in which it induced cytopathic effects and cell killing. Next, we investigated the oncolytic effects of HF10 in ear tumor models with human or mouse tumor cells. We detected HF10-infected cells within the ear tumors based on their expression of green fluorescent protein. HF10 injection suppressed ear tumor growth and prolonged overall survival. In the syngeneic model, HF10 infection induced tumor necrosis with infiltration of CD8-positive cells. Moreover, the splenocytes of HF10-treated mice released antitumor cytokines, IL-2, IL-12, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha, after stimulation with tumor cells in vitro. The HF10-treated mice that survived their original tumor burdens rejected tumor cells upon re-challenge. These results suggested that HF10 killed HNSCC cells and induced antitumoral immunity, thereby establishing it as a promising agent for the treatment of HNSCC patients.

The efficacy of combination therapy with oncolytic herpes simplex virus HF10 and dacarbazine in a mouse melanoma model.

Advanced melanoma has long been treated with chemotherapy using cytotoxic agents like dacarbazine (DTIC), but overall survival rates with these drugs have been generally low. Recently, immunoregulatory monoclonal antibodies and molecularly targeted therapy with a BRAF inhibitor and/or a MEK inhibitor, have been used to treat malignant melanoma and have improved the survival rate of patients with advanced melanoma. However, high prices of these drugs are problematic. In this study, we evaluated the oncolytic efficacy of HF10, an attenuated, replication-competent HSV, with DTIC in immunocompetent mice model of malignant melanoma. For in vitro studies, cytotoxicity assays were conducted in clone M3 mouse melanoma cells. For the in vivo studies, subcutaneous melanoma models were prepared in DBA/2 mice with clone M3 cells, and then HF10 was intratumorally inoculated with/without intraperitoneal DTIC injection. The efficacy of the therapies was evaluated by survival, growth of subcutaneous tumor, and histopathological and immunological analyses. Both HF10 infection and DTIC treatment showed cytotoxic effects in melanoma cells, but combination treatment with HF10 and DTIC showed a rapid and strong cytotoxic effect compared with monotherapy. In the subcutaneous melanoma model, intratumoral HF10 inoculation significantly inhibited tumor growth. HF10 also inhibited the growth of non-inoculated contralateral tumors when it was injected into the ipsilateral tumors of mice. In histologic and immunohistochemical analysis, tumor lysis and inflammatory cell infiltration were observed after intratumoral HF10 inoculation. When mice were treated with HF10 and DTIC, the combination therapy induced a robust systemic anti-tumor immune response and prolonged survival. IFN-γ secretion from splenocytes of the HF10-DTIC combination therapy group showed more IFN-γ secretion than did the other groups. These data showed the efficacy of HF10 and DTIC combination therapy in a mouse melanoma model.

Oncotarget Strategies For Herpes Simplex Virus-1.

The high level of manipulability of viral genome has set up HSV-1 to be an ideal viral vector for oncolytic virotherapy. In the past two decades, several oncolytic HSV-1 viruses have been successfully developed and assessed in animal studies. Accumulated evidences show that oncolytic HSV- 1 can efficiently infect many tumor cells and augment anti-tumor effect by induction of systemic innate and adaptive immune responses. Inspiring results have been accomplished in several phase I clinical trials for glioma, head and neck squeous cells carcinoma and Melanoma using oncolytic HSV- 1 viruses. More recently, oncovey, one of oncolytic HSV-1 viruses has been approved by FDA for the comprehensive evolution of its anti-tumor effects in phase III clinical trials. These promising studies encourage more efforts to be devoted to craft the new generation of oncolytic HSV-1. Herein, we will review and summarize the basic strategies to construct oncolytic HSV-1 viruses and their applications in cancer therapy.

Oncolytic viral therapy with a combination of HF10, a herpes simplex virus type 1 variant and granulocyte-macrophage colony-stimulating factor for murine ovarian cancer.

Ovarian cancer is the most frequent cause of gynecological cancer-related mortality as a majority of patients are diagnosed at an advanced stage with intraperitoneal dissemination because of the absence of initial symptoms. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the maturation of specialized antigen-presenting cells. In this study, we utilized a herpes simplex virus (HSV) amplicon expressing murine GM-CSF combined with HF10 (mGM-CSF amplicon), a highly attenuated HSV type 1 strain functioning as a helper virus to strengthen anti-tumor immune response, for the treatment of ovarian cancer with intraperitoneal dissemination. A mouse ovarian cancer cell line, OV2944-HM-1 (HM-1), was intraperitoneally injected, following which HF10 only or the mGM-CSF amplicon was injected intraperitoneally three times. HF10 injection prolonged survival and decreased intraperitoneal dissemination, but to a lesser extent than the mGM-CSF amplicon. Although HF10 replication was not observed in HM-1 cells, expression of VP5, a late gene coding the major capsid protein of HSV, was detected. Moreover, mGM-CSF production was detected in transfected HM-1 cells. Immunohistochemical staining revealed the infiltration of CD4- and CD8-positive cells into the peritoneal tumor(s). A significantly increased CD4+ T cell concentration was observed in the spleen. Murine splenic cells after each treatment were stimulated with HM-1 cells, and the strongest immune response was observed in the mice that received mGM-CSF amplicon injections. These results suggested that the mGM-CSF amplicon is a promising agent for the treatment of advanced ovarian cancer with intraperitoneal dissemination.

Phase I Dose-escalation Clinical Trial of HF10 Oncolytic Herpes Virus in 17 Japanese Patients with Advanced Cancer.

Oncolytic virus therapy is a promising new therapeutic method, one of an eagerly anticipated class of biological therapies against cancer. There are many different classes of oncolytic virus. One of these, herpes oncolytic virus, is strongly oncolytic and has a large DNA genome as 150k bp. HF10 is a spontaneous mutant of herpes simplex virus -1 (HSV-1) that replicates within tumors and destroys cancers without damaging normal tissue and organs. Clinical trials of HF10 are underway in Japan and the United States. The first pilot study of HF10 was initiated in Japan in 2003. This study examined the safety and efficacy of HF10 in the treatment of breast cancer and head and neck cancers; the trial also included careful dose escalation studies. In 2005, a clinical trial using HF10 to treat pancreatic cancer was initiated. screened In this Japanese study, 17 patients received HF10 in their tumor sites. A clinical trial in the United States is also ongoing to evaluate safety, tolerability and evidence of antitumor activity in patients with refractory superficial solid tumors. Here, we report the evaluation of the 17 patients treated in Japan. Among the patients, 6 had recurrent breast cancer, 3 had recurrent head and neck cancer, and 8 had non-resectable pancreatic cancer. No severe adverse side effects have been observed, and some therapeutic potential has been reported based on pathological findings, tumor markers, and diagnostic radiography. Those results should encourage further clinical trials of HF10 around the world.

Mast cells play a key role in host defense against herpes simplex virus infection through TNF-α and IL-6 production.

The essential contribution of mast cells (MCs) to bacterial host defense has been well established; however, little is known about their role in viral infections in vivo. Here, we found that intradermal injection with herpes simplex virus 2 (HSV-2) into MC-deficient Kit(W/Wv) mice led to increased clinical severity and mortality with elevated virus titers in HSV-infected skins. Ex vivo HSV-specific tetramer staining assay demonstrated that MC deficiency did not affect the frequency of HSV-specific cytotoxic T lymphocytes (CTLs) in draining lymph nodes. Moreover, the high mortality in Kit(W/W-v) mice was completely reversed by intradermal reconstitution with bone marrow-derived MCs (BMMCs) from wild-type, but not TNF(-/-) or IL-6(-/-) mice, indicating that MCs or, more specifically, MC-derived tumor necrosis factor (TNF) and IL-6 can protect mice from HSV-induced mortality. However, HSV did not directly induce TNF-α or IL-6 production by BMMCs; supernatants from HSV-infected keratinocytes induced the production of these cytokines by BMMCs without degranulation. Furthermore, IL-33 expression was induced in HSV-infected keratinocytes, and blocking the IL-33 receptor T1/ST2 on BMMCs significantly reduced TNF-α and IL-6 production by BMMCs. These results indicate the involvement of MCs in host defense at HSV-infected sites through TNF-α and IL-6 production, which is induced by keratinocyte-derived IL-33.

Enhanced antitumoral activity of oncolytic herpes simplex virus with gemcitabine using colorectal tumor models.

To enhance the oncolytic activity of herpes simplex viruses (HSVs) control of immune-suppression and immune-resistance by cancer cells is important. Myeloid-derived suppressor cells (MDSCs), which interfere with tumor-suppressive environments, are inhibited by gemcitabine (GEM) treatment. We investigated the oncolytic activity and systemic antitumor immunity induced by oncolytic HSVs in combination with GEM treatment. A mouse model with subcutaneous tumors on both sides of the lateral flanks was used. A highly attenuated HSV type 1, strain HF10, was inoculated into one side of each tumor three times following intraperitoneal injection of GEM. Histopathological changes and IFN-γ secretion of the tumor and leukocytes in the spleen were analyzed. These treatments were repeated to enhance oncolytic activity. HF10 inoculation reduced tumor growth only on the HF10-treated side. HF10 inoculation following GEM treatment resulted in greater reduction of tumor growth on the HF10-treated tumor; furthermore, reduction of tumors on the contralateral untreated side was also observed. Necrosis of the tumor was observed in areas where HSV-infected cells were detected. F4/80(+) macrophages around the tumor were eliminated, and CD4(+) T and CD8(+) T cells increased in the spleen. A single injection of GEM decreased CD11b(+) /Gr-1(+) MDSCs while retaining CD4(+) T cells and CD8(+) T cells. Repetition of this treatment regimen resulted in even greater reduction of tumor growth on both sides and complete rejection in some of the mice. Intratumoral injection of oncolytic HSVs following GEM injection reduced MDSCs. Repeated treatment with oncolytic HSVs following GEM resulted in enhanced oncolytic activity.

Generation and Characterization of UL21-Null Herpes Simplex Virus Type 1.

UL21 of herpes simplex virus type 1 (HSV-1) is an accessory gene that encodes a component of the tegument. Homologs of this protein have been identified in the alpha, beta, and gamma herpesvirus subfamilies, although their functions are unclear. To clarify the functions of UL21, we generated a UL21-null HSV-1 mutant. Growth analysis showed that the synthesis of infectious UL21-null HSV-1 in glial cells was delayed and that the overall yield was low. The plaque sizes of the UL21-null mutant were smaller than those of wild-type HSV-1. We identified several candidate UL21-interacting proteins, including intermediate filaments, by yeast two-hybrid screening. The distribution of glial fibrillary acidic protein (GFAP), which is the main component of intermediate filaments, was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells. These results will help clarify the function of UL21 and broaden our understanding of the life cycle of HSV.

Diabetic mice show an aggravated course of herpes-simplex virus-induced facial nerve paralysis.

HYPOTHESIS: Bell's palsy is highly associated with diabetes mellitus. BACKGROUND: The cause of Bell's palsy in diabetes mellitus is not completely understood. Diabetic mononeuropathy or reactivation of herpes simplex virus type 1 (HSV-1) may be responsible for the facial paralysis seen in diabetic patients. We previously reported transient and ipsilateral facial paralysis in mice inoculated with HSV-1. In this study, we examined the neuropathogenesis of HSV-1 in diabetic mice to clarify the relationship between Bell's palsy and diabetes mellitus. METHODS: We compared the incidence and course of facial paralysis after HSV-1 inoculation in diabetic and nondiabetic mice groups. Diabetic mice were prepared by intraperitoneal streptozotocin injection. Facial nerve damage was assessed by electrophysiologic and histopathologic examinations. RESULTS: Compared with the nondiabetic group, the incidence of facial nerve paralysis was significantly increased in the diabetic mice. Electrophysiologic examinations and histopathologic changes also revealed that the facial nerve damage was more severe in the diabetic group. CONCLUSION: The aggravated course of HSV-1 infection in diabetes suggests that HSV-1 may be the main causative factor for the increased incidence of facial paralysis in diabetic patients.

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.

Application of flow cytometric in situ hybridization assay to Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.

Epstein-Barr virus (EBV) infects various types of lymphocytes and is associated with not only B cell-origin lymphoma, but also T or natural killer cell lymphoproliferative diseases (T/NK LPD). Recently, we established a novel assay to identify EBV-infected cells using FISH. Using this assay, dual staining with antibodies to both surface antigens and an EBV-encoded small RNA (EBER) probe can be performed. In the present study, we applied this recently developed FISH assay to EBV-associated T/NK LPD to confirm its diagnostic utility. Using FISH, we prospectively analyzed peripheral blood from patients with suspected EBV-associated T/NK LPD. The results were compared with those obtained using immunobead sorting followed by quantitative PCR. In all, 26 patients were included study. Using FISH, 0.15-67.0% of peripheral blood lymphocytes were found to be positive for EBER. Dual staining was used to determine EBER-positive cell phenotypes in 23 of 26 subjects (88.5%). In five of seven patients with hydroa vacciniforme-like lymphoma (an EBV-positive cutaneous T cell lymphoma), EBER-positive cells were identified as CD3(+) CD4(-) CD8(-) TCRγδ(+) T cells. Furthermore, in a 25-year-old male patient with systemic EBV-positive T cell LPD, two lymphocyte lineages were positive for EBER: CD4(+) CD8(-) and CD4(-) CD8(+) T cells. Thus, we confirmed that our newly developed assay is useful for quantifying and characterizing EBV-infected lymphocytes in EBV-associated T/NK LPD and that it can be used not only to complement the pathological diagnosis, but also to clarify the pathogenesis and to expand the spectrum of EBV-associated diseases.

Development of a human herpesvirus 6 species-specific immunoblotting assay.

In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

Herpes simplex virus requires poly(ADP-ribose) polymerase activity for efficient replication and induces extracellular signal-related kinase-dependent phosphorylation and ICP0-dependent nuclear localization of tankyrase 1.

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) which localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Poly(ADP-ribosyl)ation of the nuclear mitotic apparatus (NuMA) protein by tankyrase 1 during mitosis is essential for sister telomere resolution and mitotic spindle pole formation. In interphase cells, tankyrase 1 resides in the cytoplasm, and its role therein is not well understood. In this study, we found that herpes simplex virus (HSV) infection induced extensive modification of tankyrase 1 but not tankyrase 2. This modification was dependent on extracellular signal-regulated kinase (ERK) activity triggered by HSV infection. Following HSV-1 infection, tankyrase 1 was recruited to the nucleus. In the early phase of infection, tankyrase 1 colocalized with ICP0 and thereafter localized within the HSV replication compartment, which was blocked in cells infected with the HSV-1 ICP0-null mutant R7910. In the absence of infection, ICP0 interacted with tankyrase 1 and efficiently promoted its nuclear localization. HSV did not replicate efficiently in cells depleted of both tankyrases 1 and 2. Moreover, XAV939, an inhibitor of tankyrase PARP activity, decreased viral titers to 2 to 5% of control values. We concluded that HSV targets tankyrase 1 in an ICP0- and ERK-dependent manner to facilitate its replication.

Antitumor activities of valproic acid on Epstein-Barr virus-associated T and natural killer lymphoma cells.

Epstein-Barr virus (EBV), which infects B cells, T cells, and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, histone deacetylase (HDAC) inhibitors have been reported to have anticancer effects against various tumor cells. In the present study, we evaluated the killing effect of valproic acid (VPA), which acts as an HDAC inhibitor, on EBV-positive and -negative T and NK lymphoma cells. Treatment of multiple T and NK cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3 and KHYG1) with 0.1-5 mM of VPA inhibited HDAC, increased acetylated histone levels and reduced cell viability. No significant differences were seen between EBV-positive and -negative cell lines. Although VPA induced apoptosis in some T and NK cell lines (SNT16, Jurkat and KHYG1) and cell cycle arrest, it did not induce lytic infection in EBV-positive T or NK cell lines. Because the killing effect of VPA was modest (1 mM VPA reduced cell viability by between 22% and 56%), we tested the effects of the combination of 1 mM of VPA and 0.01 µM of the proteasome inhibitor bortezomib. The combined treated of cells with VPA and bortezomib had an additive killing effect. Finally, we administered VPA to peripheral blood mononuclear cells from three patients with EBV-associated T or NK lymphoproliferative diseases. In these studies, VPA had a greater killing effect against EBV-infected cells than uninfected cells, and the effect was increased when VPA was combined with bortezomib. These results indicate that VPA has antitumor effects on T and NK lymphoma cells and that VPA and bortezomib may have synergistic effects, irrespective of the presence of EBV.

Identification of Epstein-Barr virus-infected CD27+ memory B-cells in liver or stem cell transplant patients.

To analyse the phenotype of Epstein-Barr virus (EBV)-infected lymphocytes in EBV-associated infections, cells from eight haematopoietic stem cell/liver transplantation recipients with elevated EBV viral loads were examined by a novel quantitative assay designed to identify EBV-infected cells by using a flow cytometric detection of fluorescent in situ hybridization (FISH) assay. By this assay, 0.05-0.78% of peripheral blood lymphocytes tested positive for EBV, and the EBV-infected cells were CD20+ B-cells in all eight patients. Of the CD20+ EBV-infected lymphocytes, 48-83% of cells tested IgD positive and 49-100% of cells tested CD27 positive. Additionally, the number of EBV-infected cells assayed by using FISH was significantly correlated with the EBV-DNA load, as determined by real-time PCR (r2  = 0.88, P < 0.0001). The FISH assay enabled us to characterize EBV-infected cells and perform a quantitative analysis in patients with EBV infection after stem cell/liver transplantation.

Herpes simplex virus 1 protein kinase Us3 and major tegument protein UL47 reciprocally regulate their subcellular localization in infected cells.

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.

Herpes simplex virus type 1 UL14 tegument protein regulates intracellular compartmentalization of major tegument protein VP16.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) has a complicated life-cycle, and its genome encodes many components that can modify the cellular environment to facilitate efficient viral replication. The protein UL14 is likely involved in viral maturation and egress (Cunningham C. et al), and it facilitates the nuclear translocation of viral capsids and the tegument protein VP16 during the immediate-early phase of infection (Yamauchi Y. et al, 2008). UL14 of herpes simplex virus type 2 exhibits multiple functions (Yamauchi Y. et al, 2001, 2002, 2003). METHODS: To better understand the function(s) of UL14, we generated VP16-GFP-incorporated UL14-mutant viruses with either single (K51M) or triple (R60A, R64A, E68D) amino acid substitutions in the heat shock protein (HSP)-like sequence of UL14. We observed the morphology of cells infected with UL14-null virus and amino acid-substituted UL14-mutant viruses at different time points after infection. RESULTS: UL14(3P)-VP16GFP and UL14D-VP16GFP (UL14-null) viruses caused similar defects with respect to growth kinetics, compartmentalization of tegument proteins, and cellular morphology in the late phase. Both the UL14D-VP16GFP and UL14(3P)-VP16GFP viruses led to the formation of an aggresome that incorporated some tegument proteins but did not include nuclear-egressed viral capsids. CONCLUSIONS: Our findings suggest that a cluster of charged residues within the HSP-like sequence of UL14 is important for the molecular chaperone-like functions of UL14, and this activity is required for the acquisition of functionality of VP16 and UL46. In addition, UL14 likely contributes to maintaining cellular homeostasis following infection, including cytoskeletal organization. However, direct interactions between UL14 and VP16, UL46, or other cellular or viral proteins remain unclear.

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.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter.

Auditory and vestibular defects induced by experimental labyrinthitis following herpes simplex virus in mice.

CONCLUSION: Our herpes simplex virus (HSV) labyrinthitis mouse model suggests that HSV infection induces vestibular neuritis and sudden deafness. OBJECTIVE: Viral labyrinthitis has been postulated to play a role in vestibular neuritis and sudden deafness. We established a mouse model to investigate the pathogenesis of HSV-induced labyrinthitis. The relationship between HSV infection and apoptosis in the labyrinth was assessed. METHODS: HSV types 1 and 2 were inoculated into the middle ear of mice, and the function of the cochlear and vestibular nerves was assessed. Histopathological changes were examined with hematoxylin and eosin staining. Anti-HSV immunohistochemistry staining and TUNEL staining were done to investigate the relationship between HSV-infected cells and apoptotic cells. RESULTS: Hearing loss and vestibular dysfunction were observed in all mice after inoculation of HSV type 1 or 2. In the cochlear duct, columnar epithelial cells in the stria vascularis were infected with HSV, but only a portion of the infected cells underwent apoptosis. In contrast, many uninfected cells in the spiral organ of Corti were apoptotic. Vestibular dysfunction was observed when vestibular ganglion cells were largely infected, but not apoptotic. These findings recapitulate sudden deafness and vestibular neuritis described in patients.