Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

OBJECTIVES: Vesicular stomatitis virus (VSV) is under development as an oncolytic virus due to its preferential replication in cancer cells and oncolytic activity, however the viral components responsible have not yet been determined. In this study the effects of VSV wild-type (wt) and M51R-mutant matrix proteins (M51R-mMP) on apoptosis, pyroptosis, necroptosis, and autophagy pathways, in an esophagus cancer cell line (KYSE-30) were investigated. METHODS: The KYSE-30 cells were transfected with pcDNA3.1 plasmids encoding wt or M51R-mMP, and apoptosis, pyroptosis, necroptosis, and autophagy were evaluated 48 and 72 hours after transfection. RESULTS: KYSE-30 cells transfected with VSV wt and M51R-mMPs significantly reduced cell viability to < 50% at 72 hours post-transfection. M51R-MP significantly increased the concentration of caspase-8 and caspase-9 at 48 and 72 hours post-transfection, respectively ( p < 0.05). In contrast, no significant changes were detected following transfection with the VSV wt plasmid. Moreover, VSV wt and M51R-mMP transfected cells did not change the expression of caspase-3. VSV wt and M51R-mMPs did not mMP change caspase-1 expression (a marker of pyroptosis) at 48 and 72 hours post-transfection. However, M51R-mMP and VSV wt transfected cells significantly increased RIP-1 (a marker of necroptosis) expression at 72 hours post-infection ( p < 0.05). Beclin-1, a biomarker of autophagy, was also induced by transfection with VSV wt or M51R-mMPs at 48 hours post-transfection. CONCLUSION: The results in this study indicated that VSV exerts oncolytic activity in KYSE-30 tumor cells through different cell death pathways, suggesting that M51R-mMP may potentially be used to enhance oncolysis.
Apoptosis ; Autophagy ; Carcinoma, Squamous Cell ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cell Death ; Cell Line ; Cell Survival ; Epithelial Cells ; Esophageal Neoplasms ; Oncolytic Viruses ; Plasmids ; Pyroptosis ; Transfection ; Vesicular Stomatitis ; Viral Structures

Interferons (IFNs) have been known as antiviral genes and they are classified by type 1, type 2, and type 3 IFN. The type 1 IFN consists of IFNα, IFNβ, IFNτ, and IFNω whereas the type 2 IFN consists of only IFNγ, which is a key cytokine driving T helper cell type 1 immunity. IFNλ belongs to the type 3 IFN, which is also known as IL-28 and IL-29 possessing antiviral activities. Type 1 IFN is produced by viral infection whereas type 2 IFN is induced by mitogenic or antigenic T-cell stimuli. The IFNτ of bovine was first discovered in an ungulate ruminant recognition hormone. IFNτ belongs to the type 1 IFN with the common feature of type 1 IFN such as antiviral activity. IFNs have been mostly studied for basic research and clinical usages therefore there was no effort to investigate IFNs in industrial animals. Here we cloned porcine IFNα8 from peripheral blood mononuclear cells of Korean domestic pig (Sus scrofa domestica). The newly cloned IFNα8 amino acid sequence from Korean domestic pig shares 98.4% identity with the known porcine IFNα8 in databank. The recombinant porcine IFNα8 showed potent antiviral activity and protected bovine Madin-Darby bovine kidney epithelial (MDBK) cells from the cytopathic effect of vesicular stomatitis virus, but it failed to protect human Wistar Institute Susan Hayflick (WISH) cells and canine Madin-Darby canine kidney epithelial-like (MDCK) cells. The present study demonstrates species specific antiviral activity of porcine IFNα8.
Amino Acid Sequence ; Animals ; Clone Cells ; Humans ; Interferons ; Kidney ; Ruminants ; Sus scrofa ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer ; Vesicular Stomatitis

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.
Cell Fusion ; Cell Line ; Coculture Techniques ; Connexin 43 ; Cytarabine ; Gap Junctions ; Genetic Therapy ; Glycoproteins* ; Homicide* ; K562 Cells ; Leukemia, Myeloid, Acute ; Membrane Proteins ; Simplexvirus* ; Suicide ; Thymidine* ; Tretinoin ; Up-Regulation ; Vesicular Stomatitis*

To clone porcine bone marrow stromal antigen-2 (BST-2) gene, construct its recombinant eukaryotic expression plasmid and induce the expression of the fusion antiviral protein, we amplified BST-2 gene by RT-PCR from the total RNA extracted from PK15 cells. The recombinant expression plasmid pcDNA-BST-2 was constructed and then was transfected into HEK293T cells to expresse the BST-2 fusion protein. Western blot and indirect immunofluorescence assay (IFA) were performed, and the biological activity was detected. The results showed that the construction of recombinant plasmid pcDNA-BST-2 was confirmed by restriction enzyme digestion and sequencing. The expressed product had antiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) and Porcine reproductive and respiratory syndrome virus (PRRSV). In conclusion, the research paves the way for further research on bioactivity assayand antiviral medication.
Animals ; Antigens, CD ; genetics ; immunology ; Cell Line ; Chickens ; Cloning, Molecular ; Gene Expression ; Humans ; Influenza in Birds ; immunology ; virology ; Orthomyxoviridae ; physiology ; Porcine Reproductive and Respiratory Syndrome ; immunology ; virology ; Porcine respiratory and reproductive syndrome virus ; physiology ; Swine ; Vesicular Stomatitis ; immunology ; virology ; Vesicular stomatitis Indiana virus ; physiology ; Virus Replication

BACKGROUND: Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) and induces inflammation. In this study we attempted to ascertain if there are endogenous host molecules controlling the production of cytokines and chemokines. Two candidates, ribosomal protein L19 and L22, were analyzed to determine if they influence cytokine production followed by TLR3 activation. In this study we report that L19 acts upon production of IP-10 or IL-8 differently in glioblastoma cells. METHODS: L19 or L22 was transfected into HEK293-TLR3, A549 or A172 cells. After treatment with several inhibitors of NF-kB, PI3K, p38 or ERK, production of IL-8 or IP-10 was measured by ELISA. siRNA was introduced to suppress expression of L19. After Vesicular stomatitis virus infection, viral multiplication was measured by western blot. RESULTS: L19 increased ERK activation to produce IL-8. In A172 cells, in which TLR3 is expressed at endosomes, L19 inhibited interferon regulatory factor 3 (IRF3) activation and IP-10 production to facilitate viral multiplication, whereas L19 inhibited viral multiplication in A549 cells bearing TLR3 on their cell membrane. CONCLUSION: Our results suggest that L19 regulates TLR3 signaling, which is cell type specific and may be involved in pathogenesis of autoimmune diseases and chronic inflammatory diseases.
Autoimmune Diseases ; Chemokines ; Cytokines ; Endosomes ; Enzyme-Linked Immunosorbent Assay ; Glioblastoma ; Inflammation ; Interferon Regulatory Factor-3 ; Interleukin-8 ; NF-kappa B ; Ribosomal Proteins ; RNA, Double-Stranded ; RNA, Small Interfering ; Toll-Like Receptor 3 ; Ursidae ; Vesicular Stomatitis ; Viruses

In the last decade, we have gained significant understanding of the mechanism by which vesicular stomatitis virus (VSV) specifically kills cancer cells. Dysregulation of translation and defective innate immunity are both thought to contribute to VSV oncolysis. Safety and efficacy are important objectives to consider in evaluating VSV as a therapy for malignant disease. Ongoing efforts may enable VSV virotherapy to be considered in the near future to treat drug-resistant ovarian cancer when other options have been exhausted. In this article, we review the development of VSV as a potential therapeutic approach for recurrent or drug-resistant ovarian cancer.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Humans ; Neoplasm Recurrence, Local ; Oncolytic Virotherapy ; methods ; Ovarian Neoplasms ; pathology ; therapy ; virology ; Vesicular stomatitis Indiana virus ; physiology ; Virus Replication

OBJECTIVETo obtain the functional information of AY358935 gene.

METHODSThe properties, subcellular location, and structure of AY358935 protein, and the expression profile of AY358935 gene were analyzed by bioinformatics software and the biological functions of the gene were predicted. AY358935 expression was detected by Western blot analysis in early virus infection.

RESULTSAY358935 was evolutionally conserved. The human AY358935 protein had an amino acid similarity of 74%, 60%, 38% and 33% with its counterpart in horses, mice, zebrafish and Xenopus laevis, respectively. Bioinformatics analysis indicated that AY358935 protein was located likely in the mitochondria. There was a N-terminal signal peptide and single transmembrane structure in AY358935 protein, which contained several phosphorylation sites. The secondary structure mainly comprised of alpha helices and random coils. AY358935 was ubiquitously expressed in normal tissues and carcinomas and regulated by the expression of double-stranded RNA-dependent protein kinase. AY358935 protein expression was obviously upregulated in cells 2 h after infection by vesicular stomatitis virus.

CONCLUSIONAs a predicted secretary protein with a small molecular weight, AY358935 might have important functions in cellular proliferation and anti-viral innate immune regulation.

Amino Acid Sequence ; Chromosomes, Human, Pair 11 ; genetics ; Computational Biology ; methods ; Humans ; Molecular Sequence Data ; Proteins ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Software ; Vesicular Stomatitis ; metabolism

Equine interferon-gamma (eIFN-gamma) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-gamma. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1 x 10(3) AU/mL and 1 x 10(5) AU/mL of eIFN-gamma expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-gamma were highly efficient and specific, as it was blocked by mAbs against eIFN-gamma.
Animals ; Antibodies, Monoclonal ; immunology ; Antiviral Agents ; metabolism ; pharmacology ; Baculoviridae ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; metabolism ; Horses ; Interferon-gamma ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; Vesicular stomatitis Indiana virus ; drug effects ; metabolism

Transgenic chicken and oviduct bioreactor are growing to be one of the hotspot of scientific study in the field of biology. The most successful method of producing transgenic chicken is pseudotyped retrovirus vector system, but no one has reported the production of transgenic chicken by retrovirus system recently in our country. In order to accelerate our study in this field, we introduced the relevant technical methods such as packaging retrovirus and vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus, optimizing the conditions of packaging retrovirus, concentrating VSV-G pseudotyped retrovirus, helper virus assays, and microinjection of retrovirus. Furthermore, we successfully conducted in vivo study for detecting the marker gene EGFP of chicken embryo as well as in vitro study for detecting that gene of chicken embryo myoblast (CFM), thus we have provided an applied technical platform for studies of transgenic chicken in the future.
Animals ; Animals, Genetically Modified ; Chick Embryo ; Chickens ; genetics ; DNA Primers ; Genetic Vectors ; genetics ; Retroviridae ; genetics ; Vesicular stomatitis Indiana virus ; genetics