The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia coli induces virus immunity in animals.

Vaccinia virus (VV) was successfully used as a live vaccine to eradicate smallpox, but the nature of viral proteins involved in eliciting viral immunity has not yet been identified. A potential candidate is a 14-kDa VV envelope protein that is involved in virus penetration at the level of virus-cell fusion, in cell-cell fusion late in infection, and in virus dissemination. The 14-kDa envelope protein has been produced in Escherichia coli, with properties similar to those of the native protein found in the virus particle and in infected cells (C. Lai, S. Gong, and M. Esteban, J. Biol. Chem. 256:22174-22180, 1990). In this investigation, we showed that mice immunized with purified VV 14-kDa protein synthesized in E. coli in the form of a monomer or a trimer develop high-titer neutralizing antibodies and are protected when challenged with lethal doses of wild-type VV. Our findings demonstrate that it is possible to confer protection against VV through immunization with the 14-kDa envelope protein.     

Insertional inactivation of the vaccinia virus 32-kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells.

The mechanism of poxvirus attachment to cells is poorly understood. We have identified a 32-kDa envelope protein of vaccinia virus which binds to the surface of cultured cells. This binding is specific and selective (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:22174-22180, 1990; C. Lai, S. Gong, and M. Esteban, J. Virol. 65:499-504, 1991). In this investigation, we studied the effect of inactivating the 32-kDa gene (32K gene) on the biology of vaccinia virus. We show that inactivation of the 32K gene decreases by 80% the mortality of mice infected with 32K- vaccinia virus. This reduction in mortality correlates with diminished viral gene expression in target tissues. In highly polarized epithelial cells, viral gene expression of 32K- virus was reduced (50 to 60%) at both the apical and basolateral surfaces in comparison with a 32K+ virus. Restriction of virus gene expression in polarized cell surfaces occurs for both intracellular and extracellular forms of infectious 32K- vaccinia virus. The two infectious forms of vaccinia virus 32K+ infect polarized cells preferentially by the basolateral surface. Our findings provide evidence of the importance of the 32-kDa protein in viral pathogenesis.     

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.     

Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.     

Inducible expression of the vaccinia virus A17L gene provides a synchronized system to monitor sorting of viral proteins during morphogenesis.

The vaccinia virus (VV) A17L gene encodes a 21- to 23-kDa virion component that forms a stable complex with the 14-kDa envelope protein (A27L gene). In a previous report, we described the construction of a VV recombinant, VVindA17L, in which the expression of the A17L gene is inducibly regulated by isopropyl-beta-D-thiogalactoside (IPTG). We demonstrated that shutoff of the A17L gene results in a blockade of virion morphogenesis at a very early stage (D. Rodríguez, M. Esteban, and J. R. Rodríguez, J. Virol. 69:4640-4648, 1995). In the present study, we show that virus growth is restored if the inducer is provided not later than 6 h postinfection. Immunofluorescence and immunoelectron microscopy analysis of VVindA17L-infected cells revealed that in the absence of the 21- to 23-kDa protein, the 14-kDa protein is distributed throughout the cytoplasm. After IPTG addition, the 14-kDa protein can be detected around viral factories and immature virions; at later times, it localizes in the external membranes of intracellular mature virions. Immunoelectron microscopy with anti-21- to 23-kDa antibodies showed that soon after induction, the protein accumulates in membranes of the rough endoplasmic reticulum and in the nuclear envelope. With time, the protein localizes in viral crescents and subsequently associates to the membranes of immature and intracellular mature virions. These results are consistent with a model in which the 21- to 23-kDa protein would be synthesized at the endoplasmic reticulum, from where the protein could be translocated to the membranes of the intermediate compartment to generate the precursors of the viral membranes. Also, these results argue that 14-kDa envelope protein becomes posttranslationally associated to viral membranes through its interaction with the 21-kDa protein.     

Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis.

Vaccinia virus (VV) A17L gene encodes a 23-kDa protein that is proteolytically cleaved to generate a 21-kDa product that is incorporated into the viral particles. We have previously shown that the 21-kDa protein forms a stable complex with the VV 14-kDa envelope protein and suggested that the 21-kDa protein may serve to anchor the 14-kDa protein to the envelope of the virion (D. Rodríguez, J. R. Rodríguez, and M. Esteban, J. Virol. 67:3435-3440, 1993). To study the role of the 21-kDa protein in virion assembly, in this investigation we generated a VV recombinant, VVindA17L, that contains an inducible A17L gene regulated by the E. coli repressor/operator system. In the absence of the inducer, shutoff of the A17L gene was complete, and this shutoff correlated with a reduction in virus yields of about 3 log units. Although early and late viral polypeptides are normally synthesized in the absence of the A17L gene product, proteolytic processing of the major p4a and p4b core proteins was clearly impaired under these conditions. Electron microscopy examination of cells infected in the absence of isopropylthiogalactopyranoside (IPTG) revealed that virion morphogenesis was completely arrested at a very early stage, even prior to the formation of crescent-shaped membranes, which are the first distinguishable viral structures. Only electron-dense structures similar to rifampin bodies, but devoid of membranes, could be observed in the cytoplasm of cells infected with VVindA17L under nonpermissive conditions. Considering the most recent assembly model presented by Sodeik et al. (B. Sodeik, R. W. Doms, M. Ericsson, G. Hiller, C. E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths, J. Cell Biol. 121:521-541, 1993), we propose that this protein is targeted to the intermediate compartment and is involved in the recruitment of these membranes to the viral factories, where it forms the characteristic crescent structures that subsequently result in the formation of virions.     

Virus attenuation and identification of structural proteins of vaccinia virus that are selectively modified during virus persistence.

To investigate the genetic stability of vaccinia virus DNA, we have tested whether alterations occurred in the polypeptide composition of this complex virus during persistent infections. We found that variants isolated at various passages in Friend erythroleukemia cells persistently infected with vaccinia virus contained, in addition to an 8-megadalton (MDa) deletion on the left terminus of the viral genome, major alterations in the sizes of three structural proteins with molecular masses of about 39, 21, and 14 kDa. Alterations in isoelectric points were also observed in proteins of 48, 27, and 14 kDa. The 14-kDa protein is part of the virus envelope, and the variants increased the size of this protein from 0.5 to 3 kDa with increasing passage number. Alteration in size of the 14-kDa protein is a dominant trait since it appeared in the whole virus population by passage 48. With more passages, some variants were found to increase or decrease the size of a 39-kDa core protein by about 2 kDa and to decrease the size of an envelope protein of 21 kDa by about 2 kDa. These three proteins were immunogenic in mice and elicited a strong host immune response. Major alterations in the sizes of these proteins were prevented by continuous treatment of the persistently infected cultures with interferon. However, after interferon was removed, protein modifications appeared with increasing passage number. Generation of the 8-MDa deletion and alterations in the size of the 14-kDa protein correlated with a marked decrease in virulence of these variants. Our findings suggest that during virus persistence, specific mutations are introduced in the vaccinia virus genome that lead to protein alterations and to highly attenuated viruses.     

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.     

Structural and functional characterization of a cell surface binding protein of vaccinia virus

The nature of the interaction between the enveloped DNA-containing poxviruses and the surfaces of host cells as a first step in virus infection is not known. In this investigation we have identified and defined structural and functional properties of a 32-kDa protein of vaccinia virus. This protein is part of the virus envelope and binds to the cell surface of various cultured cells. The gene encoding the 32-kDa viral protein was mapped and sequenced. It was found to code a 35,426-Da protein with a large N-terminal domain with sequence homology to carbonic anhydrases and a C-terminal domain with sequences similar to those of the attachment glycoprotein VP7 of rotavirus and to transmembrane proteins. A potential cell surface binding domain was within the last 50 amino acid residues of the C terminus. The 32-kDa protein is basic, predicted pI 8.67, is synthesized at late times post-infection, may form dimers held by disulfide bonds at the single cysteine 262, and is apparently non-glycosylated. The 32-kDa protein is a vaccinia virus antigen, with predicted antigenic sites located near amino acids 108-110 (carbonic anhydrase domain) and 298-299 (transmembrane domain). Several lines of evidence suggest that the 32-kDa protein is needed for efficient virus replication in cultured cells but that in addition to this protein other viral proteins are involved in the process of virus entry into cells.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Vaccinia virus 4c (A26L) protein on intracellular mature virus binds to the extracellular cellular matrix laminin

Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG(-) sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.     

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.     

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.     

An orthopoxvirus serpinlike gene controls the ability of infected cells to fuse.

Most orthopoxviruses encode a functional hemagglutinin (HA), which is nonessential for virus growth in cell culture. However, inactivation of the HA gene leads to the formation of polykaryocytes (syncytia) by fusion of infected cells at neutral pH. Fusion is not observed when a functional HA gene is present. Deletion of open reading frames (ORFs) K2, K3, and K4 within the HindIII K fragment of the HA-positive (HA+) vaccinia virus strain WR also led to fusion of cells upon infection at neutral pH. A novel ORF inactivation procedure utilizing the polymerase chain reaction was used to specifically implicate the K2 ORF in this phenomenon. The K2 ORF (the viral SPI-3 gene) encodes a protein resembling serine protease inhibitors (serpins). Inactivation of the SPI-3 gene in any of the HA+ orthopoxviruses tested caused infected cells to fuse in a manner which appeared identical to that seen for HA- mutants, although fusion was most pronounced with cowpox virus. SPI-3-negative strains fused despite the fact that the HA was expressed and processed normally, i.e., cells infected with SPI-3 mutants remained functionally hemadsorption positive, and analysis of the HA protein by Western immunoblot suggested that posttranslational modifications of the HA protein appeared normal. Fusion triggered by SPI-3 mutants, like that for HA- mutants, was inhibited by the monoclonal antibody C3 directed against the vaccinia virus 14-kDa envelope protein. Therefore SPI-3- and HA-mediated fusion share a requirement for the 14-kDa protein, suggesting linkage of the seemingly disparate SPI-3 and HA genes through a common pathway which normally acts to prevent fusion of cells infected with wild-type virus.     

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.     

Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein.

A library of rabbit poxvirus DNA fragments contained in the expression cloning vector lambda gt11 was screened with monoclonal antibodies that react specifically against a 14-kilodalton envelope protein of vaccinia virus and rabbit poxvirus. The 14-kilodalton protein appears to play an important role in virus penetration at the level of cell fusion; it also elicits neutralizing antibodies, and it forms covalently linked trimers on the surface of virions and in infected cells (Rodriguez et al., J. Virol. 56:482-488, 1985; Rodriguez et al., J. Virol. 61:395-404, 1987). Two recombinant bacteriophages expressing beta-galactosidase fusion proteins were isolated. Restriction enzyme analysis and hybridization studies mapped the 14-kilodalton encoding sequences in the middle of vaccinia virus HindIII A DNA fragment. Nucleotide sequence analysis revealed an open reading frame (ATG) preceded by a characteristic TAA sequence of late genes. The sequence spans 330 nucleotides and codes for a protein with a molecular weight of 12,500 and an isoelectric point of 6.3. There are two small hydrophobic regions, one at the C terminus (11 amino acids) and the other at the N terminus (5 amino acids). The protein contains two cysteines for oligomer formation and one glycosylation site. Inspection of the deduced amino acid sequence of the 14-kilodalton protein revealed consensus sites with the hemagglutinin precursor of influenza A virus and with adenylate kinase and cytochrome c of various species.     

Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH

Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.     

Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB).

The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus. E. coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells. Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55. Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E. coli-produced protein. Immunization of mice with either E. coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies. In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E. coli-derived protein induced complement-independent neutralizing antibodies.     

Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.

With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.     

Vaccinia Virus Envelope D8L Protein Binds to Cell Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells

We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L(+) D8L(+)) to be defective in expression of either the A27L or the D8L gene (A27L(+) D8L(-) or A27L(-) D8L(+)) or both (A27L(-) D8L(-)). The A27L(+) D8L(+) and A27L(-) D8L(+) mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L(+) D8L(-) and A27L(-) D8L(-) viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L(+) D8L(-) and A27L(-) D8L(-) mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L(+) D8L(-) and A27L(-) D8L(-) mutant virions were 6 to 10% of that of the A27L(+) D8L(+) control virus. Virion binding assays revealed that A27L(+) D8L(-) and A27L(-) D8L(-) mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.