Effect of glycosylation inhibitors on the release of enveloped vaccinia virus.

Addition of 1 to 10 mM 2-deoxy-D-glucose (2-dg) or glucosamine (gln) to the growth medium of vaccinia virus-infected cells inhibited the release of extracellular enveloped vaccinia virus (EEV) without affecting the production of intracellular naked vaccinia virus (INV) particles. In contrast, INV infectivity (particles per PFU) was decreased sevenfold by 50 mM 2-dg. Treatment with 2-dg reduced but did not eliminate glycosylation of the INV 37,000-molecular-weight glycoprotein. The kinetics of sensitivity to inhibitor addition experiments and inhibitor reversal experiments indicated that EEV release was dependent on glycosylation before 8 h postinfection. This was supported by polyacrylamide gel electrophoretic analysis of the synthesis kinetics for cell membrane-associated vaccinia glycoproteins in 2-dg-inhibited infected cells. The dependence of vaccinia protein glycosylation before 8 h postinfection for efficient EEV release was observed in spite of the fact that the period of greatest glycoprotein synthesis was 8 to 12 h postinfection. The presence of 2-dg resulted in an incompletely glycosylated 89,000-molecular-weight glycoprotein, as indicated by a reduction in the apparent glycoprotein molecular weight. The morphological event affected by the inhibitors was the acquisition by INV of a double-membrane structure from the Golgi apparatus. This morphological intermediate is necessary for release of EEV.     

Role of cell-associated enveloped vaccinia virus in cell-to-cell spread.

The roles of intracellular naked (INV), cell-associated enveloped (CEV), and extracellular enveloped (EEV) forms of vaccinia virus in cell-to-cell and longer-range spread were investigated by using two closely related strains of vaccinia virus, WR and IHD-J. We confirmed previous results that WR and IHD-J produced similar amounts of INV and formed similar-size primary plaques but that IHD-J produced 10 to 40 times more EEV and spread to distant cells much more efficiently than did WR. Nevertheless, cells infected with WR and IHD-J had similar amounts of CEV, indicating that wrapping and transport of WR virions were unimpaired. A WR mutant with a deletion in VP37, the major outer envelope protein, formed normal amounts of INV; however, the generation of CEV was blocked and plaque formation was inhibited. These results suggested that CEV is the form of virus that mediates cell-to-cell spread. Marker rescue experiments indicated that the differences in EEV production by WR and IHD-J were not due to sequence differences in VP37. The low amount of WR EEV could be attributed to retention of CEV on the cell membrane. In support of this hypothesis, mild treatment with trypsin released as much or more infectious virus from cells infected with WR as it did with cells infected with IHD-J. Most of the virus released by trypsin sedimented with the buoyant density of EEV. Also, addition of trypsin to cells following inoculation with WR led to a comet-shaped distribution of secondary plaques characteristic of IHD-J. These results demonstrated that the release of CEV from the cell surface was limiting for extracellular virus formation and affirmed the role of EEV in long-range spread.     

Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination.

The structure, formation, and function of the virion membranes are among the least well understood aspects of vaccinia virus replication. In this study, we investigated the role of gp42, a glycoprotein component of the extracellular enveloped form of vaccinia virus (EEV) encoded by the B5R gene. The B5R gene was deleted by homologous recombination from vaccinia virus strains IHD-J and WR, which produce high and low levels of EEV, respectively. Isolation of recombinant viruses was facilitated by the insertion into the genome of a cassette containing the Escherichia coli gpt and lacZ genes flanked by the ends of the B5R gene to provide simultaneous antibiotic selection and color screening. Deletion mutant viruses of both strains formed tiny plaques, and those of the IHD-J mutant lacked the characteristic comet shape caused by release of EEV. Nevertheless, similar yields of intracellular infectious virus were obtained whether cells were infected with the B5R deletion mutants or their parental strains. In the case of IHD-J, however, this deletion severely reduced the amount of infectious extracellular virus. Metabolic labeling studies demonstrated that the low extracellular infectivity corresponded with a decrease in EEV particles in the medium. Electron microscopic examination revealed that mature intracellular naked virions (INV) were present in cells infected with mutant virus, but neither membrane-wrapped INV nor significant amounts of plasma membrane-associated virus were observed. Syncytium formation, which occurs in cells infected with wild-type WR and IHD-J virus after brief low-pH treatment, did not occur in cells infected with the B5R deletion mutants. By contrast, syncytium formation induced by antibody to the viral hemagglutinin occurred, suggesting that different mechanisms are involved. When assayed by intracranial injection into weanling mice, both IHD-J and WR mutant viruses were found to be significantly attenuated. These findings demonstrate that the 42-kDa glycoprotein of the EEV is required for efficient membrane enwrapment of INV, externalization of the virus, and transmission and that gp42 contributes to viral virulence in strains producing both low and high levels of EEV.     

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.     

Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus.

HeLa, SIRC, and RK-13 cells were compared as to their production of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) after infection with vaccinia strains WR and IHD-J. IHD-J produced more EEV from all three cell lines than did WR, although both strains produced approximately the same quantity of INV. The most efficient EEV release was from RK-13 cells infected with IHD-J, which was 200 times more than from WR-infected SIRC cells. This permitted for the first time the purification of milligram quantities of EEV that contained much fewer cell protein contaminants than could be obtained from HeLa or SIRC cells. The INV surface proteins 200K, 95K, 65K, and 13K were present in both HeLa and RK-13 cell-derived INV but were absent in SIRC cell INV. These proteins were absent in EEV from all three cell lines. Four glycoproteins of molecular weights 210 x 10(3) (210K), 110K, 89K, and 42K and five glycoproteins in the 23K to 20K range plus a nonglycosylated protein of 37K were detected in EEV from the hemagglutinin-positive IHD-J vaccinia strain. The 89K glycoprotein was not present in EEV or membranes from cells infected with the hemagglutinin-negative vaccinia strain IHD-W. Antisera to IHD-W lacking hemagglutinin-inhibiting antibodies did not precipitate the 89K glycoprotein of IHD-J. The only glycoprotein that specifically attached to rooster erythrocytes was the 89K glycoprotein. This evidence indicates that the 89K glycoprotein is the vaccinia hemagglutinin.     

Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.     

Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane.

The membrane fusion activities of the isolated single-envelope intracellular form of vaccinia virus (INV) and the double-envelope extracellular (EEV) form were studied by using a lipid-mixing assay based on the dilution of a fluorescent probe. Fluorescently labeled INV and EEV from both the IHD-J and WR strains of vaccinia virus fused with HeLa cells at neutral pH, suggesting that fusion occurs with the plasma membrane during virus entry. EEV fused more efficiently and with faster kinetics than INV: approximately 50% of bound EEV particles fused over the course of 1 h, compared with only 25% of the INV particles. Fusion of INV and EEV was strongly temperature dependent, being decreased by 50% at 34 degrees C and by 90% at 28 degrees C. A monoclonal antibody to a 14-kilodalton envelope protein of INV that has been implicated in the fusion reaction (J. F. Rodriguez, E. Paez, and M. Esteban, J. Virol. 61:395-404, 1987) completely suppressed the initial rate of fusion of INV but had no effect on the fusion activity of EEV, suggesting that vaccinia virus encodes two or more membrane fusion proteins. Finally, cells infected with the WR strain of vaccinia virus formed syncytia when briefly incubated at pH 6.4 or below, indicating that an acid-activated viral fusion protein is expressed on the cell surface. However, WR INV and EEV did not display increased fusion activity at acid pH, suggesting that the acid-dependent fusion factor is not incorporated into virions or that its activity there is masked.     

Polypeptide composition of extracellular enveloped vaccinia virus.

Extracellular enveloped vaccinia (EEV) virus grown in SIRC and in HeLa cells was purified by consecutive equilibrium centrifugations in sucrose and cesium chloride gradients. A higher degree of purity was obtained with virus material prepared in SIRC cells. The polypeptides of purified EEV and INV (intracellular naked vaccinia) virus were compared in polyacrylamide slab gel electrophoresis. Three proteins (200,000 molecular weight [200K], 95K, and 13K) detected in HeLa-derived INV were absent in EEV. In addition, two INV proteins (65K and 30K) occurred in reduced concentrations in EEV, white another INV protein (27K) was increased in EEV. INV from SIRC cells showed similar alterations of these proteins (with the exception of the 30K and 13K proteins). Detergent treatment, ether extraction, and Pronase treatment showed that these six proteins are located at the surface of INV and are not cecessary for infectivity. Eight proteins (210K, 110K, 89K, 42K, 37K, 21.5K, 21K, and 20K) were detected in EEV that were absent from inv. Brij-58 treatment was employed to remove the envelope from EEV, resulting in the formation of naked particles and an envelope fraction which were separated on cesium chloride gradients. The envelope fractions contained all eight proteins. Seven of the eight proteins were glycoproteins, with the 37K protein being the only unglycosylated protein. It is concluded that a processing of surface INV particle proteins occurs during evelopment. The resultant EEV particle is comprised of an INV particle with a modified surface composition enclosed in an envelope containing virus-specific proteins unique to EEV.     

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.     

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.     

Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network.

During the assembly of vaccinia virus, the intracellular mature virus becomes enwrapped by a cellular cisterna to form the intracellular enveloped virus (IEV), the precursor of the extracellular enveloped virus (EEV). In this study, we have characterized the origin of this wrapping cisterna by electron microscopic immunocytochemistry using lectins, antibodies against endocytic organelles, and recombinant vaccinia viruses expressing proteins which behave as Golgi resident proteins. No labelling for endocytic marker proteins could be detected on the wrapping membrane. However, the wrapping membrane labelled significantly for a trans Golgi network (TGN) marker protein. The recycling pathway from endosomes to the TGN appears to be greatly increased following vaccinia virus infection, since significant amounts of endocytic fluid-phase tracers were found in the lumen of the TGN, Golgi complex, and the wrapping cisternae. Using immunoelectron microscopy, we localized the vaccinia virus membrane proteins VV-p37, VV-p42, VV-p21, and VV-hemagglutinin (VV-HA) in large amounts in the wrapping cisternae, in the outer membranes of the IEV, and in the outermost membrane of the EEV. The bulk of the cellular VV-p37, VV-p21, and VV-p42 were in the TGN, whereas VV-HA was also found in large amounts on the plasma membrane and in endosomes. Collectively, these data argue that the TGN becomes enriched in vaccinia virus membrane proteins that facilitate the wrapping event responsible for the formation of the IEV.     

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.     

Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.     

Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress.

Sequence analysis of the vaccinia virus strain Western Reserve genome revealed the presence of an open reading frame (ORF), SalL4R, which has the potential to encode a transmembrane glycoprotein with homology to C-type animal lectins (G. L. Smith, Y. S. Chan, and S. T. Howard, J. Gen. Virol. 72:1349-1376, 1991). Here we show that the SalL4R gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified three glycoprotein species of Mr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is present in purified extracellular enveloped virus particles but not in intracellular naked virus (INV). A mutant virus was constructed by placing a copy of the SalL4R ORF downstream of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible vaccinia virus promoter within the thymidine kinase locus and subsequently deleting the endogenous SalL4R gene. The growth kinetics of this virus demonstrated that SalL4R was nonessential for the production of infectious INV but was required for virus dissemination. Consistent with this finding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed that without SalL4R expression, the inability of the virus to spread is due to a lack of envelopment of INV virions by Golgi-derived membrane, a morphogenic event required for virus egress.     

An attenuated LC16m8 smallpox vaccine: analysis of full-genome sequence and induction of immune protection

The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood. Here, we determined the full-genome sequences of the m8, parental LC16mO (mO), and grandparental Lister (LO) strains and analyzed their evolutionary relationships. Sequence data and PCR analysis indicated that m8 was a progeny of LO and that m8 preserved almost all of the open reading frames of vaccinia virus except for the disrupted EEV envelope gene B5R. In accordance with this genomic background, m8 induced 100% protection against a highly pathogenic vaccinia WR virus in mice by a single vaccination, despite the lack of anti-B5R and anti-EEV antibodies. The immunogenicity and priming efficacy with the m8 vaccine consisting mainly of IMV were as high as those with the intact-EEV parental mO and grandparental LO vaccines. Thus, mice vaccinated with 10(7) PFU of m8 produced low levels of anti-B5R antibodies after WR challenge, probably because of quick clearance of B5R-expressing WR EEV by strong immunity induced by the vaccination. These results suggest that priming with m8 IMV provides efficient protection despite undetectable levels of immunity against EEV.     

Intracellular localization of vaccinia virus extracellular enveloped virus envelope proteins individually expressed using a Semliki Forest virus replicon

The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.     

Differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice

The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.     

The vaccinia virus 14-kilodalton fusion protein forms a stable complex with the processed protein encoded by the vaccinia virus A17L gene.

The mechanism by which the 14-kDa fusion protein of vaccinia virus (VV) is anchored in the envelope of intracellular naked virions (INV) is not understood. In this investigation, we demonstrate that the 14-kDa protein interacts with another virus protein with an apparent molecular mass of 21 kDa. Microsequence analysis of the N terminus of the 21-kDa protein revealed that this protein is encoded by the VV A17L gene. The 21-kDa protein is processed from a 23-kDa precursor, by cleavage at amino acid position 16, at the consensus motif Ala-Gly-Ala, previously identified as a cleavage site for several VV structural proteins. The 21-kDa protein contains two large internal hydrophobic domains characteristic of membrane proteins. Pulse-chase analysis showed that within 1 h after synthesis, the 14-kDa protein forms a stable complex with the 21-kDa protein. Formation of the complex was not inhibited by rifampin, indicating that the interaction between these two proteins occurs prior to virion morphogenesis. Immunoprecipitation analysis of disrupted virions showed the presence of the 21-kDa protein in the viral particle. Release of the 14-kDa-21-kDa protein complex from INV required treatment with the nonionic detergent Nonidet P-40 and a reducing agent. The protein complex consisted of 14-kDa trimers and of 21-kDa dimers. Since the 14-kDa fusion protein lacks a signal sequence and a large hydrophobic domain characteristic of membrane proteins, our findings suggest that the 21-kDa protein serves to anchor the 14-kDa protein to the envelope of INV.