Protein Composition of the Structural Components of Vesicular Stomatitis Virus

Digitonin, a sterol glycoside which complexes with cholesterol, stripped off the envelope of vesicular stomatitis (VS) virions and liberated two viral structural proteins, 83% of P6 and 53% of P4. Deoxycholate also disrupted VS virions but released nucleocapsid cores which could be identified by higher buoyant density, ratio of incorporated (3)H-uridine to (14)C-protein, and electron microscopy. The major nucleocapsid protein was P5 but varying amounts of the minor protein aggregate P2 were present, depending on the concentration of urea used for extraction. P2 appeared to be a polymer of P5. Two other minor structural proteins, P1 and P3, could not be located in the virion. From these data, we conclude that the three microscopically identifiable structures of VS virions are each composed primarily of a single major protein, as follows: P6 = envelope protein, P4 = protein of underlying "shell," and P5 = nucleocapsid protein.     

Combined Administration with DNA Encoding Vesicular Stomatitis Virus G Protein Enhances DNA Vaccine Potency

DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8⁺ T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)—together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen—elicited robust E7-specific CD8⁺ T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.Due to their safety, low manufacturing cost, and ease of production, DNA vaccines have emerged as one of the most attractive approaches to prevent and treat viral infections, as well as cancers with defined tumor-associated antigens. Several studies have demonstrated that DNA vaccines can produce significant CD8⁺ T-cell-mediated immunity—which is often essential in the elimination of pathogens and malignancies—together with therapeutic benefit in various animal models of disease (4, 21-23, 29). However, DNA vaccines have achieved limited success in the clinic, since they generally elicit limited CD8⁺ T-cell responses in humans despite repeated high-dose administration (15, 28). Therefore, there is a critical need to develop DNA vaccines which generate these types of responses.DNA vaccines are commonly delivered by inoculation into skeletal muscle, where myocytes uptake them and express their encoded antigen. Myocytes, however, lack major histocompatibility complex (MHC) class II and costimulatory molecules and, as a result, have poor ability to prime naive T cells. Thus, the potency of DNA vaccines depends on sequential phases of antigen transfer from myocytes to sentinel dendritic cells (DCs), followed in close succession by the maturation of these DCs. The DCs then migrate to lymphoid organs, where they can present the antigen to and activate cognate naive CD8⁺ T cells (11). In the absence of either of these phases, either a null or a tolerogenic immune response is generated.One of the major challenges to the generation of CD8⁺ T-cell responses is therefore the design of vaccines that efficiently target antigen to and stimulate the maturation of DCs in a concerted manner. We hypothesized that this could be accomplished through the induction of an inflammatory form of myocyte death with concentrated antigen release to DCs upon DNA immunization. Viral fusogenic membrane glycoproteins (FMGs) represent ideal agents for eliciting such an effect. It has been shown that FMGs induce the fusion of cells into large multinucleated syncytia, which are lysed rapidly by nonapoptotic mechanisms (1) and in the process shed exosomes from the membrane (2). We reasoned that, since nonapoptotic death has been linked to stimulation of the innate immune system (25) and since exosomes have been reported to contain antigen that can be readily taken up by DCs (30), FMGs may bolster the release of antigens encoded by the DNA vaccine, leading to enhanced antigen-specific CD8⁺ T-cell responses.We have previously created and characterized a panel of DNA vaccines targeting the E7 oncoprotein of human papillomavirus (HPV) type 16, a model cervical cancer antigen. Because E7 is required for the maintenance of the transformed cellular phenotype and is expressed in virtually all cases of cervical cancers, it provides an ideal molecular target against which to apply our vaccination approach. In head-to-head comparisons of these vaccines, we showed that one particular construct, CRT/E7, could generate the most potent immunological and therapeutic effect against E7-expressing tumors (14). However, although CRT/E7 can cure mice with small tumors, it invariably fails to control more advanced forms of disease. In light of this obstacle, we sought to determine whether VSV-G could render CRT/E7 therapeutically effective against otherwise refractory E7-expressing tumors. We have previously characterized the growth kinetics of one such aggressive tumor model, TC-1, in mice and observed consistently that 1 week after subcutaneous challenge with 5 × 10⁵ cancerous cells, the mass becomes clearly detectable by palpation and visual inspection, and vaccination with CRT/E7—even with repeated high dose administration—has negligible beneficial effect on disease progression or animal survival. At this point, we considered the cancer to be sufficiently advanced, and this timeline was therefore adopted for the tumor treatment experiments in the present study.In the present study, we explored whether combined administration with VSV-G could successfully control the growth of aggressive TC-1 tumors refractory to treatment with CRT/E7 alone, and potential mechanisms by which this might be achieved. Collectively, our data indicate that incorporation of FMGs into DNA vaccines represents a therapeutically promising strategy for the generation of antigen-specific T-cell-mediated immunity against viral infections and cancer.     

Comparison of Membrane Protein Glycopeptides of Sindbis Virus and Vesicular Stomatitis Virus

A comparison has been made of the membrane glycoproteins and glycopeptides from two enveloped viruses, Sindbis virus and vesicular stomatitis virus (VSV). Glycopeptides isolated from Sindbis virus and VSV grown in the same host appear to differ principally in the number of sialic acid residues per glycopeptide; when sialic acid is removed by mild acid treatment, the glycopeptides of the two viral proteins are indistinguishable by exclusion chromatography. Preliminary evidence argues that the carbohydrate moiety covalently bound to different virus-specified membrane proteins may be specified principally by the host.     

Model for Vesicular Stomatitis Virus

Vesicular stomatitis virus contains single-stranded ribonucleic acid of molecular weight 3.6 x 10(6) and three major proteins with molecular weights of 75 x 10(3), 57 x 10(3), and 32.5 x 10(3). The proteins have been shown to be subunits of the surface projections, ribonucleoprotein, and matrix protein, respectively. From these values and from estimates of the proportions of the individual proteins, it has been calculated that the virus has approximately 500 surface projections, 1,100 protein units on the ribonucleoprotein strand, and 1,600 matrix protein units. Possible models of the virus are proposed in which the proteins are interrelated.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Infective Virus Substructure from Vesicular Stomatitis Virus

Treatment of suspensions of vesicular stomatitis virus with Tween-ether results in a rapid and considerable loss of infectivity (ca. 4 logs in 2 min), but the residual infectivity is comparatively stable to further treatment with ether. The infectivity remaining after the short exposure to Tween-ether is not due to virus for the following reasons. (i) It is much less infective for tissue cultures than for mice, whereas the intact virion is equally infective for both hosts. (ii) The residual infectivity is much less stable than virus infectivity in both sucrose and tartrate gradients. (iii) Virus immune serum does not neutralize its activity. (iv) The infectivity is associated with material which sediments further in sucrose gradients and has a greater buoyant density in tartrate gradients than the virion. Experiments with (32)P-labeled virion showed that the infective substructure contains ribonucleic acid with the same sedimentation characteristics as that extracted from the virion. Electron microscopy shows that the infective component has the same overall bullet-like structure as the virion but lacks the outer envelope and fringe structure.     

Carbohydrate Composition of Vesicular Stomatitis Virus

Analysis by gas-liquid chromatography of the trimethylsilylated sugar residues of purified vesicular stomatitis virus grown in L cells or chick embryo cells revealed the presence in the whole virion of four hexoses (glucose, galactose, mannose, and fucose), two hexosamines (glucosamine and galactosamine), and 34 to 40% neuraminic acid. The isolated viral glycoprotein was devoid of galactosamine and fucose, both of which sugars were present in whole virions presumably as part of the membrane glycolipids.     

Inhibition of Protein Synthesis in L Cells Infected with Vesicular Stomatitis Virus

The inhibition of protein synthesis in L cells by vesicular stomatitis virus (VSV) requires the synthesis of new protein subsequent to virus infection. However, two mechanisms may be involved in the inhibition of cell protein synthesis by VSV: an initial, multiplicity-dependent, ultraviolet-insensitive inhibition and a progressive, ultraviolet-sensitive inhibition.     

Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS₁, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.     

Differential Targeting of Vesicular Stomatitis Virus G Protein and Influenza Virus Hemagglutinin Appears During Myogenesis of L6 Muscle Cells

Exocytic organelles undergo profound reorganization during myoblast differentiation and fusion. Here, we analyzed whether glycoprotein processing and targeting changed during this process by using vesicular stomatitis virus (VSV) G protein and influenza virus hemagglutinin (HA) as models. After the induction of differentiation, the maturation and transport of the VSV G protein changed dramatically. Thus, only half of the G protein was processed and traveled through the Golgi, whereas the other half remained unprocessed. Experiments with the VSV tsO45 mutant indicated that the unprocessed form folded and trimerized normally and then exited the ER. It did not, however, travel through the Golgi since brefeldin A recalled it back to the ER. Influenza virus HA glycoprotein, on the contrary, acquired resistance to endoglycosidase H and insolubility in Triton X-100, indicating passage through the Golgi. Biochemical and morphological assays indicated that the HA appeared at the myotube surface. A major fraction of the Golgi-processed VSV G protein, however, did not appear at the myotube surface, but was found in intracellular vesicles that partially colocalized with the regulatable glucose transporter. Taken together, the results suggest that, during early myogenic differentiation, the VSV G protein was rerouted into developing, muscle-specific membrane compartments. Influenza virus HA, on the contrary, was targeted to the myotube surface.     

Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein

Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.     

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.