Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway

The VP40 matrix protein of Ebola virus is able to bud from mammalian cells as a virus-like particle (VLP). Interactions between L-domain motifs of VP40 and host proteins such as Tsg101 and Nedd4 serve to facilitate budding of VP40 VLPs. Since intracellular levels of calcium are known to influence localization and function of host proteins involved in virus budding, we sought to determine, whether alterations of calcium or calmodulin levels in cells would affect budding of VP40 VLPs. VP40 VLP release was assessed in cells treated with BAPTA/AM, a calcium ion chelator, or with ionomycin, a calcium ionophore. In addition, VLP budding was assessed in cells treated with W7, W13, or TFP; all calmodulin antagonists. Results from these experiments indicated that: (i) budding of VP40 VLPs was reduced in a dose-dependent manner in the presence of BAPTA/AM, and slightly enhanced in the presence of ionomycin, (ii) VP40 VLP budding was reduced in a dose-dependent manner in the presence of W7, whereas VP40 VLP budding was unaffected in the presence of cyclosporine-A, (iii) budding of VSV-WT and a VSV recombinant (M40 virus) possessing the L-domains of Ebola VP40 was inhibited in the presence of W7, W13, or TFP, (iv) inhibition of virus budding by W7, W13, and TFP appears to be L-domain independent, and (v) the mechanism of calcium/calmodulin-mediated inhibition of Ebola VLP budding may involve the Ras/Raf/MEK/ERK signaling pathway.     

Phosphorylation of the HTLV-1 matrix L-domain-containing protein by virus-associated ERK-2 kinase

L-domain-containing proteins from animal retroviruses play a critical role in the recruitment of the host cell endocytic machinery that is required for retroviruses budding. We recently demonstrated that phosphorylation of the p6(gag) protein containing the L-domain of the human immunodeficiency virus type 1 regulates viral assembly and budding. Here, we investigated whether or not the L-domain-containing protein from another human retrovirus, namely the matrix protein of the human T-cell leukemia virus type 1, that contains the canonical PTAP and PPPY L-domain motifs, shares similar functional properties. We found that MA is phosphorylated at several sites. We identified one phosphorylated amino acid in the HTLV-1 MA protein as being S105, located in the close vicinity to the L-domain sequence. S105 phosphorylation was found to be mediated by the cellular kinase ERK-2 that is incorporated within HTLV-1 virus particles in an active form. Mutation of the ERK-2 target S105 residue into an alanine was found to decrease viral release and budding efficiency of the HTLV-1(ACH) molecular clone from transfected cells. Our data thus support the postulate that phosphorylation of retroviral L-domain proteins is a common feature to retroviruses that participates in the regulation of viral budding.     

A luciferase-based budding assay for Ebola virus

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.     

Double-layered membrane vesicles released from mammalian cells infected with Sendai virus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-M(VSV)). When cells were infected with SeV-M(VSV), VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (>/= 0.8 microm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M.     

Filovirus assembly and budding

Filoviruses belong to the order of negative-stranded non-segmented RNA viruses and are classified into two genera, Ebola and Marburg viruses. They have a characteristic filamentous shape, which is largely determined by the matrix protein VP40. Although VP40 is the main driving force for assembly and budding from the host cell, the production of infectious virus involves an intricate interplay between all viral structural proteins in addition to cellular factors, e.g., those that normally function in multi-vesicular body biogenesis. As a consequence, assembly and budding steps are defined to specific cellular compartments, and the recent progress in understanding how the different components are assembled into stable enveloped virus particles is reviewed.     

A novel method for analysis of membrane microdomains: vesicular stomatitis virus glycoprotein microdomains change in size during infection,and those outside of budding sites resemble sites of virus budding

Membrane proteins, including viral envelope glycoproteins, may be organized into areas of locally high concentration, commonly referred to as membrane microdomains. Some viruses bud from detergent-resistant microdomains referred to as lipid rafts. However, vesicular stomatitis virus (VSV) serves as a prototype for viruses that bud from areas of plasma membrane that are not detergent resistant. We developed a new analytical method for immunoelectron microscopy data to determine whether the VSV envelope glycoprotein (G protein) is organized into plasma membrane microdomains. This method was used to quantify the distribution of the G protein in microdomains in areas of plasma membrane that did not contain budding sites. These microdomains were compared to budding virus envelopes to address the question of whether G protein-containing microdomains were formed only at the sites of budding. At early times postinfection, most of the G protein was organized into membrane microdomains outside of virus budding sites that were approximately 100-150 nm, with smaller amounts distributed into larger microdomains. In contrast to early times postinfection, the increased level of G protein in the host plasma membrane at later times postinfection led to distribution of G protein among membrane microdomains of a wider variety of sizes, rather than a higher G protein concentration in the 100- to 150-nm microdomains. VSV budding occurred in G protein-containing microdomains with a range of sizes, some of which were smaller than the virus envelope. These microdomains extended in size to a maximum of 300-400 nm from the tip of the budding virion. The data support a model for virus assembly in which G protein organizes into membrane microdomains that resemble virus envelopes prior to formation of budding sites, and these microdomains serve as the sites of assembly of internal virion components.     

Neurovirulence of influenza virus in mice I. Neurovirulence of recombinants between virulent and avirulent virus strains

The neurovirulence of influenza A/WSN (HONI) virus in mice was studied using recombinants between neurovirulent WSN and nonneurovirulent A/Hong Kong (HK, H3N2) viruses. Parental derivation of genes in recombinants were analyzed by the electrophoresis of viral RNA in urea-polyacrylamide gel. The neurovirulence was tested by intracerebral inoculation of recombinants into mice. It was found that five large genes, P₃, P₁, P₂, HA, and NP, were not essential for the virulence, because recombinants having these five genes derived from HK parent were virulent. Recombinants in which any of three remaining WSN genes, NA, M, and NS, was replaced with the one from HK parent, failed to kill mice. Therefore, these three genes were responsible for the difference of neurovirulence between the two virus strains. However, when tested in mice immunosuppressed by the administration of cyclophosphamide, recombinants containing either M or NS protein from HK parent were virulent, but viruses containing HK neuraminidase were still avirulent. Viruses containing HK neuraminidase appeared incapable of multiplying in the mouse brain, while those containing either M or NS protein derived from HK virus multiplied to a limited extent. It was suggested that WSN neuraminidase was the principal determining factor of the neurovirulence of WSN virus, without which no virus multiplication occurred, while M and probably NS proteins of WSN virus played a role of helper or accessory virulence factor(s), enabling the efficient virus replication.     

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.     

Parvoviral target cell specificity: acquisition of fibrotropism by a mutant of the lymphotropic strain of minute virus of mice involves multiple amino acid substitutions within the capsid

Unlike the prototype strain of minute virus of mice, MVM(p), the lymphotropic strain, MVM(i), cannot form plaques on monolayers of mouse A9 fibroblasts. At very low frequency, mutants arise in MVM(i) stocks which are able to plaque on A9 cells, and we report here the isolation and mapping of such a mutant, designated hr101. Analysis of intratypic recombinants containing regions of the hr101 genome substituted into the infectious clone of its parent MVM(i) shows that the ability to form plaques on fibroblast monolayers maps to the same small region of the coat protein gene which we had previously shown, by constructing intertypic recombinants, to contain the fibrotropic determinant of MVM(p) (Gardiner and Tattersall, J. Virol. 62, 2605-2613, 1988). DNA sequencing of the hr101 regions in virus stocks derived from these recombinants identified four single-base changes between the mutant coat protein gene and that of its parent. Each of these changes occurs in the same position as a similar change found between MVM(i) and MVM(p), and each of them change the amino acid encoded at that position. Three of the four changes substitute the same amino acid as found in MVM(p), and the fourth change substitutes an alanine in hr101 for a glutamic acid residue in MVM(i), in a position occupied by glycine in MVM(p). Analysis of the recombinants within this region shows that plaque formation on A9 monolayers is dependent upon the latter change plus one adjacent, MVM(p)-like change. This observation was confirmed by recreating this double mutant in the infectious clone of MVM(i) via site-directed mutagenesis. In addition to extending the host range of MVM(i) into A9 fibroblasts, the hr101 mutations have a complex effect on the virus' ability to grow lytically in a series of different T-lymphocyte cell lines.     

Vesicular stomatitis virus as an oncolytic vector

Recent data has shown that viruses such as vesicular stomatitis virus (VSV), a relatively non-pathogenic, negative-stranded RNA virus, can preferentially replicate in malignant cells and less so in normal cells. VSV appears able to carry out this function in transformed cells since these hosts exhibit the hallmarks of flawed host defense, probably involving the interferon system, which is essential for preventing virus replication. The simple genetic constitution of VSV, lack of any known transforming, integrating or reassortment properties, extensive knowledge relating to its interaction with the immune system and the ability to genetically manipulate this agent affords an ideal opportunity to exploit the oncolytic and gene targeting potential of this innocuous virus. Thus, aside from preferentially targeting malignant cells VSV recombinants could be generated that could increase a tumor's susceptibility to chemotherapeutic agents and/ or importantly, the host immune response. Collectively, our data and others demonstrate that VSV as well as other RNA viruses could provide a promising and exciting approach to cancer therapy.     

Paramyxovirus Sendai virus C proteins are essential for maintenance of negative-sense RNA genome in virus particles

The Sendai virus (SeV) C proteins are a nested set of four accessory proteins, C', C, Y1, and Y2, encoded on the P mRNA from an alternate reading frame. The C proteins are multifunctional proteins involved in viral pathogenesis, inhibition of viral RNA synthesis, counteracting the innate immune response of the host cell, inhibition of virus-induced apoptosis, and facilitating virus-like particle (VLP)/virus budding. Among these functions, the roles for pathogenesis and counteracting host cell interferon (IFN) responses have been studied extensively, but the others are less well understood. In this paper, we found that the C proteins contributed in many ways to the efficient production of infectious virus particles by using a series of SeV recombinants without one or more C protein expression. Knockout of both C' and C protein expression resulted in reduced virus release despite higher viral protein synthesis in the cells. Interestingly, for the viruses without C' and C, or all four C protein expression, non-infectious virions containing antigenomic RNAs were produced predominantly compared to genomic RNA-containing infectious virions, due to aberrant viral RNA synthesis. Our results demonstrate for the first time that the C proteins regulate balance of viral genome and antigenome RNA synthesis for efficient production of infectious virus particles in the course of virus infection.     

Matrix protein of VSV New Jersey serotype containing methionine to arginine substitutions at positions 48 and 51 allows near-normal host cell gene expression

The matrix (M) protein of vesicular stomatitis virus (VSV) plays significant roles in the replication of VSV through its involvement in the assembly of virus particles as well as by facilitating the evasion of innate host cell defense mechanisms. The presence of methionine at position 51 (M51) of the matrix (M) protein of the VSV Indiana serotype (VSV(Ind)) has been proven to be crucial for cell rounding and inhibition of host cell gene expression. The M protein of VSV(Ind) with the substitution of M51 with arginine (R:M51R) results in the loss of inhibitory effects on host cell gene expression. The VSV(Ind) expressing the M(M51R) protein became the attractive oncolytic virus which is safer and more tumor-specific because the normal cells can clear the mutant VSV(Ind) easily but tumor cells are susceptible to the virus because a variety of tumor cells lack innate antiviral activities. We have studied the role of the methionines at positions 48 and 51 of the M protein of the New Jersey serotype of VSV (VSV(NJ)) in the induction of cytopathic effects (CPE) and host cell gene expression. We have generated human embryonic kidney 293 cell lines inducibly expressing M proteins with M to R mutations at positions 48 and 51, either separately or together as a double mutant, and examined expression of heat shock protein 70 (HSP70) as an indicator of host cell gene expression. We have also generated recombinant VSV(NJ) encoding the mutant M proteins M(M48R) or M(M48R+M51R) for the first time and tested for the expression of HSP70 in infected cells. Our results demonstrated that the M51 of VSV(NJ) M proteins has a major role in cell rounding and in suppressing the host cell gene expression either when the M protein was expressed alone in inducible cell lines or when expressed together with other VSV proteins by the recombinant VSV(NJ). Amino acid residue M48 may also have some role in cell rounding and in the inhibitory effects of VSV(NJ) M, which was demonstrated by the fact that the cell line expressing the double substitution mutant M(M48R+M51R) exhibited the least cytopathic effects and the least inhibitory effect on host cell gene expression.     

Productive replication of Ebola virus is regulated by the c-Abl1 tyrosine kinase

Ebola virus causes a fulminant infection in humans resulting in diffuse bleeding, vascular instability, hypotensive shock, and often death. Because of its high mortality and ease of transmission from human to human, Ebola virus remains a biological threat for which effective preventive and therapeutic interventions are needed. An understanding of the mechanisms of Ebola virus pathogenesis is critical for developing antiviral therapeutics. Here, we report that productive replication of Ebola virus is modulated by the c-Abl1 tyrosine kinase. Release of Ebola virus-like particles (VLPs) in a cell culture cotransfection system was inhibited by c-Abl1-specific small interfering RNA (siRNA) or by Abl-specific kinase inhibitors and required tyrosine phosphorylation of the Ebola matrix protein VP40. Expression of c-Abl1 stimulated an increase in phosphorylation of tyrosine 13 (Y(13)) of VP40, and mutation of Y(13) to alanine decreased the release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain was inhibited by c-Abl1-specific siRNAs or by the Abl-family inhibitor nilotinib by up to four orders of magnitude. These data indicate that c-Abl1 regulates budding or release of filoviruses through a mechanism involving phosphorylation of VP40. This step of the virus life cycle therefore may represent a target for antiviral therapy.     

Development of an HIV vaccine using a vesicular stomatitis virus vector expressing designer HIV-1 envelope glycoproteins to enhance humoral responses

Vesicular stomatitis virus (VSV), like many other Rhabdoviruses, have become the focus of intense research over the past couple of decades based on their suitability as vaccine vectors, transient gene delivery systems, and as oncolytic viruses for cancer therapy. VSV as a vaccine vector platform has multiple advantages over more traditional viral vectors including low level, non-pathogenic replication in diverse cell types, ability to induce both humoral and cell-mediate immune responses, and the remarkable expression of foreign proteins cloned into multiple intergenic sites in the VSV genome. The utility and safety of VSV as a vaccine vector was recently demonstrated near the end of the recent Ebola outbreak in West Africa where VSV pseudotyped with the Ebola virus (EBOV) glycoprotein was proven safe in humans and provided protective efficacy against EBOV in a human phase III clinical trial. A team of Canadian scientists, led by Dr. Gary Kobinger, is now working with International AIDS Vaccine Initiative (IAVI) in developing a VSV-based HIV vaccine that will combine unique Canadian research on the HIV-1 Env glycoprotein and on the VSV vaccine vector. The goal of this collaboration is to develop a vaccine with a robust and potent anti-HIV immune response with an emphasis on generating quality antibodies to protect against HIV challenges.     

Assembly of nucleocapsids with cytosolic and membrane-derived matrix proteins of vesicular stomatitis virus.

During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid-M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Assembly and budding of influenza virus

Influenza viruses are causative agents of an acute febrile respiratory disease called influenza (commonly known as "flu") and belong to the Orthomyxoviridae family. These viruses possess segmented, negative stranded RNA genomes (vRNA) and are enveloped, usually spherical and bud from the plasma membrane (more specifically, the apical plasma membrane of polarized epithelial cells). Complete virus particles, therefore, are not found inside infected cells. Virus particles consist of three major subviral components, namely the viral envelope, matrix protein (M1), and core (viral ribonucleocapsid [vRNP]). The viral envelope surrounding the vRNP consists of a lipid bilayer containing spikes composed of viral glycoproteins (HA, NA, and M2) on the outer side and M1 on the inner side. Viral lipids, derived from the host plasma membrane, are selectively enriched in cholesterol and glycosphingolipids. M1 forms the bridge between the viral envelope and the core. The viral core consists of helical vRNP containing vRNA (minus strand) and NP along with minor amounts of NEP and polymerase complex (PA, PB1, and PB2). For viral morphogenesis to occur, all three viral components, namely the viral envelope (containing lipids and transmembrane proteins), M1, and the vRNP must be brought to the assembly site, i.e. the apical plasma membrane in polarized epithelial cells. Finally, buds must be formed at the assembly site and virus particles released with the closure of buds. Transmembrane viral proteins are transported to the assembly site on the plasma membrane via the exocytic pathway. Both HA and NA possess apical sorting signals and use lipid rafts for cell surface transport and apical sorting. These lipid rafts are enriched in cholesterol, glycosphingolipids and are relatively resistant to neutral detergent extraction at low temperature. M1 is synthesized on free cytosolic polyribosomes. vRNPs are made inside the host nucleus and are exported into the cytoplasm through the nuclear pore with the help of M1 and NEP. How M1 and vRNPs are directed to the assembly site on the plasma membrane remains unclear. The likely possibilities are that they use a piggy-back mechanism on viral glycoproteins or cytoskeletal elements. Alternatively, they may possess apical determinants or diffuse to the assembly site, or a combination of these pathways. Interactions of M1 with M1, M1 with vRNP, and M1 with HA and NA facilitate concentration of viral components and exclusion of host proteins from the budding site. M1 interacts with the cytoplasmic tail (CT) and transmembrane domain (TMD) of glycoproteins, and thereby functions as a bridge between the viral envelope and vRNP. Lipid rafts function as microdomains for concentrating viral glycoproteins and may serve as a platform for virus budding. Virus bud formation requires membrane bending at the budding site. A combination of factors including concentration of and interaction among viral components, increased viscosity and asymmetry of the lipid bilayer of the lipid raft as well as pulling and pushing forces of viral and host components are likely to cause outward curvature of the plasma membrane at the assembly site leading to bud formation. Eventually, virus release requires completion of the bud due to fusion of the apposing membranes, leading to the closure of the bud, separation of the virus particle from the host plasma membrane and release of the virus particle into the extracellular environment. Among the viral components, M1 contains an L domain motif and plays a critical role in budding. Bud completion requires not only viral components but also host components. However, how host components facilitate bud completion remains unclear. In addition to bud completion, influenza virus requires NA to release virus particles from sialic acid residues on the cell surface and spread from cell to cell. Elucidation of both viral and host factors involved in viral morphogenesis and budding may lead to the development of drugs interfering with the steps of viral morphogenesis and in disease progression.     

Assembly of membrane glycoproteins studied by phenotypic mixing between mutants of vesicular stomatitis virus and retroviruses

Temperature sensitive (ts) mutations of vesicular stomatitis virus (VSV), Indiana serotype, which belong to complementation group V (tsV) have been shown to affect the viral envelope glycoprotein, or G protein. When ts V mutants are grown in cells producing avian leukosis viruses, the titers of infectious VSV obtained at the nonpermissive temperature are 10⁴-fold higher than in control cells. Cells releasing murine leukemia viruses or avian reticuloendotheliosis virus rescue VSV ts V mutants much less efficiently. The rescued virions have the properties of envelope pseudotypes in that their host range is restricted to that of the helper retrovirus, they are neutralized by anti-retrovirus antibodies but not anti-VSV antibodies, and they are not thermolabile. Sensitive serological techniques, including the use of complement-mediated virolysis, immunoprecipitation, and monoclonal antibody reacting with G protein, show that VSV pseudotypes produced at the nonpermissive temperature have no detectable G protein, whereas VSV particles released from retrovirus infected cells at the permissive temperature have mosaic envelopes bearing both VSV G protein and retrovirus glycoprotein. In mixed infections of Rous sarcoma virus (RSV) and VSV ts V mutants, pseudotype particles with RSV genomes and VSV envelope antigens are produced only at the permissive temperature. In contrast, substantial yields of RSV(VSV) pseudotypes but no VSV(RSV) pesudotypes are obtained at the nonpermissive temperature with VSV carrying mutations in complementation group III, which affect M protein. Thermolabile VSV tsV mutants form RSV(VSV) pseudotypes which also are thermolabile. The kinetics of heat inactivation of G protein function in tsV mutants is the same in VSV particles with unmixed envelopes and with mosaic envelopes. From these studies of phenotypic mixing we draw the following conclusions: (i) The synthesis of functional M protein but not G protein is essential for the maturation of VSV virions. (ii) VSV M protein is not required for the assembly of G protein into retrovirus virions. (iii) The thermolabile nature of tsV VSV mutants is an intrinsic property of the G protein, independent of the type of virion into which it is incorporated and of other viral glycoproteins which may be assembled into the envelope of the same virion.