ABSTRACT
BACKGROUND: A major challenge in cervical cancer radiotherapy is tailoring the radiation doses efficiently to eliminate malignant cells and reduce the side effects in normal tissues. Oncolytic adenovirus drug H101 was recently tested and approved as a topical adjuvant treatment for several malignancies. OBJECTIVE: This study aimed to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 in combination with radiation in different cervical cancer cells. METHODS: Human cervical cancer cell lines C33a, SiHa, CaSki, and HeLa were treated with varying concentrations of H101 alone or in combination with radiation (2 Gy or 4 Gy). Cell viability and apoptosis were measured at the indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration was measured by qRT-PCR. In situ RNA scope was used to determine HPV E6 status. P53 protein alterations were detected by Western blot. RESULTS: Cell viability and apoptosis assays revealed that the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anticancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV-negative C33a cells (P<0.05). Low-HPV16-viral-load SiHa cells were more sensitive to the combination therapy than high-HPV16- viral-load CaSki cells (P<0.05). The combined treatment reduced HPV16 E6 expression and increased cellular P53 levels compared to those observed with radiation alone in SiHa and CaSki cells (P<0.05). CONCLUSION: Oncolytic adenovirus H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.
Subject(s)
Adenoviridae , Oncolytic Virotherapy/methods , Uterine Cervical Neoplasms/therapy , Adenoviridae/physiology , Adenoviridae/radiation effects , Apoptosis/radiation effects , Cell Line, Tumor , Combined Modality Therapy , Female , Gene Expression Regulation, Viral/radiation effects , Humans , Oncogene Proteins, Viral/genetics , Oncolytic Viruses/physiology , Oncolytic Viruses/radiation effects , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/virology , Repressor Proteins/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/radiotherapy , Uterine Cervical Neoplasms/virology , Viral Load , Virus ReplicationABSTRACT
Epstein-Barr virus, a ubiquitous human herpes virus with oncogenic activity, can be found in 6%-16% of gastric carcinomas worldwide. In Epstein-Barr virus-associated gastric carcinoma, only a few latent genes of the virus are expressed. Ionizing irradiation was shown to induce lytic Epstein-Barr virus infection in lymphoblastoid cell lines with latent Epstein-Barr virus infection. In this study, we examined the effect of ionizing radiation on the Epstein-Barr virus reactivation in a gastric epithelial cancer cell line (SNU-719, an Epstein-Barr virus-associated gastric carcinoma cell line). Irradiation with X-ray (dose = 5 and 10 Gy; dose rate = 0.5398 Gy/min) killed approximately 25% and 50% of cultured SNU-719 cells, respectively, in 48 h. Ionizing radiation increased the messenger RNA expression of immediate early Epstein-Barr virus lytic genes (BZLF1 and BRLF1), determined by real-time reverse transcription polymerase chain reaction, in a dose-dependent manner at 48 h and, to a slightly lesser extent, at 72 h after irradiation. Similar findings were observed for other Epstein-Barr virus lytic genes (BMRF1, BLLF1, and BcLF1). After radiation, the expression of transforming growth factor beta 1 messenger RNA increased and reached a peak in 12-24 h, and the high-level expression of the Epstein-Barr virus immediate early genes can convert latent Epstein-Barr virus infection into the lytic form and result in the release of infectious Epstein-Barr virus. To conclude, Ionizing radiation activates lytic Epstein-Barr virus gene expression in the SNU-719 cell line mainly through nuclear factor kappaB activation. We made a brief review of literature to explore underlying mechanism involved in transforming growth factor beta-induced Epstein-Barr virus reactivation. A possible involvement of nuclear factor kappaB was hypothesized.
Subject(s)
Epstein-Barr Virus Infections/radiotherapy , Herpesvirus 4, Human/genetics , Stomach Neoplasms/radiotherapy , Transcription Factor RelA/genetics , Transcription Factor RelB/genetics , Cell Line , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/virology , Gene Expression Regulation, Viral/radiation effects , Herpesvirus 4, Human/pathogenicity , Humans , Immediate-Early Proteins/genetics , Promoter Regions, Genetic , Radiation, Ionizing , Stomach Neoplasms/genetics , Stomach Neoplasms/virology , Trans-Activators/geneticsSubject(s)
Hemagglutinin Glycoproteins, Influenza Virus/genetics , Nicotiana/genetics , Tobamovirus/genetics , Gene Expression Regulation, Viral/radiation effects , Genetic Vectors/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Light , Plant Leaves/genetics , Plant Leaves/virology , Plants, Genetically Modified , Temperature , Nicotiana/virologyABSTRACT
Gamma irradiation is a non-thermal processing technique used to disinfect harmful microorganisms in agriculture. This technology has been shown to be an effective method to control bacterial and fungal plant pathogens. However, its effect on viral plant pathogen is less understood. Gamma irradiation was evaluated for the inactivation of tobacco mosaic virus (TMV). TMV infectivity has gradually decreased following irradiation in a dose-dependent manner and virus was completely inactivated at a dose over 40 kGy. Transmission electron microscopy revealed that increased gamma irradiation disrupts the virion structure and degrades viral proteins, which results in TMV inactivation. The mechanisms, through which gamma irradiation inactivates TMV, can be directly associated with the damage to the virus constituents.
Subject(s)
Capsid Proteins , Gamma Rays , Tobacco Mosaic Virus/radiation effects , Dose-Response Relationship, Radiation , Gene Expression Regulation, Viral/radiation effectsABSTRACT
BACKGROUND: The combined effects of Human papillomavirus (HPV) and Herpes simplex type 1 (HSV-1) infections and their effects on cancer cell radioresistance are unexplored. MATERIALS AND METHODS: An HPV16-positive hypopharyngeal carcinoma cell line (UD-SCC-2) was infected with wt-HSV-1 at low multiplicity of infection (MOI) and irradiated with 2 Gy at 24 h postinfection. Viability assays and quantitative reverse-transcriptase PCR for HPV16 E6, E7, nuclear factor kappa B1, B-cell CLL/lymphoma 2 (BCL2), and caspases 3, 8 and 9 at 24, and 72 h, as well as immunocytochemistry for BCL2, caspase 3, cyclin E, mouse double minute 2 homolog (MDM2), HSV-1 and Ki-67 were performed at 144 h postirradiation. RESULTS: At 144 h, cell viability was significantly lowered by irradiation only in uninfected cells. Infection combined with irradiation resulted in increased expression of E6, E7, BCL2 and NF-κB1 at 144 h. Simultaneously, E6 and E7 were down-regulated in non-irradiated infected cells. Irradiation and infection with 0.00001 MOI separately up-regulated caspase 3 but infection with 0.0001 MOI halved its expression in irradiated cells. CONCLUSION: HSV-1 infection modulates radioresistance of HPV16-positive hypopharyngeal carcinoma cells.
Subject(s)
Carcinoma, Squamous Cell/radiotherapy , Gamma Rays , Head and Neck Neoplasms/radiotherapy , Herpesvirus 1, Human/radiation effects , Human papillomavirus 16/radiation effects , Papillomavirus Infections/radiotherapy , Viral Load/radiation effects , Animals , Blotting, Western , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/virology , Gene Expression Regulation, Viral/radiation effects , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/virology , Humans , Immunoenzyme Techniques , Mice , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Optimal treatment strategies for primary central nervous system lymphoma (PCNSL) have not been established. In this study, we investigated the treatment outcomes and prognostic factors of high-dose methotrexate, vincristine, and procarbazine (MVP) chemotherapy followed by an interim response-adapted intensification strategy in immunocompetent patients with PCNSL. We evaluated the evidence of infection with Epstein-Barr virus (EBV) in both brain tumor tissue and whole blood. Forty patients were retrospectively reviewed. Ten (25 %) patients who achieved complete response (CR) in the interim analysis did not receive any additional consolidation treatment after completion of planned high-dose MVP chemotherapy. Additional radiotherapy (n = 9) or autologous stem cell transplantation (ASCT) (n = 7) was performed in patients who did not achieve CR in the interim analysis. The median age was 55 years. The overall CR rate was 62.5 % (n = 25), and the objective response rate was 75.0 %. Two-year overall survival (OS) was 59.8 %, and 2-year progression-free survival was 47.1 %. Grade 3 or 4 neutropenia and thrombocytopenia occurred in 47.5 and 32.5 % of patients, respectively. Treatment-related mortality was 15.0 % (n = 6), and four patients developed delayed neurotoxicity. There was no evidence of EBV-encoded RNA expression in brain tumor tissue. Ten (29.4 %) of 34 patients showed detectable EBV-DNA in whole blood. Poor performance status and EBV-DNA positivity in whole blood were significantly associated with inferior OS (p = 0.032, p = 0.023, respectively). We suggest that high-dose MVP chemotherapy followed by an early response-adapted intensification strategy may be effective and minimize the number of patients who receive radiotherapy or ASCT in the early course of treatment.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Brain Neoplasms/therapy , Epstein-Barr Virus Infections/therapy , Herpesvirus 4, Human , Lymphoma/therapy , Stem Cell Transplantation , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Autografts , Brain Neoplasms/metabolism , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Brain Neoplasms/virology , DNA, Viral/metabolism , Disease-Free Survival , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/mortality , Epstein-Barr Virus Infections/pathology , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/radiation effects , Humans , Lymphoma/metabolism , Lymphoma/mortality , Lymphoma/pathology , Lymphoma/virology , Male , Methotrexate/administration & dosage , Methotrexate/adverse effects , Middle Aged , Procarbazine/administration & dosage , Procarbazine/adverse effects , RNA, Viral/metabolism , Retrospective Studies , Survival Rate , Vincristine/administration & dosage , Vincristine/adverse effectsSubject(s)
Antigens, Viral/metabolism , Cell Phone , Gene Expression Regulation, Viral/radiation effects , Herpesvirus 4, Human/physiology , Herpesvirus 4, Human/radiation effects , Viral Proteins/metabolism , Antigens, Viral/genetics , Cell Line , Humans , Immunohistochemistry , Viral Proteins/geneticsABSTRACT
Cyprinid herpesvirus-3 (CyHV-3) is a member of the Alloherpesviridae, in the order Herpesvirales. It causes a fatal disease in carp and koi fish. The disease is seasonal and is active when water temperatures ranges from 18 to 28 °C. Little is known about how and where the virus is preserved between the permissive seasons. The hallmark of the herpesviruses is their ability to become latent, persisting in the host in an apparently inactive state for varying periods of time. Hence, it could be expected that CyHV-3 enter a latent period. CyHV-3 has so far been shown to persist in fish maintained under restrictive temperatures, while shifting the fish to permissive conditions reactivates the virus. Previously, we demonstrated that cultured cells infected with CyHV-3 at 22 °C and subsequently transferred to a restrictive temperature of 30 °C preserve the virus for 30 days. The present report shows that cultured carp cells maintained and exposed to CyHV-3 at 30 °C are abortively infected; that is, autonomous viral DNA synthesis is hampered and the viral genome is not multiplied. Under these conditions, 91 of the 156 viral annotated ORFs were initially transcribed. These transcripts were down-regulated and gradually shut off over 18 days post-infection, while two viral transcripts encoded by ORFs 114 and 115 were preserved in the infected cells for 18 days p.i. These experiments, carried out in cultured cells, suggest that fish could be infected at a high non-permissive temperature and harbor the viral genome without producing viral particles.
Subject(s)
Carps/virology , Down-Regulation , Gene Expression Regulation, Viral/radiation effects , Herpesviridae/physiology , Herpesviridae/radiation effects , Virus Latency/radiation effects , Animals , Cells, Cultured , Herpesviridae/genetics , Hot TemperatureABSTRACT
BACKGROUND: We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated. METHODS: Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8. RESULTS: Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin. CONCLUSIONS: Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.
Subject(s)
Genetic Vectors/radiation effects , Promoter Regions, Genetic , TATA Box , Transcriptional Activation/radiation effects , Animals , Dimethyl Sulfoxide/pharmacology , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/radiation effects , Genes, Transgenic, Suicide , HeLa Cells , Humans , Mannitol/pharmacology , Mice , Neoplasms, Experimental/metabolism , Oxidative Stress , Promoter Regions, Genetic/radiation effects , Protons , Retroviridae , TATA Box/genetics , TATA Box/radiation effects , Transcriptional Activation/genetics , X-RaysABSTRACT
PURPOSE: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. METHODS AND MATERIALS: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. RESULTS: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. CONCLUSIONS: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.
Subject(s)
Adenoviruses, Human/radiation effects , DNA Repair , Defective Viruses/radiation effects , Gene Expression/radiation effects , Genetic Therapy/methods , Genetic Vectors/radiation effects , Transgenes/radiation effects , Adenoviruses, Human/genetics , Cell Line, Tumor , Combined Modality Therapy/methods , DNA Breaks, Double-Stranded , Defective Viruses/genetics , Gene Expression/genetics , Gene Expression Regulation, Viral/genetics , Gene Expression Regulation, Viral/radiation effects , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/metabolism , Humans , Luciferases/genetics , Protein Kinase Inhibitors/pharmacology , Radiation Dosage , Topoisomerase I Inhibitors/pharmacology , Transgenes/genetics , Up-Regulation/genetics , Up-Regulation/radiation effects , Virus Replication/genetics , Virus Replication/radiation effectsABSTRACT
Human endogenous retroviruses (HERVs) represent a cellular reservoir of potentially pathogenic retroviral genes. A growing body of evidence indicates that the activation of endogenous retroviral sequences might be involved in the transformation of melanocytes. In this study, we investigated the effects of ultraviolet radiation (UVR) on the expression of human endogenous retrovirus type K (HERV-K) in melanoma cells and non-melanoma cells in vitro. Solely in melanoma cell lines, irradiation with UVB (200 mJ/cm(2)) resulted in a significant transcriptional activation of the retroviral pol gene as well as in an enhanced expression of the retroviral envelope protein (env). In addition, UVB treatment induced the production of retroviral particles in the supernatants of melanoma cell lines. These data indicate that HERV-K expression can be activated by UVB irradiation and suggest an involvement of HERV-K in UVR-related melanoma pathogenesis.
Subject(s)
Endogenous Retroviruses/genetics , Endogenous Retroviruses/radiation effects , Melanoma/virology , Skin Neoplasms/virology , Ultraviolet Rays , Base Sequence , Cell Line, Tumor , Gene Expression Regulation, Viral/radiation effects , Gene Products, pol/genetics , Gene Products, pol/metabolism , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptional Activation/radiation effects , Virion/metabolism , Virion/radiation effects , Virus Activation/radiation effectsABSTRACT
The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.
Subject(s)
DNA Replication/radiation effects , DNA, Viral/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Gene Expression Regulation, Viral/radiation effects , Herpesvirus 1, Human/physiology , Recombination, Genetic/radiation effects , Ultraviolet Rays , Virus Replication/radiation effects , Cell Line , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair/physiology , DNA Repair/radiation effects , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA Replication/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Gene Expression Regulation, Viral/physiology , Herpes Simplex/genetics , Herpes Simplex/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Poly-ADP-Ribose Binding Proteins , Rad52 DNA Repair and Recombination Protein/genetics , Rad52 DNA Repair and Recombination Protein/metabolism , Recombination, Genetic/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Virus Replication/physiology , Xeroderma Pigmentosum Group A Protein/genetics , Xeroderma Pigmentosum Group A Protein/metabolismABSTRACT
The Ski-interacting protein SKIP/SNW1 associates with the P-TEFb/CDK9 elongation factor and coactivates inducible genes, including HIV-1. We show here that SKIP also associates with c-Myc and Menin, a subunit of the MLL1 histone methyltransferase (H3K4me3) complex and that HIV-1 Tat transactivation requires c-Myc and Menin, but not MLL1 or H3K4me3. RNAi-ChIP experiments reveal that SKIP acts downstream of Tat:P-TEFb to recruit c-Myc and its partner TRRAP, a scaffold for histone acetyltransferases, to the HIV-1 promoter. By contrast, SKIP is recruited by the RNF20 H2B ubiquitin ligase to the basal HIV-1 promoter in a step that is bypassed by Tat and downregulated by c-Myc. Of interest, we find that SKIP and P-TEFb are dispensable for UV stress-induced HIV-1 transcription, which is strongly upregulated by treating cells with the CDK9 inhibitor flavopiridol. Thus, SKIP acts with c-Myc and Menin to promote HIV-1 Tat:P-TEFb transcription at an elongation step that is bypassed under stress.
Subject(s)
Gene Expression Regulation, Viral/physiology , Nuclear Receptor Coactivators/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins/metabolism , Transcriptional Activation/physiology , tat Gene Products, Human Immunodeficiency Virus/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cyclin T/genetics , Cyclin T/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , DNA Damage/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Flavonoids/pharmacology , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/radiation effects , HIV Long Terminal Repeat/physiology , HeLa Cells , Histone-Lysine N-Methyltransferase , Histones/metabolism , Humans , Methylation , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Receptor Coactivators/genetics , Piperidines/pharmacology , Positive Transcriptional Elongation Factor B/antagonists & inhibitors , Positive Transcriptional Elongation Factor B/metabolism , Promoter Regions, Genetic/physiology , Protein Binding/physiology , Protein Interaction Domains and Motifs/physiology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA, Small Interfering/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/radiation effects , Ubiquitin-Protein Ligases/genetics , Ultraviolet RaysABSTRACT
In a previous study of experimental murine encephalitis induced by Junín virus (JV), an arenavirus, we showed increased expression of iNOS by unidentified cells, concomitant with the astrocyte reaction. The specific inhibition of iNOS was associated with greater mortality but lower astrocytosis, suggesting that the protective role of nitric oxide (NO) synthesized by iNOS was related to enhanced astrocyte activation, representing a beneficial cellular response to virus-induced central nervous system damage. In the present work, cultured astrocytes were used to study whether JV infection could trigger iNOS expression and assess its eventual relationship with viral replication, glial fibrilary acidic protein (GFAP) expression levels and the presence of apoptosis. We found that JV infection of astrocytes did not induce apoptosis but produced both increased iNOS synthesis, detected by immunocytochemistry and fluorescence activated cell sorting (FACS) analysis, and increased NO, which was indirectly measured by nitrite/nitrate levels. These changes occurred early relative to the increases in GFAP expression, as detected by immunocytochemistry, FACS analysis and RT-PCR. The fact that iNOS inhibition abolished enhanced GFAP expression in infected monolayers suggests that NO was directly involved. In addition, iNOS inhibition enhanced virus replication. Together with data from confocal microscopy, these results suggest that JV induces iNOS expression in infected astrocytes and that the resulting NO has an important role both in reducing viral replication and in enhancing subsequent astrocyte activation.
Subject(s)
Astrocytes/metabolism , Astrocytes/virology , Junin virus/physiology , Animals , Animals, Newborn , Astrocytes/radiation effects , Brain/cytology , Cells, Cultured , Clobetasol/analogs & derivatives , Enzyme Inhibitors/pharmacology , Flow Cytometry/methods , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/radiation effects , Glial Fibrillary Acidic Protein/metabolism , Lysine/analogs & derivatives , Lysine/pharmacology , Mice , Mice, Inbred BALB C , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Virus Replication/physiologyABSTRACT
BACKGROUND: Photodynamic therapy (PDT) involves the use of a photosensitizing agent, which may require metabolic synthesis (i.e. a prodrug), followed by light activation. Numerous studies have advanced PDT as a means for treating bacteria, fungi and viruses. In this study, the photoinactivation of Herpes simplex virus type 1 (HSV-1) in human keratinocytes using 5-aminolaevulinic acid (5-ALA) was investigated. METHODS: HaCat cells were infected with HSV-1 and treated with 5-ALA to verify its antiviral effect during the stages of adsorption and penetration to host cells. Immunoblot analysis was used to estimate the effect of ALA-PDT on the production of viral proteins glycoprotein D (gD), infected cell proteins (ICP) 27 and virion protein (VP) 16. We also investigated whether the effect of ALA-PDT was associated with a cellular apoptotic mechanism through DNA fragmentation and the study of p53, PARP and caspase-3 protein expression. RESULTS: While the treatment of ALA-PDT after the viral adsorption period reduced HSV-1 replication by about 70%, it did not act on the virus in the first phase of infection. The viral proteins' expressions were reduced by ALA-PDT treatments. There was no evidence of ALA-PDT-induced apoptosis. CONCLUSION: Our data suggest that the target of photoinactivation appears to be viral replication and not a cellular response.
Subject(s)
Aminolevulinic Acid/pharmacology , Apoptosis/drug effects , Herpes Simplex/drug therapy , Herpesvirus 1, Human/metabolism , Photochemotherapy , Photosensitizing Agents/pharmacology , Prodrugs/pharmacology , Virus Replication/drug effects , Aminolevulinic Acid/therapeutic use , Apoptosis/radiation effects , Cell Line , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/radiation effects , Herpes Simplex/metabolism , Humans , Keratinocytes/virology , Photosensitizing Agents/therapeutic use , Prodrugs/therapeutic use , Viral Proteins/biosynthesis , Virus Replication/radiation effectsABSTRACT
PURPOSE: The Epstein Barr virus (EBV) is intimately associated with nasopharyngeal cancer (NPC) in a latent state expressing a limited number of genes. The process of switching from latency to replication is not well understood, particularly in response to DNA stress; hence, the focus of this study is on an EBV-positive NPC model. EXPERIMENTAL DESIGN: C666-1 cells were exposed to radiation (2-15 Gy) or cisplatin (0.1-50 microg/mL) assayed subsequently for relative EBV copy number (BamHI) and lytic gene expression (BRLF1 and BZLF1) using quantitative real-time PCR. Chromatin immunoprecipitation was conducted to assess the interaction of the transcription factor nuclear factor-Y (NF-Y) with promoter sequences. RESULTS: Radiation-induced and cisplatin-induced BamHI expression, along with increased levels of BRLF1 and BZLF1 in a dose-dependent and time-dependent manner, associated with the immediate nuclear transactivation of the transcription factor NF-Y and its own increased transcription of NF-Y subunits 8 h posttreatment. In silico analysis revealed three putative NF-Y consensus-binding sequences in the promoter region of BRLF1, which all interacted with NF-Y in response to radiation and cisplatin, confirmed using chromatin immunoprecipitation. Introduction of dominant-negative NF-YA reduced BRLF1 expression after radiation and cisplatin by 2.8-fold; in turn, overexpression of NF-YA resulted in a 2-fold increase in both BRLF1 and BZLF1 expression. CONCLUSIONS: These results show that NF-Y is an important mediator of EBV stress response in switching from a latent to lytic state. This novel insight could provide a potential therapeutic strategy to enhance NPC response to radiation and cisplatin.
Subject(s)
CCAAT-Binding Factor/physiology , Gene Expression Regulation, Viral , Nasopharyngeal Neoplasms/genetics , Nasopharyngeal Neoplasms/virology , Transcription Factors/physiology , Virus Activation/physiology , Virus Latency/physiology , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Cell Line, Tumor , Cisplatin/pharmacology , DNA-Binding Proteins/metabolism , Enzyme-Linked Immunosorbent Assay , Epstein-Barr Virus Infections/metabolism , Gamma Rays , Gene Expression/drug effects , Gene Expression/physiology , Gene Expression/radiation effects , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/radiation effects , Genes, Viral , Herpesvirus 4, Human/drug effects , Herpesvirus 4, Human/physiology , Herpesvirus 4, Human/radiation effects , Humans , Immediate-Early Proteins/metabolism , Immunoprecipitation , Mice , Mice, SCID , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/metabolism , Viral Proteins/metabolism , Virus Activation/drug effects , Virus Activation/radiation effects , Virus Latency/drug effects , Virus Latency/radiation effectsABSTRACT
The 3'-untranslated regions (UTRs) of human papillomavirus 16 (HPV16) and bovine papillomavirus 1 (BPV1) contain a negative regulatory element (NRE) that inhibits viral late gene expression. The BPV1 NRE consists of a single 9-nucleotide (nt) U1 small nuclear ribonucleoprotein (snRNP) base pairing site (herein called a U1 binding site) that via U1 snRNP binding leads to inhibition of the late poly(A) site. The 79-nt HPV16 NRE is far more complicated, consisting of 4 overlapping very weak U1 binding sites followed by a poorly understood GU-rich element (GRE). We undertook a molecular dissection of the HPV16 GRE and identify via UV cross-linking, RNA affinity chromatography, and mass spectrometry that is bound by the CUG-binding protein 1 (CUGBP1). Reporter assays coupled with knocking down CUGBP1 levels by small interfering RNA and Dox-regulated shRNA, demonstrate CUGBP1 is inhibitory in vivo. CUGBP1 is the first GRE-binding protein to have RNA interfering knockdown evidence in support of its role in vivo. Several fine-scale GRE mutations that inactivate GRE activity in vivo and GRE binding to CUGBP1 in vitro are identified. The CUGBP1.GRE complex has no activity on its own but specifically synergizes with weak U1 binding sites to inhibit expression in vivo. No synergy is seen if the U1 binding sites are made weaker by a 1-nt down-mutation or made stronger by a 1-nt up-mutation, underscoring that the GRE operates only on weak sites. Interestingly, inhibition occurs at multiple levels, in particular at the level of poly(A) site activity, nuclear-cytoplasmic export, and translation of the mRNA. Implications for understanding the HPV16 life cycle are discussed.
Subject(s)
3' Untranslated Regions/metabolism , Gene Expression Regulation, Viral/physiology , Human papillomavirus 16/genetics , RNA 3' Polyadenylation Signals/physiology , RNA, Viral/metabolism , 3' Untranslated Regions/genetics , Binding Sites/physiology , Bovine papillomavirus 1/genetics , Bovine papillomavirus 1/metabolism , CELF1 Protein , Gene Expression Regulation, Viral/radiation effects , HeLa Cells , Human papillomavirus 16/metabolism , Humans , Point Mutation , RNA 3' Polyadenylation Signals/radiation effects , RNA, Small Interfering/genetics , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , RNA, Viral/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ultraviolet RaysABSTRACT
Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.
Subject(s)
Immunity, Innate , Interferon Regulatory Factor-3/physiology , Sin Nombre virus/immunology , Sin Nombre virus/metabolism , Toll-Like Receptors/physiology , Virus Internalization , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , DEAD Box Protein 58 , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/physiology , Gene Expression Regulation, Viral/immunology , Gene Expression Regulation, Viral/radiation effects , Humans , Interferon Regulatory Factor-7/physiology , Interferons/physiology , Receptors, Immunologic , Receptors, Virus/physiology , Sin Nombre virus/pathogenicity , Sin Nombre virus/radiation effects , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 3/physiology , Ultraviolet Rays , Virion/immunology , Virus Replication/immunologyABSTRACT
Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the (299)Gly and (399)Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence ((299)Asp/(399)Thr), or the (299)Gly or (399)Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing (299)Gly or (399)Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-kappaB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-alpha. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus.
Subject(s)
Lipopolysaccharides/immunology , Lipopolysaccharides/metabolism , Polymorphism, Genetic/immunology , Respiratory Syncytial Viruses/immunology , Toll-Like Receptor 4/genetics , Bronchi/immunology , Bronchi/radiation effects , Bronchi/virology , Cell Line , Cell Membrane/genetics , Cell Membrane/immunology , Cell Membrane/metabolism , Cells, Cultured , Child , Gene Expression Regulation, Viral/immunology , Gene Expression Regulation, Viral/radiation effects , Glycine/genetics , Humans , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Isoleucine/genetics , Lipopolysaccharides/antagonists & inhibitors , Monocytes/cytology , Monocytes/immunology , Monocytes/metabolism , Mutagenesis, Site-Directed , Protein Transport/genetics , Protein Transport/immunology , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Respiratory Mucosa/radiation effects , Respiratory Mucosa/virology , Respiratory Syncytial Viruses/radiation effects , Toll-Like Receptor 4/physiology , Transfection , Ultraviolet RaysABSTRACT
Human cytomegalovirus (HCMV) establishes and maintains a latent infection in myeloid cells and can reactivate to cause serious disease in allograft recipients. To better understand the molecular events associated with the establishment of latency, we tracked the virus following infection of primary human myeloid progenitor cells at days 1, 2, 3, 5, and 11. At all time points, the viral genome was maintained in most cells at approximately 10 copies. Infectious virus was not detected, but virus could be reactivated by extended fibroblast coculture. In contrast to wild-type HCMV, the viral genome was rapidly lost from myeloid progenitors infected with ultraviolet (UV)-inactivated virus, suggesting viral gene expression was required for efficient establishment of latency. To identify viral genes associated with the establishment phase, RNA from each time point was interrogated using custom-made HCMV gene microarrays. Using this approach, we detected expression of viral RNAs at all time points. The pattern of expression differed from that which occurs during productive infection, and decreased over time. This study provides evidence that a molecular pathway into latency is associated with expression of a unique subset of viral transcripts. Viral genes expressed during the establishment phase may serve as targets for therapies to interrupt this process.