Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semiquantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope, and increased LRP1 expression is likely a defense response to infection.     

The vaccinia virus 4c and A-type inclusion proteins are specific markers for the intracellular mature virus particle.

Gel analysis of vaccinia virus particles purified by buoyant [correction of bouyant] density demonstrates a protein with an estimated molecular mass of 59 kDa, which is apparently restricted to the intracellular mature virion (IMV) form. Western blotting (immunoblotting) and immunoprecipitation procedures identify the protein as the vaccinia virus 4c protein, which facilitates occlusion of poxvirus particles within cowpox cytoplasmic inclusions. Western blotting procedures also identify the truncated A-type inclusion protein of vaccinia virus as a specific marker for IMV particles. Kinetic analyses of virion maturation and 4c production suggest that peak enveloped virion production occurs before peak IMV production in the virus replication cycle and that 4c production is concomitant with maturation of IMV. The implications for a distinct and evolutionarily conserved function of IMV in viral pathogenesis are discussed.     

Vaccinia virus morphogenesis and dissemination

Vaccinia virus is the smallpox vaccine. It is the most intensively studied poxvirus, and its study has provided important insights about virus replication in general and the interactions of viruses with the host cell and immune system. Here, the entry, morphogenesis and dissemination of vaccinia virus are considered. These processes are complicated by the existence of two infectious vaccinia virus particles, called intracellular mature virus (IMV) and extracellular enveloped virus (EEV). The IMV particle is surrounded by one membrane, and the EEV particle comprises an IMV particle enclosed within a second lipid membrane containing several viral antigens. Consequently, these virions have different biological properties and play different roles in the virus life cycle.     

Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.     

Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly

Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.     

Disparity between levels of in vitro neutralization of vaccinia virus by antibody to the A27 protein and protection of mice against intranasal challenge

Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates.     

The product of the vaccinia virus L5R gene is a fourth membrane protein encoded by all poxviruses that is required for cell entry and cell-cell fusion

The L5R gene of vaccinia virus is conserved among all sequenced members of the Poxviridae but has no predicted function or recognized nonpoxvirus homolog. Here we provide the initial characterization of the L5 protein. L5 is expressed following DNA replication with kinetics typical of a viral late protein, contains a single intramolecular disulfide bond formed by the virus-encoded cytoplasmic redox pathway, and is incorporated into intracellular mature virus particles, where it is exposed on the membrane surface. To determine whether L5 is essential for virus replication, we constructed a mutant that synthesizes L5 only in the presence of an inducer. The mutant exhibited a conditional-lethal phenotype, as cell-to-cell virus spread and formation of infectious progeny were dependent on the inducer. Nevertheless, all stages of replication occurred in the absence of inducer and intracellular and extracellular progeny virions appeared morphologically normal. Noninfectious virions lacking L5 could bind to cells, but the cores did not enter the cytoplasm. In addition, virions lacking L5 were unable to mediate low-pH-triggered cell-cell fusion from within or without. The phenotype of the L5R conditional lethal mutant is identical to that of recently described mutants in which expression of the A21, A28, and H2 genes is repressed. Thus, L5 is the fourth component of the poxvirus cell entry/fusion apparatus that is required for entry of both the intracellular and extracellular infectious forms of vaccinia virus.     

The vaccinia virus I1 protein is essential for the assembly of mature virions.

The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.     

Effects of deletion or stringent repression of the H3L envelope gene on vaccinia virus replication

The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Delta) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Delta or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Delta or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.     

Vaccinia Virus Protein Complex F12/E2 Interacts with Kinesin Light Chain Isoform 2 to Engage the Kinesin-1 Motor Complex

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Vaccinia virus A28L gene encodes an essential protein component of the virion membrane with intramolecular disulfide bonds formed by the viral cytoplasmic redox pathway

We report the initial characterization of the product of the vaccinia virus A28L gene, which is highly conserved in all sequenced poxviruses. Our studies showed that the A28 protein is expressed at late times during the virus replication cycle and is a membrane component of the intracellular mature virion. An N-terminal hydrophobic sequence, present in all poxvirus A28 orthologs, anchors the protein in the virion surface membrane so that most of it is exposed to the cytoplasm. The cytoplasmic domain contains four conserved cysteines, which form two intramolecular disulfide bonds. Disulfide bond formation depended on the expression of three viral proteins, E10, A2.5, and G4, which together comprise a conserved cytoplasmic redox pathway. A28 is the third identified substrate of this pathway; the others are the L1 and F9 proteins. We constructed a conditional-lethal recombinant vaccinia virus with an inducible A28L gene. The recombinant virus was propagated in the presence of inducer but was unable to replicate and spread in its absence. During a single round of an abortive infection in the absence of inducer, the synthesis and processing of viral proteins, assembly of intra- and extracellular virions, and formation of actin tails occurred normally. In another paper (T. Senkevich, B. M. Ward, and B. Moss, J. Virol. 78:2357-2366, 2004), we have demonstrated that virions assembled without A28 cannot carry out a second round of infection because they are defective in cell penetration.     

Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [(35)S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.     

Herpes Simplex Virus 1 Counteracts Tetherin Restriction via Its Virion Host Shutoff Activity

The interferon-inducible membrane protein tetherin (Bst-2, or CD317) is an antiviral factor that inhibits enveloped virus release by cross-linking newly formed virus particles to the producing cell. The majority of viruses that are sensitive to tetherin restriction appear to be those that acquire their envelopes at the plasma membrane, although many viruses, including herpesviruses, envelope at intracellular membranes, and the effect of tetherin on such viruses has been less well studied. We investigated the tetherin sensitivity and possible countermeasures of herpes simplex virus 1 (HSV-1). We found that overexpression of tetherin inhibits HSV-1 release and that HSV-1 efficiently depletes tetherin from infected cells. We further show that the virion host shutoff protein (Vhs) is important for depletion of tetherin mRNA and protein and that removal of tetherin compensates for defects in replication and release of a Vhs-null virus. Vhs is known to be important for HSV-1 to evade the innate immune response in vivo. Taken together, our data suggest that tetherin has antiviral activity toward HSV-1 and that the removal of tetherin by Vhs is important for the efficient replication and dissemination of HSV-1.     

Mechanism of Vaccinia Virus Release and Its Specific Inhibition by N1-Isonicotinoyl-N2-3-Methyl-4- Chlorobenzoylhydrazine

The release of vaccinia virus from RK-13 cells and its specific inhibition by N(1)-isonicotinoyl-N(2)-3-methyl-4- chlorobenzoylhydrazine (IMCBH) was studied. Intracellular naked vaccinia virus (INV) was wrapped by intracytoplasmic membranes, forming an intracellular double-membraned virion. Wrapped virions migrated to the cell surface, where the outer virion membrane presumably fused with the plasma membrane, releasing virus surrounded by the inner membrane, referred to as extracellular enveloped vaccinia virus (EEV). At no time was there any evidence that vaccinia virus acquired an envelope by budding of naked virus from the cytoplasmic membrane. Naked virus and double-membraned virus each constituted about one-third of intracellular virus at 8 and 12 h postinfection (p.i.). Beginning at 16 h p.i., the proportion of intracellular virus occurring as double-membraned virus steadily decreased to 1% at 24 h while the proportion of naked virus rose to 87%. IMCBH inhibited the formation of the double-membraned virion and the appearance of EEV while not affecting the production of INV. IMCBH had no effect on INV infectivity or polypeptide composition, on vaccinia virus-specified membrane-associated proteins or glycoproteins, or on hemadsorption. The presence of IMCBH until 4 h p.i. did not decrease the amount of EEV at 48 h p.i., whereas less than 10% of the normal 48-h EEV yield was obtained if the drug was present during the first 16 h p.i. Cell cultures infected at very low multiplicities showed a rapid virus dissemination in the absence of the drug, whereas the presence of IMCBH very effectively inhibited this spread. We conclude that vaccinia virus is liberated via a double-membraned intermediate as an enveloped virion and that it is this extracellular enveloped virus that is responsible for dissemination of infection.     

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by golgin-97

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.     

Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system

The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level.     

Vaccinia virus WR gene A5L is required for morphogenesis of mature virions

The vaccinia virus WR A5L open reading frame (corresponding to open reading frame A4L in vaccinia virus Copenhagen) encodes an immunodominant late protein found in the core of the vaccinia virion. To investigate the role of this protein in vaccinia virus replication, we have constructed a recombinant virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major vaccinia virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion.     

Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein.

There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.