Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein.

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

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.     

Homology between the glycoproteins of vesicular stomatitis virus and rabies virus

We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found.     

Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin.

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Inactivation of a human norovirus surrogate, human norovirus virus-like particles, and vesicular stomatitis virus by gamma irradiation

Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.     

PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses.

By employing improved techniques it has been possible to produce and characterize a representative spectrum of mammalian and primate retrovirus pseudotypes of vesicular stomatitis virus (VSV). Selection of appropriate cell lines for both the production and subsequent detection of the VSV pseudotypes has been the most important factor in permitting their demonstration. The host range for penetration of these retrovirus pseudotypes of VSV has been defined and found to differ from that reported for the replication of the corresponding retroviruses. Additionally, retroviruses having an identical host range for replication were distinguishable by differences in their host range for penetration, implying that restriction of replication may be occurring by different mechanisms. Studies of the plaque-forming efficiency of retrovirus pseudotypes of VSV in cell lines nonpermissive for replication of the corresponding retroviruses permitted a distinction to be made between the restriction of replication occurring as a consequence of postpenetration events and that occurring as a consequence of a block of penetration itself. The demonstration of primate retrovirus pseudotypes of VSV permits the use of VSV as a probe for the detection of this group of viruses.     

Infectivity-deficient vesicular stomatitis virus produced in the presence of interferon has a functional virion core

Interferon induces two antiviral actions against vesicular stomatitis virus by (i) inhibiting viral protein synthesis which leads to a reduction in virion production, and (ii) producing progeny which are deficient in infectivity (VSVIF). At low or physiological concentrations of interferon, while the virion production was decreased by less than 10-fold, the virion infectivity yield was suppressed more than 1000-fold. The VSVIF was found to be deficient (quantitatively) in envelop glycoprotein G and protein M. Tryptic peptide mapping indicated that there was no detectable structural abnormality in the G, M, and N proteins of VSVIF. The virion cores, lacking only the envelop G protein, isolated from VSVIF and control VSV have essentially identical specific infectivity. This indicated that the virion proteins L, N, NS, and M, as well as viral RNA that make up the virion core, must be functionally normal, and the observed deficiency in G protein was likely to be the cause of the functional deficiency of the virion. Low concentrations of DEAE-dextran, which is known to partially overcome the virion's dependence on the G protein for adsorption to the cell during infection, were found to enhance the infectivity of VSVIF more than the control virion. These results together indicated that the loss of infectivity in the VSVIF was due to the deficiency of the surface glycoprotein G.     

Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein.

MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mx1, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mx1, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.