The formation of vesicular stomatitis virus (San Juan strain) becomes temperature-sensitive when glucose residues are retained on the oligosaccharides of the glycoprotein

We have studied the effects of inhibiting the initial steps in processing of asparagine-linked oligosaccharides on the formation of vesicular stomatitis virus (VSV). Our data show that conditions which prevent the removal of glucose can block the growth of this virus. Our conclusion that inhibition of VSV synthesis is due specifically to an effect on the ability of the virus glycoprotein, G, to mature to a correct functional conformation is based on the following observations: (i) two drugs, deoxynojirimycin and castanospermine , both of which selectively inhibit the processing glucosidases, affected virus growth; (ii) only one of the two strains (San Juan and Orsay ) of VSV tested was affected and that strain, VSV(San Juan), is known to have a G protein highly sensitive to alterations in oligosaccharide structure; (iii) the effect was to make the formation of VSV(San Juan) temperature-sensitive, a result previously observed with alterations in the oligosaccharides on G protein; (iv) a cell variant missing glucosidase II activity also became temperature-sensitive in its ability to produce VSV(San Juan) but not VSV( Orsay ). Although inhibition of glucosidase activity by 1- deoxynojirimycin caused a 10-fold drop in virion formation, transport of G protein to the plasma membrane was not altered. The growth of VSV(San Juan) at 40 degrees C was not affected when subsequent steps in the processing pathway were blocked. These data indicate that by the time the glucose residues are removed G has attained a stable conformation.     

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.     

Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.

Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.     

Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.     

Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential

Brain tumors classified as glioblastomas have proven refractory to treatment and generally result in death within a year of diagnosis. We used seven in vitro tests and one in vivo trial to compare the efficacy of nine different viruses for targeting human glioblastoma. Green fluorescent protein (GFP)-expressing vesicular stomatitis (VSV), Sindbis virus, pseudorabies virus (PRV), adeno-associated virus (AAV), and minute virus of mice i-strain (MVMi) and MVMp all infected glioblastoma cells. Mouse and human cytomegalovirus, and simian virus 40 showed only low levels of infection or GFP expression. VSV and Sindbis virus showed strong cytolytic actions and high rates of replication and spread, leading to an elimination of glioblastoma. PRV and both MVM strains generated more modest lytic effects and replication capacity. VSV showed a similar oncolytic profile on U-87 MG and M059J glioblastoma. In contrast, Sindbis virus showed strong preference for U-87 MG, whereas MVMi and MVMp preferred M059J. Sindbis virus and both MVM strains showed highly tumor-selective actions in glioblastoma plus fibroblast coculture. VSV and Sindbis virus were serially passaged on glioblastoma cells; we isolated a variant, VSV-rp30, that had increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that four (VSV, Sindbis virus, MVMi, and MVMp) of the nine viruses studied merit further analysis for potential therapeutic actions on glioblastoma.     

Immune response in the absence of neurovirulence in mice infected with m protein mutant vesicular stomatitis virus

Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors.     

A mathematical model for cell cycle-specific cancer virotherapy

Oncolytic viruses preferentially infect and replicate in cancerous cells, leading to elimination of tumour populations, while sparing most healthy cells. Here, we study the cell cycle-specific activity of viruses such as vesicular stomatitis virus (VSV). In spite of its capacity as a robust cytolytic agent, VSV cannot effectively attack certain tumour cell types during the quiescent, or resting, phase of the cell cycle. In an effort to understand the interplay between the time course of the cell cycle and the specificity of VSV, we develop a mathematical model for cycle-specific virus therapeutics. We incorporate the minimum biologically required time spent in the non-quiescent cell cycle phases using systems of differential equations with incorporated time delays. Through analysis and simulation of the model, we describe how varying the minimum cycling time and the parameters that govern viral dynamics affect the stability of the cancer-free equilibrium, which represents therapeutic success.     

Molecular characterization of China human rabies vaccine strains

To understand the molecular characteristics of China human rabies vaccine strains, we report the full-length genome of the aG strain and present a comprehensive analysis of this strain and almost all available lyssavirus genomes (58 strains) from GenBank (as of Jan 6, 2011). It is generally considered that the G protein plays a predominant role in determining the pathogenicity of the virus, to this end we predicted the tertiary structure of the G protein of aG strain, CTN181 strain and wild type strain HN10 based on the crystal structure of Vesicular stomatitis virus (VSV) G. The predicted RABV G structure has a similar topology to VSV G and the ectodomain can be divided into 4 distinct domains DI — DIV. By mapping the characterized mutations to this structure between China vaccine strains and their close street strains, we speculate that the G303(P-H) mutations of CTN181 and HN10 causing DII 3D change may be associated with the II attenuated virulence in both strains. Specifically, the two signature mutations (G165P and G231P) in the aG strain are withinßsheets, suggesting that both sites are of structural importance.     

Replication-Competent Rhabdoviruses with Human Immunodeficiency Virus Type 1 Coats and Green Fluorescent Protein: Entry by a pH-Independent Pathway

We describe a replication-competent, recombinant vesicular stomatitis virus (VSV) in which the gene encoding the single transmembrane glycoprotein (G) was deleted and replaced by an env-G hybrid gene encoding the extracellular and transmembrane domains of a human immunodeficiency virus type 1 (HIV-1) envelope protein fused to the cytoplasmic domain of VSV G. An additional gene encoding a green fluorescent protein was added to permit rapid detection of infection. This novel surrogate virus infected and propagated on cells expressing the HIV receptor CD4 and coreceptor CXCR4. Infection was blocked by SDF-1, the ligand for CXCR4, by antibody to CD4 and by HIV-neutralizing antibody. This virus, unlike VSV, entered cells by a pH-independent pathway and thus supports a pH-independent pathway of HIV entry. Additional recombinants carrying hybrid env-G genes derived from R5 or X4R5 HIV strains also showed the coreceptor specificities of the HIV strains from which they were derived. These surrogate viruses provide a simple and rapid assay for HIV-neutralizing antibodies as well as a rapid screen for molecules that would interfere with any stage of HIV binding or entry. The viruses might also be useful as HIV vaccines. Our results suggest wide applications of other surrogate viruses based on VSV.     

Neutralized vesicular stomatitis virus binds to host cells by a different “receptor”

The addition of immune serum to vesicular stomatitis virus (VSV) results in the formation of VSV/antibody complexes which are non-infectious. Despite their being neutralized, these complexes still bind to host cells and are internalized at a rate equivalent to or greater than that observed for infectious virus. However, in contrast to infectious virus, the binding and uptake of neutralized VSV is not inhibited by phosphatidylserine and is partially sensitive to trypsin treatment. These results suggest that neutralized VSV binds to a different or additional cell binding site. By altering the VSV binding site, neutralizing antibodies may interfere with the fusogenic activity of G protein.     

Antibodies against the two serotypes of vesicular stomatitis virus measured by enzyme-linked immunosorbent assay: immunodominance of serotype-specific determinants and induction of asymmetrically cross-reactive antibodies.

The serological relationship between the two vesicular stomatitis virus (VSV) strains Indiana (VSV-Ind) and New Jersey (VSV-NJ) were analyzed by using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G responses, defined by their resistance to treatment with 2-mercaptoethanol, were assessed by ELISA by using sucrose gradient-purified VSV or purified VSV glycoproteins (G) as antigens. When low doses (10(6) PFU) of live VSV or 10(8) PFU of UV-inactivated virus were given intraperitoneally (i.p.), only non-cross-reactive antibody responses were observed in a primary immune response. However, when 10(6) PFU of live VSV were injected intravenously (i.v.), cross-reactive antibodies were generated; anti-VSV-NJ antibodies cross-reacted more against VSV-Ind than did anti-VSV-Ind antibodies against VSV-NJ. When 10(8) PFU of live VSV or UV-inactivated VSV mixed with complete Freund adjuvant was given i.p., high levels of cross-reactive antibodies detectable by ELISA were induced in primary and secondary responses. When purified G protein was used instead of purified whole virus in the ELISA, the cross-reactivity was found to be asymmetrical after immunization with live VSV given i.v. but not after i.p. inoculation; anti-VSV-NJ sera bound almost equally well to VSV-Ind G protein, whereas anti-VSV-Ind sera bound virtually exclusively to the G protein of the homologous serotype. The data suggest that immunization with VSV given i.p. results in a more specific, i.e., less cross-reactive, response than that either after i.v. infection or after the virus antigen is made available in great amounts or if it persists for prolonged periods when given i.p. together with complete Freund adjuvant. The unique determinants were immunodominant because they induced antibodies preferentially, whereas partially shared determinants induced antibody responses asymmetrically, more slowly, and with lower titers. Interestingly, the asymmetric cross-reactivity of anti-VSV antibodies, as measured by ELISA, against purified VSV G was opposite that observed for cytotoxic T cells.     

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.     

Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA.     

Basolateral protein transport in streptolysin O-permeabilized MDCK cells

We have reconstituted polarized protein transport in streptolysin O- permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross- linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin- 2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.     

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

人集落刺激因子-1(CSF-1)对常见呼吸道病毒的抑制作用

本文观察集落刺激因子-1(CSF-1)及其诱生剂:内毒素(LPS)、胞壁二肽(MDP)和干扰素(IFN-α)对下列病毒所致细胞病变的抑制,包括不同型别腺病毒5株,单疱病毒Ⅰ型和Ⅱ型,流感病毒A_3型,鼻病毒,ECHO11型各1株,在人胚肺传代细胞株上病变抑制的结果如下:对HSV-1、HSV-2、腺病毒6、11、22型,流感A_3型,鼻病毒。(CSF-1)和IFN-α,一样有明显抑毒效果,LPS MDP联合使用对以上病毒有明显增强抑毒作用。CSF-1和IFN-α的抑毒作用能分别被CSF-1和IFN-α的抗体所解除。 CSF-1在人胚肺传代细胞和人胚皮肤肌肉传代细胞上对VSV的抑毒效果在人胚肺细胞中效果比人胚皮肤肌肉细胞更明显。LPS10ng/ml作用48小时比作用24小时效果更强。LPS MDP和IFN-α对二种细胞都有同样高效的抑毒作用。     

Susceptibility of influenza viruses to interferon and to poly(I) . Poly(C) determined by the plaque reduction method

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) . poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) . poly(C) for influenza A and B viruses were as high as 3.0- to 4.5-fold and 6- to 18-fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0-6.2 and 7.3-15.2 units/ml, respectively. The ratio of PD50 of poly(I) . poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly(I) . poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) . poly(C).