Vesicular Stomatitis Virus Matrix Protein Research Articles

The Role of Vesicular Stomatitis Virus Matrix Protein in Autophagy in the Breast Cancer.

Background:Breast cancer is one of the most difficult malignancies to treat. Therapeutics is used to target and kill the cancer cells. Non-human oncolytic viruses have the ability to cause cell death directly to cancers. The objective here was to investigate the role of Vesicular Stomatitis Virus (VSV) Matrix (M) protein in autophagy in the breast cancer cell line. Methods: Two different VSV wild type and mutant (M51R) M protein constructs were produced. Breast cancer cell line BT-20 was transfected by either wild type or mutant vectors. Transfection efficiency was measured using a fluorescent microscopy. Expression of VSV M protein was investigated at protein level. Cell cytotoxicity was measured using an MTT assay. The autophagy pathway was studied by Beclin-1 immunoassay. Data were statistically analyzed between different transfected groups. Results:It has been shown that the VSV M protein induced higher levels of Beclin-1 than the M51R mutant in the BT-20 cell line. Increased levels of Beclin-1 were also associated with VSV M cell-induced cytotoxicity. Conclusion:It has been shown here that VSV wild type or mutant M proteins can cause autophagy-induced cell death by increasing Beclin-1 expression. This includes the possible role of VSV to be used as an oncolytic virus in breast cancer treatment.

Polymer Vector Loading Vesicular Stomatitis Virus Matrix Protein Gene for Colon Cancer Therapy.

Gene therapy has been widely studied in colon cancer treatment. However, effective delivery of genes is a limitation for clinical applications. In the research, DOATP/mPEG-PLA-mPEG (DPLP) nanoparticles carrying the vesicular stomatitis virus matrix protein plasmid (pVSVMP) was used for colon cancer therapy, resulting in high transfection efficiency and expression efficiency in CT26 cells. Moreover, the DPLP-pVSVMP complex was provide with apoptosis induction and proliferation suppression of CT26 cells in vitro and can efficiently inhibit tumor growth in murine colon cancer model by inducing apoptosis, suppressing proliferation and angiogenesis. These results suggest that DPLP nanoparticles delivering pVSVMP might be a latent therapeutic avenue for colon cancer.

The VSV matrix protein inhibits NF-κB and the interferon response independently in mouse L929 cells

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22–20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22–20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein.

Cell Death Mechanisms in Esophageal Squamous Cell Carcinoma Induced by Vesicular Stomatitis Virus Matrix Protein.

ObjectivesVesicular 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.MethodsThe 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.ResultsKYSE-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.ConclusionThe 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.

Targeted Nanoparticle‐Mediated Gene Therapy Mimics Oncolytic Virus for Effective Melanoma Treatment

AbstractOncolytic virus has potential applications in cancer therapy. However, its clinical application is restricted by the virus‐associated biosafety issues. Here, inspired by the key role of vesicular stomatitis virus matrix protein (VSVMP) in the oncolytic vesicular stomatitis virus (VSV) induced apoptosis, a targeted nanoparticle‐delivered neutral VSVMP gene formulation is designed to act like the VSV for cancer therapy. This VSVMP formulation consists of a CRGDKGPDC peptide modified hybrid monomethoxy poly (ethylene glycol)‐poly(d,l‐lactide) nanoparticles complexed with VSVMP plasmid, having good blood compatibility and tumor targeting ability. The transfection efficiency is as high as that of VSV. After intravenous administration, the VSVMP formulation can efficiently target the tumor, significantly inhibit the melanoma growth and metastasis, prolong the survival time of tumor‐bearing mice, and does not cause obvious systemic toxicity. The anticancer mechanisms involve apoptosis induction, angiogenesis inhibition and some virus‐associated signal pathways activation. This work demonstrates a VSV‐inspired nonviral gene therapy that has promising clinical applications in melanoma treatment.

Delivery of modified mRNA encoding vesicular stomatitis virus matrix protein for colon cancer gene therapy.

Plasmid DNA based gene delivery has been widely utilized among both pre-clinical and clinical gene therapy studies. However, therapeutic efficiency is usually limited by the size and potential immune-stimulation issue of plasmid backbone. As an alternative form of genetic material, chemically modified messenger RNA (mRNA) provides a promising alternative to plasmid DNA. In this work, an in vitro transcription mRNA encoding vesicular stomatitis virus matrix protein (VSVMP) was delivered by a cationic liposome–protamine complex, resulting in high mRNA transporting and expression efficiency. The liposome–protamine complex delivered VSVMP mRNA strongly inhibits the growth of C26 tumor cells through inducing apoptosis, while obvious tumor regressions were achieved on both abdominal cavity metastatic and subcutaneous xenograft models in vivo with high safety. Our results also demonstrated that the liposome–protamine–mRNA complex was as potent as its plasmid DNA counterpart, showing strong potential in further colon cancer therapy.

Ovarian Cancer Therapy by VSVMP Gene Mediated by a Paclitaxel-Enhanced Nanoparticle.

Nanoparticles have great promise for gene delivery. However, the transfection efficiency of nanoparticle-based gene delivery systems is always unsatisfied to meet the requirement of effective gene therapy. Herein, we used low-dosage paclitaxel to enhance a nanoscaled gene delivery system that was self-assembled from N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate and monomethoxy poly(ethylene glycol)-poly(d,l-lactide) (DPP), creating a paclitaxel-encapsulated DPP (P-DPP) nanoparticle. The encapsulated low-dosage paclitaxel significantly improved the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells, in some of which the transfection efficiency is as high as 92%. By the P-DPP nanoparticle, vesicular stomatitis virus matrix protein (VSVMP) that could induce cell apoptosis was delivered to treat ovarian cancer. The encapsulation of paclitaxel in DPP nanoparticles increased the expression of VSVMP, enhancing VSVMP to induce antiproliferation and apoptosis in SKOV3 ovarian cancer cells. Intraperitoneal administration of P-DPP-delivered VSVMP effectively inhibited the intraperitoneal metastasis of SKOV3 ovarian cancer, which was more efficient than DPP-delivered VSVMP. Moreover, it was found that the tumor cell apoptosis induction, tumor cell proliferation inhibition, and tumor angiogenesis suppression were involved in the anticancer mechanism of this nanocomplex. Our data suggest that the encapsulation of low-dosage paclitaxel can enhance the gene delivery efficiency of the DPP nanoparticles against multiple cancer cells and exert a synergistic anticancer effect with VSVMP gene in ovarian cancer treatment. The VSVMP gene therapy delivered by the paclitaxel-enhanced nanoparticle has potential application in ovarian cancer therapy.

Curcumin promotes the oncoltyic capacity of vesicular stomatitis virus for the treatment of prostate cancers

Vesicular stomatitis virus (VSV) matrix (M) protein mutants have been studied as oncolytic agents due to their capacity to effectively kill cancer cells while exhibiting low virulence in vivo. Despite encouraging results, many cancer cells maintain resistance to oncolytic VSV mutants in part due to residual antiviral responses. We sought to determine whether combination of VSV with natural agents with anti-tumor properties, such as curcumin, resveratrol, and flavokavain B, would enhance tumor cell killing in a prostate cancer model. Our results revealed that pretreatment with curcumin potentiated VSV-induced oncolysis of PC-3 prostate cancer cells in cell culture and in a mouse model of prostate cancer. The ability of curcumin to synergize with VSV in PC-3 cells correlated with a cumulative decrease in the expression of the anti-apoptotic protein, Bcl-xl, and in the phosphorylation of NF-κB. Although curcumin did not impact the expression of type I IFN in infected cells, it inhibited the phosphorylation and activation of STAT1, a key player in the IFN response pathway, leading to an overall increase in virus-infected cells. These results suggest that curcumin sensitizes prostate cancer cells to the oncolytic effects of VSV by modulating antiviral responses and components of the intrinsic apoptotic pathway.

The vesicular stomatitis virus matrix protein inhibits NF-κB activation in mouse L929 cells

A previous study found that NF-κB activation is delayed in L929 cells infected with wild-type (wt) strains of VSV, while activation occurred earlier in cells infected with mutant strain T1026R1 (R1) that encodes a mutation in the cytotoxic matrix (M) protein. The integrity of the other R1 proteins is unknown; therefore our goal was to identify the viral component responsible for preventing NF-κB activation in L929 cells. We found that the M protein inhibits viral-mediated activation of NF-κB in the context of viral infection and when expressed alone via transfection, and that the M51R mutation in M abrogates this function. Addition of an IκB kinase (IKK) inhibitor blocked NF-κB activation and interferon-β mRNA expression in cells infected with viruses encoding the M51R mutation in M. These results indicate that the VSV M protein inhibits activation of NF-κB by targeting an event upstream of IKK in the canonical pathway.

VEGF-D-enhanced lymph node metastasis of ovarian cancer is reversed by vesicular stomatitis virus matrix protein.

Lymphatic metastasis is a poor prognostic factor in ovarian cancer, which correlates to the majority of cancer deaths. Matrix protein (MP) of vesicular stomatitis virus (VSV) exhibits potent antitumor and antiangiogenic activities through inducing apoptosis and inhibiting angiogenesis. In this study, the antitumor and antimetastatic effects of MP were further investigated. Wild-type SKOV3 (WT-SK) cells were successfully transfected with empty vector pcDNA3.1 plasmid, or pcDNA3.1-VEGF-D recombinant plasmid to construct cell lines named EV-SK, and VEGFD-SK, respectively. Inhibition of VEGFD-SK cell migration and invasion was detected by Transwell and wound healing assay. Then, lymphogenous metastatic model of ovarian cancer was established by injecting VEGFD-SK cells subcutaneously into the left hindlimb claw pad of nude mice. The inducted apoptotic effect of MP on VEGFD-SK cells were assessed by flow analysis and Hoechst-33258 staining, respectively, in vitro. The in vivo antitumor and antiangiogenic activities of MP gene were evaluated with lymphogenous metastatic model of ovarian cancer. Tumor volume and lymphatic metastasis rates were measured. Lymphatic vessels were delineated using Evan's blue and LYVE-1 staining. Expression of VEGF-D and MMP-2 were evaluated by immunostaining. Apoptosis of tumor cells was analyzed by Hoechst-33258 staining. Mice bearing VEGFD-SK tumor cells displayed more rapid tumorigenesis, higher lymphogenous metastatic tendency and increased lymphatic vessel density compared with the mice bearing WT-SK or EV-SK cells. However, VEGF-D-enhanced metastasis was evidently reversed by MP. MP significantly reduced the invasion of VEGFD-SK cells, tumor volume, lymphatic metastasis rates and lymphatic vessel density compared with control groups (P<0.05), accompanied with down-expression of VEGF-D and MMP-2 and increased apoptosis. Our data indicate that MP has strong antitumor and antimetastatic abilities, and it may be a promising therapeutic strategy against the lymphatic metastasis of human ovarian cancer.

Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2.

The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.

Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses.

Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

Complexes of Vesicular Stomatitis Virus Matrix Protein with Host Rae1 and Nup98 Involved in Inhibition of Host Transcription

Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.

Gene therapy using the human telomerase catalytic subunit gene promoter enables targeting of the therapeutic effects of vesicular stomatitis virus matrix protein against human lung adenocarcinoma

The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), is highly active in immortalized cells and more than 90% of human cancer cells, but is quiescent in the majority of normal somatic cells. Thus, the hTERT promoter has been extensively used in targeted cancer gene therapy. Vesicular stomatitis virus (VSV) matrix protein (MP) induces the apoptosis of tumor cells in the absence of other viral components. In our previous studies, we successfully constructed the pVAX-M plasmid from the pVAX plasmid, which expressed wild-type VSV MP (VSV MP is under the control of the CMV promoter) and demonstrated that pVAX-M efficiently suppresses the growth of malignant tumors via the induction of apoptosis in vitro and in vivo. The present study was designed to construct the plasmid phTERTM (VSV MP is under the control of the hTERT promoter) and investigate whether it had a targeted antitumor effect in nude mice bearing human lung adenocarcinoma. In vitro, A549 human lung adenocarcinoma cells were treated with NS, Lip-null, etoposide, Lip-pVAX-M or Lip-phTERT-M, and examined for cell viability through MTT assays or for apoptosis by flow cytometry and TUNEL assays. In vivo, A549 human lung carcinoma models in nude mice were established. Mice were treated with 10 4-weekly intravenous administrations of NS, Lip-null, etoposide (2 mg/kg), Lip-pVAX-M or Lip-phTERT-M. Subsequently, Lip-phTERT-M was found to be the most efficient inhibitor of tumor growth and inducer of tumor cell apoptosis when compared with the other groups in vivo and in vitro (P<0.05). Notably, immunohistochemical staining showed that Lip-phTERT-M significantly limited the overexpression of VSV MP to the tumor tissues and reduced VSV MP expression in other organs in comparison with Lip-pVAX-M (P<0.05). Therefore, it can be concluded that phTERT-M demonstrates a targeted antitumor effect on A549 human lung adenocarcinoma cells. These observations suggest that phTERT-M gene therapy may be a novel and potent strategy for targeting human lung adenocarcinoma.

Phenotypes of vesicular stomatitis virus mutants with mutations in the PSAP motif of the matrix protein

Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus assembly, and two motifs, (24)PPPY(27) and (37)PSAP(40), that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the (24)PPPY(27) motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the (37)PSAP(40) motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 [tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1)] through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants' phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.

Treatment of Lymphoma in Mice by Intravenous Administration of Vesicular Stomatitis Virus Matrix Protein Gene Encapsulated in Cationic Liposome

Abstract 4712 Background and Objective□FVesicular Stomatitis Virus (VSV) is an enveloped, negativesense RNA virus and prototypic member of the family rhabdoviridae. Some studies have shown that VSV can dissolve tumor cells with oncolytic effect. It has been reported that VSV can infect a variety of tumor cell lines such as glioma, melanoma, hepatocellular carcinoma, breast cancer and prostate cancer, and also demonstrated the antitumor effects on these tumors. Mechanism involving these effects was found that VSV can inhibit host gene expression, to induce cell rounding and to induce apoptosis. However, there is no report of such antitumor effect on lymphoma. Matrix Protein, the most important structural component of VSV, can also induce apoptosis of tumor cells even in absence of other component of VSV. Liposome is a promising non-viral gene vector, which can protect the DNA in vivo escape from degradation of the nuclease, and assist the DNA to penetrate the cell wall, together with the exogenous DNA into the cells. Cationic liposome has displayed high transfection efficiency, low cytotoxicity, no immunogenicity, and it also can improve the drug stability. In the present study, we established EL4 lymphoma model in mice, to explore the antitumor effects in vivo of a recombinant plasmid of pcDNA3.1-Matrix Protein of VSV complexed with cationic liposome in mice with EL4 lymphoma and potential mechanism. Methods□FThe plasmid DNA encoding Matrix Protein of VSV (P-M) has been constructed. Then the recombinant plasmid encapsulated with cationic liposomes, vectors of transfection, was generated. After the lymphoma model was established in sixty C57BL/6 mice by injecting EL4 tumor cells subcutaneouly into the right flank of mice, the mice bearing EL4 lymphoma were divided randomly into five groups including Lip-MP group, Lip-pVAX group, Lip group, ADM group and NS group, which were intravenously injected with liposome-pcDNA3.1 MP complex, liposome-pVAX complex, empty liposome, Adriamycin and normal saline, respectively, every three days in 3 weeks period of time. The side effects, tumor volumes and survival were monitered regularly. After the mice were sacrificed, microvessel density and tumor proliferation index in tumor tissues were determined by CD31, Ki-67 immunohistochemistry staining, meanwhile the tumor apoptosis index was measured by TUNEL method. Results□FSix days after treatments, the tumor volumes in Lip-MP group of C57BL/ 6 mice bearing EL4 lymphoma gradually became much smaller than those in Lip-pVAX, Lip and NS groups (P<0.05). At the 31st day after tumor cell injection, the tumor growth inhibition rates were (70.14±7.56)% in Lip-MP group, (21.67±7.66)% in Lip-pVAX group, (17.43±9.60)% in Lip group, (76.33±3.61)% in ADM group, respectively. The median survival of mice in Lip-MP group, 44 days after inoculation of tumor cells, was significantly longer than those in control groups (P<0.05), the median survival in Lip-pVAX, Lip and NS groups being 39 days, 38.5 days and 34 days, respectively. The MVD values in tumor tissues determined by CD31 staining were 11.38±2.92 in Lip-MP group, 20.13±3.40 in Lip-pVAX group, 27.50±3.16 in Lip group, 14.88±4.02 in ADM group and 33.25±3.11 in NS group, respectively. The MVD values in Lip-MP group were less than those in Lip-pVAX, Lip and NS groups (P<0.05). The proliferation index determined by Ki-67 staining in tumor tissues were (31.07±4.31)% in Lip-MP group, (42.45±4.04)% in Lip-pVAX group, (61.04±8.24)% in Lip group, (29.21±3.05)% in ADM group and (81.19±6.12)% in NS group, respectively. It revealed that Lip-MP complex apparently suppressed the proliferation of EL4 tumor cells in vivo(P<0.05). TUNEL assays showed that apoptosis index of tumor cells were 10.60±1.71 in Lip-MP group, 4.93±1.36 in Lip-pVAX group, 3.35±0.94 in Lip group, 4.51±1.10 in ADM group and 2.27±0.25 in NS group, respectively. The apoptosis index in Lip-MP group was higher than those in other control groups (P<0.05). Conclusions□FLip-MP complex, the plasmid encoding Matrix Protein of Vesicular Stomatitis Virus (VSV-MP) encapsulated in cationic liposome, may significantly inhibit the growth of lymphoma and prolong the survival of mice bearing EL4 lymphoma without any obvious side effects. Furthermore, inducing tumor cell apoptosis, inhibiting tumor angiogenesis, and suppressing tumor cell proliferation by Lip-MP complex may contribute to these antitumor effects in vivo. Disclosures:No relevant conflicts of interest to declare.

VSV-MP gene therapy strategy inhibits tumor growth in nude mice model of human lung adenocarcinoma

Vesicular stomatitis virus (VSV) matrix protein (MP) can induce in vitro apoptosis of tumor cells in the absence of other viral components. Here, the antitumor activity of VSV-MP against lung adenocarcinoma was investigated in vivo. A pVAX-plasmid DNA encoding VSV-MP and control empty vectors (pVAX) were constructed and wrapped-up with liposome. A549 and Spc-A1 human lung adenocarcinoma cells were transfected with liposomal-VSV-MP (Lip-MP) or Lip-pVAX and then examined for cell viability or apoptosis using Hoechst/propidium iodide staining by flow cytometry, and further demonstrated by caspase/poly ADP-ribose polymerase (PARP) cleavage analysis. For the in vivo study, A549 and Spc-A1 lung carcinoma models in nude mice were established and randomly assigned into three groups to receive eight 2-weekly intravenous administrations of medium alone as control, Lip-pVAX or Lip-MP, respectively. Subsequently, Lip-MP significantly reduced tumor growth and prolonged the survival of tumor-bearing mice compared with Lip-pVAX and control agents (P<0.05), with much higher apoptosis index of both in vivo and in vitro tumor cells, respectively (P<0.05). In addition, in vivo antitumoral effect was associated with natural killer-(NK) cell congregation without evidence of toxicity. These observations suggest that systemically delivering Lip-MP has a specific dual antitumor activity in human lung adenocarcinoma by inducing apoptosis and possibly stimulating NK-cell responses, it may provide a clue for developing new therapeutic approaches against human lung adenocarcinoma.

Vesicular stomatitis virus matrix protein (VSVMP) inhibits the cell growth and tumor angiogenesis in oral squamous cell carcinoma

The purpose of this study was to investigate the anticancer property of vesicular stomatitis virus matrix protein (VSVMP) in oral squamous cell carcinoma (OSCC) via in vitro and in vivo approaches. In this study, we found that OSCC cells treated with VSVMP showed retarded cell growth in vitro. The percentage of apoptotic cells in VSVMP group was much higher than that of the control groups. Moreover, our in vivo experiments showed that the growth of tumor xenografts was significantly suppressed by VSVMP treatment without any obvious side effects. Further studies revealed that the suppression of tumor growth may be caused by the synergistic effect of VSVMP related cell apoptosis enhancing and tumor angiogenesis suppression, and the latter is most likely correlated with the suppression of VEGF pathway. This study indicated that VSVMP treatment can effectively inhibited the cell growth and tumor angiogenesis in OSCC without obvious adverse effects. Therefore, VSVMP might be a potential and efficient strategy for OSCC treatment.

The protease-sensitive loop of the vesicular stomatitis virus matrix protein is involved in virus assembly and protein translation

To study the contribution of the protease-sensitive loop of the VSV M protein in virus assembly we recovered recombinant VSV (rVSV) with mutations in this region and examined virus replication. Mutations in the highly conserved LXD motif (aa 123–125) resulted in reduced virion budding, reduced virus titers and enhanced M protein exchange with M-ribonucleocapsid complexes (M-RNPs), suggesting that the mutant M proteins were less tightly associated with RNP skeletons. In addition, viral protein synthesis began to decrease at 4 h post-infection (hpi) and was reduced by ~ 80% at 8 hpi for the mutant rVSV-D125A. The reduced protein synthesis was not due to decreased VSV replication or transcription; however, translation of a reporter gene with an EMCV IRES was not reduced, suggesting that cap-dependent, but not cap-independent translation initiation was affected in rVSV-D125A infected cells. These results indicate that the LXD motif is involved in both virus assembly and VSV protein translation.

Proteomic Analyses of Gastric Cancer Cells Treated with Vesicular Stomatitis Virus Matrix Protein

Gastric cancer constitutes the second leading cause of mortality worldwide and the fourth most common cancer. While chemotherapy remains the primary treatment for both resectable and advanced gastric cancer, most gastric cancers are naturally resistant to anticancer drugs, rendering new therapeutic avenues in dire need. Vesicular stomatitis virus (VSV) was proved to preferentially replicate in many types of tumor cells and eventually induce apoptosis of host cells. The vesicular stomatitis virus matrix protein (MP) plays a major role in its effects. This study proved that expression of MP could effectively inhibit proliferation and induce cell death in gastric carcinoma MKN28 cells. Furthermore, we utilized a proteomics strategy to characterize proteome-wide alterations between MP-treated MKN28 lines and their untreated counterparts. A total of 97 spots were positively identified as differentially expressed, and of these 62 proteins were up-regulated, whereas 35 proteins were down-regulated. Functional analysis unraveled three significantly modified gene product subgroups: glycolytic enzymes, reactive oxygen species-associated proteins and the proteins regulating RNA transport and maturation. Expression of three altered proteins was further validated by semi-quantitative RT-PCR or/and western blotting. Furthermore, we demonstrated that MP expression could induce rapid intracellular ROS accumulation in MKN28 cells. These results provide evidence for the anti-cancer potential of MP, and a novel MP-mediated apoptotic signaling pathway is proposed. Our findings are considered a significant step toward a better understanding the mechanism of MP-induced anti-cancer effect.