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.     

Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor.

A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.     

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.     

Vaccinia virus J1R protein: a viral membrane protein that is essential for virion morphogenesis

Vaccinia virus, a member of the poxvirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature virus particles. This study examines the biological function of J1R protein in the vaccinia virus life cycle. A recombinant vaccinia virus was constructed to conditionally express J1R protein in an isopropyl-beta-D-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during vaccinia virus infection, the virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that vaccinia virus J1R is a membrane protein that is required for virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.     

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.     

The vaccinia virus A14.5L gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses

A short sequence, located between the A14L and A15L open reading frames (ORFs) of vaccinia virus, was predicted to encode a hydrophobic protein of 53 amino acids that is conserved in orthopoxviruses, leporipoxviruses, yatapoxiruses, and molluscipoxviruses. We constructed a recombinant vaccinia virus with a 10-codon epitope tag appended to the C terminus of the A14.5L ORF. Synthesis of the tagged protein occurred at late times and was blocked by an inhibitor of DNA replication, consistent with regulation by a predicted late promoter just upstream of the A14.5L ORF. Hydrophobicity of the protein was demonstrated by extraction into the detergent phase of Triton X-114. The protein was associated with purified vaccinia virus particles and with membranes of immature and mature virions that were visualized by electron microscopy of infected cells. Efficient release of the protein from purified virions occurred after treatment with a nonionic detergent and reducing agent. A mutant virus, in which the A14.5L ORF was largely deleted, produced normal-size plaques in several cell lines, and the yields of infectious intra- and extracellular viruses were similar to those of the parent. In contrast, with a mouse model, mutant viruses with the A14.5L ORF largely deleted were attenuated relative to that of the parental virus or a mutant virus with a restored A14.5L gene.     

Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae.

A vaccinia virus open reading frame (ORF) previously predicted to encode thymidylate kinase (TmpK) is shown to encode an active enzyme. A copy of the ORF, generated by polymerase chain reaction, was cloned into an Escherichia coli inducible expression vector. Cell extracts of E. coli expressing the vaccinia gene contained high levels of TmpK activity, whereas extracts of cells without the TmpK gene did not. The vaccinia ORF expressed from a yeast vector complemented a Saccharomyces cerevisiae cdc8 mutant, demonstrating functional compatibility of the vaccinia virus and yeast TmpK enzymes. The gene is shown to be nonessential for the replication of vaccinia virus in cultured cells by the construction of a viable virus mutant that has the coding region of the TmpK gene interrupted by the Ecogpt gene. Synthesis of the vaccinia TmpK protein in infected cells was demonstrated by the use of a polyvalent rabbit antiserum raised against the purified TmpK enzyme expressed in E. coli to immunoprecipitate a 23-kDa early polypeptide from cells infected with wild type vaccinia but not from cells infected with the TmpK mutant. Plasmid vectors that allow the construction of recombinant viruses expressing foreign gene(s) from the nonessential TmpK locus are described.     

Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability.

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.     

Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.     

A structure-activity study of the HindIII-I fragment of the L-IVP strain of vaccinia virus genome. I. Cloning of T5 gene and identification of its protein product]

The I5 gene from the HindIII-I-fragment of the vaccinia virus strain L-IVP DNA was cloned into bacterial vector pUC19. The monospecific antiserum to the expression product of this gene in E. coli was obtained. This antiserum was demonstrated to co-precipitate the virion protein p90. The vaccinia virus strain L-LVP DNA was shown to have only one ORF coding the p90 protein instead of two ORF H5 and H4 as known for vaccinia virus strain WR. This protein is associated with the core of vaccinia virion, but some of its antigenic determinants are exposed on the surface of the viral particles.     

The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus.

The mechanisms allowing vaccinia virus to spread from cell to cell are incompletely understood. The A34R gene of vaccinia virus encodes a glycoprotein that is localized in the outer membranes of extracellular virions. The small-plaque phenotype of an A34R deletion mutant was similar to that of mutants with deletions in other envelope genes that fail to produce extracellular vaccinia virions. Transmission electron microscopy, however, revealed that the A34R mutant produced numerous extracellular particles that were labeled with antibodies to other outer-envelope proteins and with protein A-colloidal gold. Fluorescence and scanning electron microscopy indicated that expression of the A34R protein was necessary for detection of vaccinia virus-induced actin tails, which provide motility to the intracellular enveloped form of vaccinia virus, and of virus-tipped specialized microvilli that project from the cell. The ability of vaccinia virus-infected cells to form syncytia after a brief exposure to a pH below 6, known as fusion from within, failed to occur in the absence of expression of the A34R protein; nevertheless, purified A34R- virions were capable of mediating low-pH-induced fusion from without. The present study provides genetic and microscopic evidence for the involvement of a specific viral protein in the formation or stability of actin-containing microvilli and for a role of these structures in cell-to-cell spread rather than in formation of extracellular virions.     

Vaccinia virus WR53.5/F14.5 protein is a new component of intracellular mature virus and is important for calcium-independent cell adhesion and vaccinia virus virulence in mice

The vaccinia virus WR53.5L/F14.5L gene encodes a small conserved protein that was not detected previously. However, additional proteomic analyses of different vaccinia virus isolates and strains revealed that the WR53.5 protein was incorporated into intracellular mature virus (IMV). The WR53.5 protein contains a putative N-terminal transmembrane region and a short C-terminal region. Protease digestion removed the C terminus of WR53.5 protein from IMV particles, suggesting a similar topology to that of the IMV type II transmembrane protein. We generated a recombinant vaccinia virus, vi53.5L, that expressed WR53.5 protein under isopropyl-beta-d-thiogalactopyranoside (IPTG) regulation and found that the vaccinia virus life cycle proceeded normally with or without IPTG, suggesting that WR53.5 protein is not essential for vaccinia virus growth in cell cultures. Interestingly, the C-terminal region of WR53.5 protein was exposed on the cell surface of infected cells and mediated calcium-independent cell adhesion. Finally, viruses with inactivated WR53.5L gene expression exhibited reduced virulence in mice when animals were inoculated intranasally, demonstrating that WR53.5 protein was required for virus virulence in vivo. In summary, we identified a new vaccinia IMV envelope protein, WR53.5, that mediates cell adhesion and is important for virus virulence in vivo.     

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.     

IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress.

A novel method has been developed to study the functional roles of individual vaccinia virus gene products that is neither limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants. The system utilises the E. coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG. It allows the generation of recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled. In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus. This approach has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane and for egress of mature extracellular viral particles. This is the first vaccinia virus protein shown to be specifically required for these processes. In vivo this system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without reducing the amount of the foreign antigen expressed in each infected cell. Attenuation of other live virus vaccines may be developed in a similar way.     

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.     

The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.     

A poxvirus protein with a RING finger motif binds zinc and localizes in virus factories.

Shope fibroma virus (SFV) is a Leporipoxvirus closely related to the highly virulent myxoma virus. The DNA sequence of the BamHI N fragment of the SFV DNA genome was determined, and the single complete open reading frame (N1R) was characterized. The protein encoded by the N1R gene was found to contain a C3HC4 RING finger motif at the C terminus. This C3HC4 motif is the hallmark of a growing family of proteins, many of which are involved in regulation of gene expression, DNA repair, or DNA recombination. Complete homologs of the SFV N1R gene were also detected in variola virus, myxoma virus, and vaccinia virus strain IHD-W. In contrast, the gene is completely absent from vaccinia virus strain Copenhagen, and in vaccinia virus strain WR, the open reading frame is truncated prior to the zinc binding domain because of an 11-bp deletion, thus producing a frameshift and premature stop codon. Recombinant N1R protein from SFV was expressed in Escherichia coli and shown to bind zinc in a specific manner. Using fluorescence microscopy to visualize a peptide epitope tag (derived from ICP27 of herpes simplex virus) fused to the N terminus of the poxvirus proteins, we observed that the N1R protein of SFV and its homologs in myxoma virus and vaccinia virus IHD-W were localized primarily to the virus factories in the cytoplasm of infected cells and, to a lesser degree, the host cell nucleus. The truncated protein of vaccinia virus strain WR failed to localize in this manner but instead was observed throughout the cytoplasm.     

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.