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Characterization of vaccinia virus glycoproteins by monoclonal antibody precipitation
Abstract
Monoclonal antibodies were used to characterize vaccinia virus glycoproteins known to be incorporated into the envelope of extracellular enveloped vaccinia (EEV) virus. The 89K hemagglutinin, 42K, and 23-28K glycoproteins were predominantly expressed as late vaccinia proteins. The 89K glycoprotein was sulfated and phosphorylated but not acylated. 89K precursor proteins of 32K, 41.5K, and 52K were detected. The former had a molecular weight expected from the deduced amino acid sequence of the hemagglutinin gene. A 76K glycoprotein that did not contain methionine and was not sulfated or phosphorylated was precipitated late in infection by the anti-hemagglutinin monoclonal antibody. The appearance of this protein was inhibited by rifampicin and it may thus result from 89K cleavage. A 220K complex contained some or all of the hemagglutinin gene products linked by disulfide bonds. The 42K glycoprotein was not sulfated or phosphorylated but was acylated. This glycoprotein was disulfide bonded with the EEV 37K nonglycosylated envelope protein. The 23-28K glycoprotein was not sulfated but was both phosphorylated and acylated. The 23-28K glycoprotein group of five proteins had a common protein backbone that was differentially glycosylated. Pulse-chase, glycosylation inhibition with tunicamycin, and glycosidase experiments established that the precursor to the 23-28K glycoproteins was a 21K protein. Members of this protein family formed dimers of approximately 55K through disulfide bonds.
... At least eight vaccinia virus-encoded proteins may be palmitoylated, as evidenced by the incorporation of 3 H-labeled palmitate in culture (11). These proteins include wrapped virus (WV)-specific protein p37 (F13L) (7,(12)(13)(14) and products of open reading frames (ORFs) A33R (15), B5R (16), F13L (17), the A22R Holliday resolvase (11), A36R, and the A56R hemagglutinin (11). ...
... Glycosylation has been clearly detected among the wrapping membrane proteins of wrapped vaccinia virus (WV): A33L protein was identified as a glycoprotein based on the mobility of diffuse bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the presence of NX(S/T) sites in the extracellular domain (15). Examination of A33L protein produced in the presence of tunicamycin or monensin (14), inhibitors of N-linked and O-linked glycosylation, respectively, indicated the possession of both N-and O-linked glycans by A33L. Use of both drugs in combination led to a discrete protein band with the predicted molecular mass of 33 kDa. ...
... WV protein modifications have also been demonstrated using monoclonal antibodies (MAbs) raised against WV (14), via the immunoprecipitation of WV surface proteins from extracts of infected cells incubated with [ 3 H]glucosamine. Again, glycosylation was confirmed using tunicamycin and monensin. ...
Importance:
Poxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry.
... A33 is one of seven enveloped virus-specific proteins encoded by orthopoxviruses and is found in all of the members of the subfamily Chordopoxvirinae, except for the members of the Avipoxvirus genus (Roper, Payne, and Moss, 1996). A33 was reported to be a disulfidebonded multimer in infected cells and has been reported to be palmitoylated (Payne, 1992;Roper et al., 1998). The six cysteine residues in A33 are highly conserved among the members of the subfamily Chordopoxvirinae. ...
... The six cysteine residues in A33 are highly conserved among the members of the subfamily Chordopoxvirinae. Of these six cysteine residues, C36 is predicted to be palmitoylated as it is the only cysteine in the cytoplasmic tail (Grosenbach, Hansen, and Hruby, 2000;Payne, 1992). Of the remaining five cysteines, C100, C109, C126, and C180 were recently reported to be involved in intramolecular disulfide bond formation ( Su et al., 2010). ...
... In addition, A33-HA C100S , A33-HA C109S , A33-HA C126S , and A33-HA C180S migrated as a doublet on SDS-PAGE (Fig. 2). A33 has been shown to be glycosylated and there are two putative N-linked glycosylation sites (Payne, 1992). However, it has not been shown if A33 is glycosylated at one or both sites. ...
The orthopoxvirus protein A33 forms a disulfide-bonded high molecular weight species that could be either a homodimer or a heteromultimer. The protein is a major target for neutralizing antibodies and the majority of antibodies raised against A33 only recognize the disulfide-bonded form. Here, we report that A33 is present as a disulfide-bonded homodimer during infection. Additionally, we examined the function of intermolecular disulfide bonding in A33 homodimerization during infection. We show that the cysteine at amino acid 62 is required for intermolecular disulfide bonding, but not dimerization as this mutant was still able to form homodimers. To investigate the role of disulfide-bonded homodimers during viral morphogenesis, recombinant viruses that express an A33R with cysteine 62 mutated to serine were generated. The recombinant viruses had growth characteristics similar to their parental viruses, indicating that intermolecular disulfide-bonded homodimerization of A33 is not required for its function.
... The outer envelope of EEV was reported to contain nine glycosylated proteins and one nonglycosylated protein (10,21,22). Recent biochemical characterization of purified EEV by using monoclonal antibodies (MAbs) suggested, however, that these nine glycoproteins might represent the products of a smaller number of genes (24). MAbs that recognized the 89-kDa hemagglutinin glycoprotein also bound to smaller proteins of 32, 41.5, 52, and 76 kDa as well as to a larger 220-kDa complex. ...
... We have taken a reverse genetic approach similar to that of Smith and coworkers (7,8) and in this report provide direct proof that the B5R ORF encodes the 42-kDa glycoprotein described by Payne (24). In addition, the signal and transmembrane peptides were identified, and the 42-kDa protein was localized within the infected cell and EEV by immunofluorescence and electron microscopy, respectively. ...
... In addition, the signal and transmembrane peptides were identified, and the 42-kDa protein was localized within the infected cell and EEV by immunofluorescence and electron microscopy, respectively. (24) except that the medium was serum free. Antibodies. ...
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.
... The development of monoclonal antibodies (MAbs) to EEVspecific proteins allows for unambiguous characterization of the proteins without the problems of cross-reactivity and background inherent in polyclonal sera (30). MAb 4 reacts with a variably glycosylated protein very similar to that encoded by the A34R gene, previously characterized by use of polyclonal sera (11,23). ...
... MAb 4 reacts with a variably glycosylated protein very similar to that encoded by the A34R gene, previously characterized by use of polyclonal sera (11,23). The MAb 4-reactive protein and the A34R-encoded protein are both EEV specific, have similar molecular weights, and are expressed predominantly late in infection and variably glycosylated (11,23,30), leading to the assumption that MAb 4 recognizes the A34R protein (37). The realization that this might not be the case arose from studies involving a recombinant VV expressing an epitope-tagged A34R gene product (35). ...
... Antibodies. Mouse MAbs 4, 66, 105, and 20 were produced as previously described (30). An antibody to the 14-kDa IMV protein (mouse MAb C3) was kindly provided by M. Esteban (34). ...
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.
... MC021-HA interacts with B5 and exhibits an altered interaction with A33. F13 has previously been reported to interact with both B5 and A33 (50)(51)(52)(53)(54). While MOCV contains an A33 homolog, it lacks a B5 homolog (33). ...
... To investigate this, we infected cells with vF13L-HA, vMC021L-HA, and vΔF13L and subjected the cell lysates to immunoprecipitation with either an anti-B5 monoclonal antibody (MAb) (Fig. 5A) or anti-A33 antiserum (Fig. 5C). Consistent with previous reports, Western blot analysis revealed an interaction between F13-HA and B5 (Fig. 5A) and between F13-HA and A33 (Fig. 5C) (53,54). Additionally, a band corresponding to MC021-HA was coimmunoprecipitated with both B5 (Fig. 5A) and A33 (Fig. 5C) from lysates of cells infected with vMC021L-HA, suggesting that MC021-HA interacts with both B5 and A33 (directly or indirectly). ...
The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in turn is essential for the subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV are produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate and showed that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small-plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV and further delineates the dual role of F13 during infection.
... Immunogenic proteins of EEV A33R: Among EEV proteins, A33R is the outer envelope type II integral transmembrane glycosylated protein having both N and O glycosylation (~23-28kDa) (Payne, 1992;Roper et al., 1996;Roper et al., 1998). A33R mainly gets localized to the golgi apparatus and helps in EEV formation and is present as a dimer unit. ...
... In contrast to A33R, the other significant EEV membrane protein B5R (~42kDa) is but type I membrane protein that is glycosylated at N-terminal and palmitylated (Isaacs et al., 1992;Payne, 1992;Smith et al., 2002). This glycosylated protein is highly conserved among many strains of VACV as well as other poxviruses (Engelstad et al., 1993). ...
Capripoxvirus (CaPV) infections are highly contagious and OIE notifiable viral diseases of sheep, goats and cattle. They are endemic in most parts of the globe associated with significant production losses due to high morbidity, high mortality rate and animal trade restrictions. Though several diagnostics including molecular tools are available, recombinant protein based diagnostic assays namely ELISA is safer and robust to handle large sample size and also to minimize labor/time. However, the genus Capripoxvirus encodes putative 147 proteins in their genome, among which some of them are reported as potential immunogenic candidate genes. Selection and use of such candidate immunogenic proteins from an array of genes located at different structures of a mature virion are the real challenge and time consuming task for researchers. Nevertheless, identification of candidate gene(s) using advanced bioinformatic tools will ease the process and can select the suitable protein(s) to use in the development of specific and sensitive diagnostic assays and also effective vaccine candidates which are vital elements in control and eradication of any infectious disease from an endemic country. In this review, we describe different structures of mature pox virion with reference to vaccinia virus (VACV), list of immunogenic candidate genes presents in CaPV genome and their potential use in diagnostic/vaccine developments.
... Specifically, recombinant viruses that have a deletion in any of these proteins, with the exception of A56 and K2, have a small-plaque phenotype, indicating a reduction in cell-to-cell spread (17,(20)(21)(22)24). Interactions between B5 and A33 (30,31), B5 and A34 (32,33), A33 and A36 (32), A34 and A36 (33), B5 and F13 (34), F12 and E2 (35), F12 and A36 (36), and K2 and A56 (37) have been reported, with some of these interactions mediating intracellular trafficking and incorporation of EV proteins into IEV, IEV transport, and subsequent EV release. ...
... Nonetheless, we cannot discount the possibility that the C-terminal residues of A34, missing in each of our recombinants, contain a site necessary for cell binding and truncation of these residues and that removal of this site is directly responsible for the reduction in binding and the small-plaque phenotype. Additionally, A34 is known to be glycosylated, and its stability, localization, and glycosylation status have been reported to be affected by the presence or absence of B5 (17,34,45,59). The glycosylation sites on A34 have not been reported. ...
Previous studies have shown that the vaccinia virus glycoproteins A34 and B5 interact, and in the absence of A34, B5 is mislocalized and not incorporated into extracellular virions. Here, using a transient-transfection assay, residues 80 to 130 of the ectodomain of A34 were determined to be sufficient for interaction with B5. Recombinant viruses expressing A34 with a full, partial, or no B5 interaction site were constructed and characterized. All of the A34 truncations interacted with B5 as predicted by the transient-transfection studies but had a small-plaque phenotype. Further analysis revealed that all of the recombinants incorporated detectable levels of B5 into released virions but were defective in cell binding and extracellular virion (EV) dissolution. This study is the first to directly demonstrate that A34 is involved in cell binding and implicate the ectodomain in this role.
... 45 In its absence, wrapping is inhibited, few EEV particles are produced and the virus is attenuated. 46 B5R has been shown to co-localize and interact with F13L 47,48 and, in the presence of ST-246, less immunoprecipitated B5R-F13L complex was found; this might be attributed to (i) mislocalization of F13L and/or B5R or (ii) disruption of the B5R -F13L complex by ST-246. 38 It remains unclear exactly how ST-246 interferes with F13L. ...
... The DB5R constructs were included to clarify a possible role of B5R in drug activity as B5R was shown to co-localize and associate with F13L in Golgi cisternae and post-Golgi vesicles. 47,48 Also, mutations in F13L affect the proper localization of B5R in post-Golgi vesicles (e.g. in the absence of F13L, B5R is mainly found in Golgi cisternae). 47 In turn, B5R mutations have been shown not to influence F13L distribution significantly. ...
ST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target.
Vaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity.
Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences.
Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.
© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
... A33 is a type-II integral membrane glycoprotein, forming a disulfide-bonded homodimer modified with N-and O-linked glycosylations and acylation and found on the surface of extracellular virions (EV) and infected cells [14][15][16][17]. This protein was shown to form a complex with the viral proteins A36, A34 and B5, thus stabilizing their presentation on EVs [16,18,19]. ...
... Recombinant Sindbis viruses were produced and expression of the modified A33 genes was confirmed by infection of BHK 21 cells followed by Western blot analysis of the cell lysates ( Figure 6B). A33 is post-translationally modified in cells (N' and O' glycosylations and acylation) resulting in a well-known multiple band pattern in SDS-PAGE [14,16,19]. All Sindbis viruses expressed the modified A33 VACV genes, yet certain differences in the multiple band pattern could be observed, mainly in the double mutant A33 (Q117K-L118S). ...
Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104--120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.
... The A33 protein is also a type II membrane protein with a C-type lectin-like fold with several glycoforms of 23-28 kDa (Roper et al., 1996;Su et al., 2010). In addition to N-glycosylations, A33 is also O-glycosylated (Payne, 1992), phosphorylated (Wolffe et al., 2001) and acylated (Payne, 1992;Grosenbach et al., 2000). Deletion of A33 leads to a reduction in plaque size (Roper et al., 1998;Law et al., 2002) and actin tail formation from the cell surface (Roper et al., 1998) but a threefold increase in EEV release (Roper et al., 1998). ...
... The A33 protein is also a type II membrane protein with a C-type lectin-like fold with several glycoforms of 23-28 kDa (Roper et al., 1996;Su et al., 2010). In addition to N-glycosylations, A33 is also O-glycosylated (Payne, 1992), phosphorylated (Wolffe et al., 2001) and acylated (Payne, 1992;Grosenbach et al., 2000). Deletion of A33 leads to a reduction in plaque size (Roper et al., 1998;Law et al., 2002) and actin tail formation from the cell surface (Roper et al., 1998) but a threefold increase in EEV release (Roper et al., 1998). ...
Vaccinia virus (VACV) has two infectious forms called intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Two of the seven viral proteins in the EEV outer envelope, A33 and A34, are type II membrane glycoproteins that each interact with another EEV protein called B5, however evidence for direct A33-A34 interaction is lacking. The localization and stability of A34 is affected by B5 and here data are presented showing that A34 is also affected by A33. In the absence of A33, just as without B5, the level, localisation and glycosylation profile of A34 was altered. However, the glycosylation profile of A34 without A33 is different to that observed in the absence of B5, and A34 accumulates in the Golgi apparatus rather than in the endoplasmic reticulum. Thus, A34 requires more than one other EEV protein for its processing and cellular transport.
... Multiple interactions between the EV and IEV proteins have been demonstrated. The B5 protein has been reported to interact with A33, A34, and F13 (3,5,6,10,(24)(25)(26)31). B5R encodes a 42-kDa type I glycoprotein that is involved in EV formation (11,12,18,47). ...
... To check expression, a Western blot assay of lysates from cells expressing the CTA mutant proteins was probed with an anti-HA MAb. With the exception of A33-HA E67-D72 , all of the constructs produced a smear between 25 and 30 kDa (Fig. 1B) that is typical of A33 and has been attributed to both N-and O-linked glycosylation (24,29). The A33-HA E67-D72 mutant protein was not included in the rest of our analysis due to the poor level of expression. ...
Two mechanisms exist for the incorporation of B5 into extracellular virions, one of which is dependent on A33. In the companion
to this paper (W. M. Chan and B. M. Ward, J. Virol. 86:8210–8220, 2012), we show that the lumenal domain of A33 is sufficient for interaction with the coiled-coil domain of B5 and capable of directing
B5-green fluorescent protein (GFP) into extracellular virions. Here, we have created a panel of charge-to-alanine mutations
in the lumenal domain of A33 to map the B5 interaction site. While none of these mutations abolished the interaction with
B5, a subset displayed an increased interaction with both B5 and B5-GFP. Both B5 and B5-GFP recombinant viruses expressing
these mutant proteins in place of normal A33 had a small-plaque phenotype. The increased interaction of the mutant proteins
was detected during infection, suggesting that normally the interaction is either weak or transient. In addition, the increased
A33-B5 interaction was detected on virions produced by recombinant viruses and correlated with reduced target cell binding.
Taken together, these results show that both B5 and B5-GFP interact with A33 during infection and that the duration of this
interaction needs to be regulated for the production of fully infectious extracellular virions.
... VV serves as an ideal model system for the study of protein processing for several reasons. VV proteins (or exogenous proteins expressed either transiently from transfected plasmids or from recombinant VV) are subject to modifications typical of mammalian systems, to include sulfation (Payne, 1992 ), phosphorylation, ADPribosylation , glycosylation, and as highlighted here, acylation (VanSlyke and Hruby, 1990 ). Its genes are expressed in the cytoplasm without mRNA processing, allowing prediction of gene products directly from the genomic sequence. ...
... It may be that replacement of a palmitate-acceptor cysteine with a serine in an optimal environment results in palmitylation of the serine residue through ester linkage. Many VV palmitylproteins are thought to interact either through disulfide bonds or noncovalently (Payne, 1992; Röttger et al., 1999 ). The exact nature of these interactions or their biological significance is not known. ...
Vaccinia virus encodes at least eight proteins that incorporate label from tritiated palmitic acid when it is added to infected cell cultures. Three of these palmitylproteins are encoded by the A33R, B5R, and F13L open reading frames and migrate by gel electrophoresis with relative molecular masses of 23–28, 42, and 37 kDa, respectively. In this report we provide evidence that the A22R and A36R open reading frames also encode palmitylproteins with apparent molecular masses of 22 and 50–55 kDa, respectively. Furthermore, the hemagglutinin protein (A56R) from the Copenhagen strain is shown to be palmitylated while the hemagglutinin protein from the WR and IHD-J strains is not. A 94-kDa VV palmitylprotein appears to be a multimeric complex composed of the B5R protein and possibly others. All vaccinia-encoded palmitylproteins are present in the membranous fraction of cells and are specific for the trans-Golgi network membrane-enveloped forms of the virus, suggesting that these proteins play a role in the envelopment and egress of virions or the infectivity of released virus.
... The remaining five were predicted from the D1701 sequencing results (270, 272, 277, 278, and 279). The NZ-2 B2L gene product had been previously identified as the homolog of the vaccinia (VACV) virus major envelope antigen gene, F13L [26,29]. The truncated D1701 B2L ORF described above resided in pool J, which did not induce the accumulation of I-A + / I-E + cells (Figure 2a). ...
... A33 targets F13 to the IEV, and these two proteins immunoprecipitate as an A33/F13 complex from infected cell lysates [39] . Proteins F13 and B5 colocalize forming a disulfide bonded complex that is required for efficient and complete wrapping of the intracellular mature virus [29,37,39]. ORFV is unique relative to VACV and other orthopoxviruses in its lack of any B5 homolog. ...
The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach.
The genome of Parapoxvirus ovis (Orf virus) was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer.
A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration of B2 may provide the opportunity to significantly impact host immunity while being itself only weakly recognized. The functional genomics method used to pinpoint B2 within an ORFeome may be more broadly applicable to screening for other biological activities in an animal.
... A33 is expressed on the EEV membrane as a 23 kDa, homodimeric type II transmembrane protein that undergoes both O-and N-glycosylation (Isaacs et al. 1992;Roper et al. 1996;Payne 1992). A33 controls the incorporation of A36 into the EEV particle and subsequently the production of actin tails. ...
Vaccinia Virus (VACV) is an enveloped double stranded DNA virus and the active ingredient of the smallpox vaccine. The systematic administration of this vaccine led to the eradication of circulating smallpox (variola virus, VARV) from the human population. As a tribute to its success, global immunization was ended in the late 1970s. The efficacy of the vaccine is attributed to a robust production of protective antibodies against several envelope proteins of VACV, which cross-protect against infection with pathogenic VARV. Since global vaccination was ended, most children and young adults do not possess immunity against smallpox. This is a concern, since smallpox is considered a potential bioweapon. Although the smallpox vaccine is considered the gold standard of all vaccines and the targeted antigens have been widely studied, the epitopes that are targeted by the protective antibodies and their mechanism of binding had been, until recently, poorly characterized. Understanding the precise interaction between the antibodies and their epitopes will be helpful in the design of better vaccines against other diseases. In this review we will discuss the structural basis of recognition of the immunodominant VACV antigens A27, A33, D8, and L1 by protective antibodies and discuss potential implications regarding their protective capacity.
... Thus, it was assumed that live vaccinia virus vaccination protected by potent anti-viral T cell responses. However, the inability of experimental inactivated vaccines to protect may have been due to denaturing of key targets (243), as well as the fact that the vaccine preparations did not contain some critical antigens that are present on a minor population of infectious virus (116,244 Furthermore, including targets against both MV and EV appears to provide the best protection from morbidity and mortality (98,110,138,160,246). Including targets against both forms of infectious virus is believed to provide a way to decrease the infecting inoculum (believed to be mainly MV) and then alter the spread and dissemination of the virus within an infected host (thought to be mainly EV) (73,(247)(248)(249)(250). Initial insights into appropriate targets against MV and/or EV proteins were based on the production of antibodies that could neutralize virus in vitro or provide passive protection against vaccinia virus challenge in vivo (28,96,109,(145)(146)(147)154,160,166,(251)(252)(253)(254). ...
Protein-based smallpox vaccines have shown to be effective alternatives to live virus vaccines in animal model challenge studies. It is believed that subunit vaccine protection is mainly mediated through the generation of antibodies. We, and others, have shown that Th1-type antibody responses are important for protection against poxvirus infections. This finding suggests that antibody-mediated protection by a protein-based smallpox vaccine may involve antibody that can fix complement and/or activate Fc receptors. Vaccinia virus, the prototype member of the poxvirus family, produces two infectious forms of virus: mature virus (MV) and extracellular virus (EV). EV is relatively resistant to neutralization by antibody, yet antibody against EV has been shown to protective in vivo. Therefore, we examined the importance of complement and Fc receptors in the protection afforded by antibody against EV. In the first part of this thesis, we found that polyclonal antibody against the EV proteins A33 and B5 can fix complement to efficiently neutralize EV in vitro. Additionally, we found that the complement activation requirements necessary for neutralization differ depending on the EV protein target used. We attribute these differences to the amount of A33 and B5 protein found on the EV outer membrane. We then show that in mice, both complement and Fc receptors are important for protection mediated by polyclonal antibody against the EV protein B5. In the last section, we found that non-human primates are better protected from monkeypox virus challenge by protein vaccination when the vaccine formulation generates more Th1-like antibody responses. Individual non-human primates receiving this vaccine had more homogeneous antibody responses that could neutralize EV in the presence of complement. Together, these studies implicate an important role for complement and Fc receptors in the protection by antibody against the EV form of poxviruses. This work highlights the importance of analyzing the mechanisms by which antibody provides protection from viral infection so that antibody-based vaccines and therapeutics can be more efficiently designed.
... Both the 85 and 68-kDa forms of A56 can be seen via western blot at late times. Based on the sequence, A56 contains putative sites for N-and Oglycosylation (14,90,111). When glycosylation is inhibited, A56 migrates at a weight of 58-kDa, which is different than the 35-kDa calculated weight (110). ...
Vaccinia replication is complex, as it involves both producing new infectious particles as well as a myriad of proteins dedicated to combating the host’s immune response. Although these proteins are often called “non-essential”, they are frequently needed for the virus to achieve maximum virulence in vivo. Two of these proteins are the vaccinia complement control protein (VCP) and A56, the vaccinia hemagglutinin. VCP had been previously described as a virulence factor that was secreted from infected cells and blocked the activation of the complement cascade; A56 was known to bind another viral protein, K2. We have found that VCP and A56 interact on the surface of infected cells, and that this interaction is important for full vaccinia virulence in vivo. The first part of this thesis focuses on showing a direct interaction between A56 and VCP on the surface of infected cells, and that the N-terminal cysteine of VCP is needed for this interaction. The next section establishes that this interaction occurs via an intermolecular disulfide bridge between the two proteins in a transfection model, and also extends this phenomenon to VCP homologs from other poxviruses. Mutagenesis shows that VCP binds to the 3rd cysteine (residue 162) of the ectodomain of A56. We also begin to show that viruses that cannot form the A56/VCP complex are attenuated in vivo, using a virus V where the N-terminal cysteine of VCP is mutated. In the last section, we create a virus with a recombinant A56 protein that cannot bind VCP. We show that this virus is attenuated in intranasal and intradermal models of infection in mice; infections done in C3-knockout mice suggest that this attenuation is complement dependent. This work shows that VCP is not only important as a secreted protein, and that the A56/VCP complex is important for the virus to achieve maximum pathogenesis in vivo. These results also provide insight into the contributions of the A56 protein to vaccinia virulence in an infection, and for the first time tests a site-directed A56 mutant in vivo.
... Among those, A33 is a 23 kDa, homodimeric type II transmembrane that undergoes both O-and N-glycosylation (N125 and N135) [32,36,39]. Both N-linked glycosylation sites are used in vaccinia but variola virus and monkeypox virus lack the equivalent N125 site [40]. ...
Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.
... Deletion of the gene that encodes any of these proteins, with the exception of A56 and K2, results in a small-plaque phenotype, implying that these proteins have a role in EV morphogenesis and/or infectivity. Several interactions among these proteins have been reported, and it appears that these interactions are required for the proper complement of viral proteins in the envelope of progeny EV (6,10,22,29,31,32,54,57). Among these proteins, the integral membrane protein A33 has been reported to interact with both A36 (52,57) and B5 (3,31). ...
There are two mechanisms for the incorporation of B5 into the envelope of extracellular virions produced by orthopoxviruses,
one that requires A33 and one that does not. We have hypothesized that the A33-dependent mechanism requires a direct interaction
between A33 and B5. In this study, chimeric constructs of A33 and B5/B5-green fluorescent protein (GFP) were used to show
that the two proteins interact through their lumenal domains and that the coiled-coil domain of B5 is sufficient for an interaction
with A33. Furthermore, our experiments reveal that a transmembrane domain, not necessarily its own, is requisite for the lumenal
domain of B5 to interact with A33. In contrast, the lumenal domain of A33 is sufficient for interaction with B5. Furthermore,
the lumenal domain of A33 is sufficient to restore the proper localization of B5-GFP in infected cells. Taken together, our
results demonstrate that the lumenal domains of A33 and B5 interact and that the interaction is required for the incorporation
of B5-GFP into extracellular virions, whereas the incorporation of A33 is independent of B5. These results suggest that viral
protein incorporation into extracellular virions is an active process requiring specific protein-protein interactions.
... Based on the A56 protein sequence, the calculated molecular mass of the protein is~35 kDa, but the protein contains putative sites for both N-and O-glycosylation and runs at an apparent molecular mass of 85 kDa when measured by SDS-PAGE (Brown et al., 1991;Payne, 1992;Shida & Dales, 1981). Thus, A56 is heavily glycosylated and this glycosylation is linked to its HA activity. ...
The vaccinia virus A56 protein was one of the earliest-described poxvirus proteins with an identifiable activity. While originally characterized as a haemagglutinin protein, A56 has other functions as well. The A56 protein is capable of binding two viral proteins, a serine protease inhibitor (K2) and the vaccinia virus complement control protein (VCP), and anchoring them to the surface of infected cells. This is important; while both proteins have biologically relevant functions at the cell surface, neither one can locate there on its own. The A56-K2 complex reduces the amount of virus superinfecting an infected cell and also prevents the formation of syncytia by infected cells; the A56-VCP complex can protect infected cells from complement attack. Deletion of the A56R gene results in varying effects on vaccinia virus virulence. In addition, since the gene encoding the A56 protein is non-essential, it can be used as an insertion point for foreign genes and has been deleted in some viruses that are in clinical development as oncolytic agents.
... The following criteria were used in choosing a candidate protein: Golgi localization during a natural infection, presence in IEV and EEV membranes, and requirement for EEV formation. The type 1 membrane glycoprotein encoded by the B5R gene (23,56,65) and the acetylated protein encoded by the F13L gene (2,31,48,58) have these characteristics. We then set another criterion: Golgi localization in the absence of other viral proteins. ...
The outer envelope of the extracellular form of vaccinia virus (EEV) is derived from the Golgi membrane and contains at least six viral proteins. Transfection studies indicated that the EEV protein encoded by the B5R gene associates with Golgi membranes when synthesized in the absence of other viral products. A domain swapping strategy was then used to investigate the possibility that the B5R protein contains an EEV targeting signal. We constructed chimeric genes encoding the human immunodeficiency virus (HIV) type 1 glycoprotein with the cytoplasmic and transmembrane domains replaced by the corresponding 42-amino-acid C-terminal segment of the B5R protein. Recombinant vaccinia viruses that stably express a chimeric B5R-HIV protein or a control HIV envelope protein with the original cytoplasmic and transmembrane domains were isolated. Cells infected with recombinant vaccinia viruses that expressed either the unmodified or the chimeric HIV envelope protein formed syncytia with cells expressing the CD4 receptor for HIV. However, biochemical and microscopic studies demonstrated that the HIV envelope proteins with the B5R cytoplasmic and transmembrane domains were preferentially targeted to the EEV. These data are consistent with the presence of EEV localization signals in the cytoplasmic and transmembrane domains of the B5R protein.
Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MV are further wrapped and secreted to spread infection within the host. However, late during infection MV accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titres. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate-limiting for EV production. The A26 gene is conserved within vertebrate poxviruses, but strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfil different functions represents a novel level of complexity in mammalian viruses with major impact on evolution, adaptation and transmission.
IMPORTANCE
Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intra-host dissemination and inter-host transmission, so virus maturation pathways must be tightly controlled. Here we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation towards the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses but those transmitted exclusively via biting arthropods reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.
Only a small subset of the hundreds of proteins encoded by the poxvirus genome have been shown to be effective as vaccine and/or therapeutic targets. One of these proteins is A33. Here we assess and dissect the ability of an anti-A33 humanized monoclonal antibody, c6C, to affect vaccinia virus infection in vitro. Enveloped virions (EV) released from infected cells can be sensitive or resistant to neutralization by c6C indicating there are different types of EV particles, extracellular enveloped virions (EEV) and released cellular-associated virions (rCEV), that are biologically distinct. Through a combination of plaque phenotype, confocal imaging, and neutralization assays, we found that c6C differentially affects EV from two different virus strains, IHD-J and WR. Evidence for an anti-A33 resistant EV particle, and strain differences in this phenotype, provides a logical answer as to why certain functional assays in the literature have been unable to detect anti-viral effects of anti-A33 antibodies.
Orthopoxviruses Pathogenic for Humans covers those viruses capable of causing disease in man, including monkeypox, smallpox, cowpox, and vaccinia. The coverage of each virus is comprehensive, covering the biology, molecular biology, and ecology of the virus as well as the clinical and epidemiological aspects of these viruses in humans and animals. In addition, this volume highlights developments in genetic engineering that are paving the way for potential therapeutic treatments of these viruses. © 2005 Springer Science+Business Media, Inc. All rights reserved.
Vaccinia virus encodes a 37-kDa palmitylated protein (p37) that is required for envelopment, translocation, and cell-to-cell spread of virions. We have analyzed the biological significance of the palmitate modification by constructing a recombinant vaccinia virus that expresses a nonpalmitylated p37 and comparing its biological activity to that of the wild-type virus. The mutant virus is inefficient at cell-to-cell spread and does not produce or release enveloped virions, although it produces normal amounts of nonenveloped virions. Furthermore, the mutant virus is not able to nucleate actin to propel itself through and out of the cell, a function requiring the indirect participation of p37. The deficiency in protein function appears to result from a lack of appropriate targeting to the membranes of the trans-Golgi network (TGN) which leaves p37 soluble in the cytoplasm. We conclude that the palmitate moiety is necessary for targeting or anchoring p37 to the TGN membrane, where, along with other vaccinia virus-encoded proteins, p37 is involved in the complex process of virion envelopment and release.
The vaccinia virus strain Western Reserve (WR) A34R gene encodes a C-type lectin-like glycoprotein, gp22-24, that is present in the outer membrane of extracellular enveloped virus (EEV) with type II membrane topology (S.A. Duncan and G.L. Smith, J. Virol. 66:1610-1621, 1992). Here we that a WR A34R deletion mutant (WR delta A34R) released 19- to 24-fold more EEV from infected cells than did WR virus, but the specific infectivity of the released virions was reduced 5- to 6-fold. Rupture of the WR delta A34R EEV outer envelope by freeze-thawing increased virus infectivity by five- to sixfold, because of the release of infectious intracellular mature virus. All other known EEV-specific proteins are incorporated into WR delta A34R EEV, and thus the loss of gp22-24 is solely responsible for the reduction of EEV specific infectivity. The WR delta A34R virus is highly attenuated in vivo compared with WR or a revertant virus in which the A34R gene was reinserted into WR delta A34R. This attenuation is consistent with the known important role of EEV in virus dissemination and virulence. Vaccinia virus strain International Health Department-J (IHD-J) produces large amounts of EEV and forms comets because of an amino acid substitution within the A34R protein (R. Blasco, R. Sisler, and B. Moss, J. Virol. 67:3319-3325, 1993), but despite this, IHD-J EEV has a specific infectivity equivalent to that of WR EEV. Substitution of the IHD-J A34R gene into the WR strain induced comet formation and greater release of EEV, while coexpression of both genes did not; hence, the WR phenotype is dominant. All orthopoxviruses tested express the A34R protein, but most viruses, including variola virus, have the WR rather than the IHD-J A34R genotype. The A34R protein affects plaque formation, EEV release, EEV infectivity, and virus virulence.
Protein A33 is a type II membrane protein present in the outer envelope of extracellular as well as cell-associated Vaccinia virus particles. A33 has been implicated in mediating cell-to-cell virus spread in an antibody-resistant manner. Here, using state-of-the-art structure prediction methods and structural modeling, we show that A33 has most likely evolved from a C-type lectin-like protein. Comparison of the three-dimensional A33 model to the X-ray structures of distant cellular homologues revealed that A33 retained the key residues required for adopting the C-type lectin-like fold. Our results provide insights into the structure and origin of protein A33.
The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative
vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus
protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus.
The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal
lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the
dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33
dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell
receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is
well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously
described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a
protective antibody.
Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Thesis (Ph. D.) -- University of Maryland, College Park, 2006. Includes bibliographical references. Text.
A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.
When cells were infected with vaccinia virus in the presence of [3H]palmitic acid, radiolabel was incorporated into six viral proteins with apparent molecular weights of 92, 41, 37, 26, 17, and 14 kDa, all of which are expressed at late times during the infection cycle. The [3H]palmitate-labeled fatty acid moieties from the modified proteins were isolated, converted to p-nitrophenacyl derivatives, and subjected to reverse phase HPLC analysis which confirmed the identity of the fatty acid group as palmitic acid. Furthermore, the radiolabeled palmitate-protein bonds were sensitive to treatment with neutral hydroxylamine, suggesting that association of the fatty acid moieties with these proteins occurs via a thioester linkage. Previous studies by other investigators have identified the 37-kDa protein as the major antigen present in the outer membrane of extracellular enveloped virions, and demonstrated that the protein is modified by palmitic acid but is not glycosylated (G. Hiller and K. Weber J. Virol. (1985) 55, 651-659). Growth of vaccinia virus in the presence of tunicamycin indicated that the 41- and 26-kDa palmitylated proteins were also subject to modification by glycosylation, whereas like the 37-kDa protein, the 92-, 17-, and 14-kDa species did not appear to be glycosylated. Subcellular fractionation studies provided evidence that all of the viral palmitylated proteins were membrane-associated. Extraction of purified vaccinia virus with NP-40 and DTT demonstrated that the palmitylated proteins were associated with one of the viral membranes rather than the core of the virion. Viewing these results together with the previous reports of myristylated VV proteins (Franke et al. J. Virol. (1989) 63, 4285-4291), suggests that acylation of VV proteins represents a major modification pathway utilized by VV proteins during the assembly of progeny virions.
Primers based on sequences flanking the vaccinia virus (VV) strain IHD hemagglutinin protein (HA) open reading frame (ORF) enabled amplification of HA DNA segments from the genome of raccoon poxvirus (RCN) and VV strain WR. The amplified segments produced unequal cross-hybridization signal intensities against each other, indicating sequence differences between the HA of RCN (in HindIII-G) and that of VV-WR (in HindIII-A). About 1.5 kb of sequences in the HA region were then determined from clones pRCN/HindIII-G and pVV/BamHI-32, a subclone of VV-WR HindIII-A. Pairwise analyses of the RCN and VV-WR HA nucleotide sequences showed 82, 66, and 86% homology, respectively, between the putative promoter, ORF, and transcript terminator regions and 53% homology between the deduced amino acid sequences of the HA of RCN (310 residues) and those of VV-WR (314 residues). The deduced HA amino acid sequences showed a putative signal peptide and transmembrane-anchor moiety of 70 and 62% homology and a rather distinct central domain (residues 146 to 254) of 32% homology. Additional hybridizations with the amplified segments described above enabled location of the HA gene in the HindIII-A fragment of the orthopoxviruses volepox virus (VPX) and skunk poxvirus (SKP); however, amplified DNA of either the entire HA ORF of RCN or that of VV-WR, or a portion, from the center to right end, did not hybridize with VPX or SKP, suggesting that the HA of RCN, VV, VPX, and SKP are rather diverged from each other. The VV HA was found to be closely related to that of ectromelia and variola viruses. The data are consistent with reports of hemagglutination-inhibition partial cross-reactivity between RCN, VPX, VV, and other orthopoxviruses and might lead to an explanation of the basis of syncytia formation by RCN, VPX, and SKP.
Comparison of the genomic organization of variola and vaccinia viruses has been carried out. Molecular factors of virulence of these viruses is the focus of this review. Possible roles of the genes of soluble cytokine receptors, complement control proteins, factors of virus replication, and dissemination in vivo for variola virus pathogenesis are discussed. The existence of "buffer" genes in the vaccinia virus genome is proposed.
The major envelope antigen of vaccinia virus is an acylated protein of M(r) 37,000 (p37K) which is required for the formation of extracellular enveloped virions (EEV). Despite its important role in the wrapping process, p37K has not been studied in much detail. In order to better characterize this protein we have undertaken a detailed biochemical analysis. Sodium carbonate treatment showed that p37K is tightly bound to the viral envelope. Its resistance to proteinase K digestion indicates that it is not exposed on the surface of EEV but lines the inner side of the envelope. Since p37K does not contain a signal peptide characteristic of most membrane proteins, we examined the possibility that the protein acquires its membrane affinity through the addition of fatty acids. Indeed, Triton X-114 phase partitioning experiments demonstrated that p37K is hydrophobic when acylated, but hydrophilic in the absence of fatty acids. Three other viral proteins have been shown to be required for virus envelopment and release from the host cell and we therefore tested whether p37K interacts with viral proteins. In EEV and in absence of reducing agents, an 80-kDa complex reacting with an anti-37K antiserum was found. Analysis of this complex showed that it most likely consists of a p37K homodimer. Interestingly, only a small amount of p37K occurs as a complex, most of it is present in the viral envelope as monomers.
A 14-kDa gene-specific probe of vaccinia virus Western Reserve (WR) hybridized to homologous sequences in the genomes of the orthopox virus species cowpox, camelpox, mousepox, and monkeypox virus. The corresponding genes were mapped and sequenced. Homologies of more than 95% were found when compared to the 14-kDa gene of vaccinia virus WR. However, point mutations which led to alterations in the amino acid sequences were mainly located between residues 26 and 40. By use of synthetic peptides, this part of the 14-kDa fusion protein could be identified as the binding site for four different neutralizing monoclonal antibodies.
Monoclonal antibodies (MAbs) were generated against vaccinia virus, cowpox virus KR2 Brighton, monkeypox virus Copenhagen, or ectromelia virus. Pairwise epitope specificity studies by competition ELISAs identified 23 distinct antigenic sites in 19 different orthopox virus strains. Six epitopes were completely independent of each other, and 17 closely related antigenic sites formed three separate epitope complexes. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes in the envelope of intracellular naked virus, 16 MAbs recognized proteins of 32, 30, 16 or 14 kDa in Western blotting (WB), and 9 MAbs neutralized virus infectivity. In rabbitpox virus (RPV) 18 epitopes were detected. A lambda gt11 expression library of RPV DNA was screened with the corresponding 18 MAbs. Fourteen recombinant bacteriophage clones (ph) were isolated. Cross-hybridizations of phage and RPV DNA demonstrated a reaction with the HindIII A, HindIII D, or HindIII H fragments, respectively. DNA of ph3D was related to the A25L gene, which corresponds to the A-type inclusion body gene of cowpox virus. Two phage clones contained sequences of the 14-kDa fusion protein gene (A27L gene). Ph1A contained nearly the entire 14-kDa gene encoding 4 neutralizing (neutr) and 2 nonneutr epitopes. Ph5, expressing only half of this gene product, encoded 1 nonneutr epitope. The fusion protein of vaccinia virus MVA was isolated by immune-affinity chromatography with a neutr. catching MAb. The protein formed hollow rods (ELMI) and the 6 antigenic sites that were present were identical to those expressed by Escherichia coli infected with ph1A. WB detection with a polyclonal hyperimmune serum detected protein bands of 54, 32, 30, 16, and 14 kDa. The catching MAb bound only to a 16-kDa band. The purified fusion protein induced neutralizing antibodies in mice and rabbits.
We identified a protein component of the intracellular mature vaccinia virion membrane that is a target of a potent neutralizing monoclonal antibody, 7D11, obtained from Alan L Schmaljohn. By immunofluorescent and electron microscopic analysis, MAb 7D11 was found to stain intracytoplasmic viral factories, virion membranes in cell sections, and the surface of negatively stained preparations of purified virions. The MAb 7D11 antigen, which is synthesized at late times in infection, has apparent molecular masses of 25 and 29 kDa under nonreducing and reducing conditions, respectively. The membrane antigen was most efficiently extracted from virions by NP40 detergent in combination with a reducing agent; in addition, the protein partitioned exclusively into the detergent phase when extracted with Triton X-114. Although the N-terminus of the immunoaffinity-purified protein was blocked, sequence analysis of trypic peptides revealed that the MAb 7D11 antigen was identical to the myristylated protein encoded by the L1R open reading frame previously described by C.A. Franke, E.M. Wilson, and D.E. Hruby (1990, J. Virol. 64, 5988-5996). Validation of this genetic assignment was provided by the ability of MAb 7D11 to immunoprecipitate a [3H]myristic acid-labeled product of the expected molecular weight from infected cells. In addition, we discovered that the previously described neutralizing monoclonal antibody 2D5 (Y. Ichihashi, T. Takahashi, and M. Oie, 1994, Virology 202, 834-843) also recognizes the L1R protein.
Introduction. Vaccinia virus is the vaccine that was used to eradicate smallpox. This task was accomplished in 1977 and its completion certified in 1979 by the World Health Organization (WHO) (Fenner et al., 1988). Since then poxvirus infections of humans have caused little disease and have been restricted to molluscum contagiosum and rare zoonoses such as cowpox (CPV), pseudocowpox, monkeypox, orf and yaba tumour viruses. Continued interest in vaccinia virus and other poxviruses has derived in part from the development of these viruses as cloning and expression vectors (Mackett et al., 1982; Panicali & Paoletti, 1982) that have the potential as live vaccines to combat diseases other than smallpox (Panicali et al., 1983; Smith et al., 1983 a, b, 1984; Paoletti et al., 1984).
The fungal metabolite Brefeldin A was found to inhibit the production of the infectious enveloped form of vaccinia virus, although production of the infectious intracellular form was not affected. Electron microscopic analysis and caesium chloride density centrifugation of progeny virions indicates that the drug block is not due to retention and accumulation of enveloped virions within the cell. Biochemical analysis of the candidate envelopment receptor for vaccinia virus, viral protein P37, shows that the drug has no discernible effect on palmitylation of this protein and does not prevent or alter its association with intracellular membranes. This suggests that P37 may not in fact be the receptor on intracellular membranes for vaccinia virus envelopment, and leaves open the question of what function this molecule performs in the envelopment process. 24AI 20563
Sequencing and computer analysis of a variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region has been carried out. Fifty-nine potential open reading frames (ORFs) of over 60 amino acid residues were identified. Structure-function organization of the VAR-IND DNA segment was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple differences between VAR-IND and the strains of VAC but the high identity of VAR-IND with VAR-HAR in the genetic maps are revealed. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.
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.
Wild-type vaccinia virus WR strain forms non-fusogenic (F-) large plaques and is hemagglutinin positive (HA+) under normal conditions of virus infection. We have analyzed a collection of spontaneous, highly attenuated mutants of vaccinia virus isolated from persistently infected Friend erythroleukemia cells (E. Paez, S. Dallo, and M. Esteban, J. Virol. 61, 2642-2647, 1987) for the ability to express HA during virus infection. After 14 cell passages, all the mutants isolated were hemadsorption negative (HAD-) and did not synthesize a HA that could be recognized by anti-HA monoclonal antibodies. All these HA- mutants induced extensive cell-cell fusion (F+), with the exception of two mutants (65-16 and 101-14) isolated from late cell passages. Nucleotide sequence analysis of the HA gene in these two mutants confirmed the HA- phenotype. A frameshift mutation very close to the initiation codon resulted in premature translational termination. The truncated gene now only encodes the first 25 amino acids. Analysis of progeny from "wild-type," like early serial passage virus (5-3) X mutant back crosses, shows that for one late passage non-fusogenic small-plaque mutant (101-14) among large plaque progeny there is good correspondence between the ability to fuse and the absence of a viral HA and that each large plaque mutant contains a normal 14 kDa membrane protein. However, with a second serial passage mutant 65-16, which, like 101-14, is a nonfusogenic small-plaque variant, there is again an excellent correlation between the inability to synthesize HA and the ability to fuse, but there is no correlation of plaque size with a normal 14 kDa viral membrane protein, as most large plaque mutants encode a larger, i.e., 17 kDa protein. Rescue experiments of 65-16 with bona fide cloned 14 kDa protein gene confirm that the ability to regulate plaque size and cell fusion in this mutant is due to a protein other than the 14 kDa protein. Marker rescue experiments indicated that the map position of the additional mutation coincided with a small deletion occurring in the HindIII F fragment. This deletion affected the 5'-end and promoter sequences of the 37 kDa envelope protein gene and produced a lack of expression of this protein, shown by others to be involved in the formation of extracellular enveloped virus in infected cells. These results shown that viruses with mutations of viral membrane proteins required for cell fusion and viral dissemination are selected during vaccinia virus persistence in cell culture.
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 strains vary considerably in the amounts of extracellular enveloped virus (EEV) that they release from infected cells. The IHD-J strain produces up to 40 times more EEV than does the related WR strain and consequently generates elongated comet-shaped virus plaques instead of sharply defined round ones in susceptible monolayer cells under liquid medium. The difference in EEV formation is due to the retention of enveloped WR virions on the cell surface (R. Blasco and B. Moss, J. Virol. 66:4170-4179, 1992). By using WR and IHD-J DNA fragments for marker transfer and analyzing the progeny virus by the comet formation assay, we determined that gene A34R and at least one other gene regulate the release of cell-associated virions. Replacement of the A34R gene of WR with the corresponding gene from IHD-J increased the amount of EEV produced by 10-fold and conferred the ability to form distinctive comet-shaped plaques. Gene A34R encodes an EEV-specific glycoprotein with homology to C-type animal lectins (S.A. Duncan and G.L. Smith, J. Virol. 66:1610-1621, 1992). The nucleotide sequences of the A34R genes of WR and IHD-J strains differed in six positions, of which four were silent. One of the codon mutations (Lys-151-->Glu), which is located in the putative carbohydrate recognition domain, was sufficient to transfer a comet-forming phenotype to WR virus. These data indicate that the A34R-encoded glycoprotein is involved, through its lectin homology domain, in the retention of progeny virus on the surface of parental cells and raise the possibility that the protein also has a role in virus attachment to uninfected cells.
Vaccinia virus gene B5R encodes a M(r) 42K glycoprotein that is expressed throughout infection and forms part of the envelope of extracellular virus. In this paper deletion mutants (delta B5R) lacking the B5R open reading frame (ORF) from the Western Reserve (WR) and IHD-J strains of vaccinia virus have been constructed and shown to form very small plaques compared with the wild-type viruses. This phenotype was directly attributable to loss of the B5R gene since re-insertion of this gene from WR or IHD-J into the WR mutant lacking B5R (W-delta B5R) restored a normal plaque phenotype. In the latter case the failure of the revertant to form comets indicated that the nine amino acid differences in the B5R ORF between the IHD-J and WR strains of virus are not responsible for comet formation by IHD-J virus. Furthermore, the B5R deletion mutant of IHD-J (I-delta B5R) still formed small comets. Despite the small plaque phenotype of the deletion mutants, normal yields of intracellular naked virus (INV) were produced. In contrast, deletion of B5R had a profound affect on the formation of the extracellular enveloped virus (EEV). Transmission electron microscopy indicated that INV particles were not wrapped by a double layer of Golgi-derived membrane and enveloped particles were not detected within the cell or on the cell surface without expression of the B5R protein. Biochemical measurement of EEV formation, by labeling infected cells with [3H]thymidine followed by cesium chloride density gradient centrifugation of particles released from the cells 24 hr postinfection, showed that only 10% of WT levels of EEV were produced by I-delta B5R. The loss of the B5R ORF caused severe attenuation in intranasally infected mice. At doses between 10(4) and 3 x 10(7) plaque-forming units there were no signs of disease in animals infected with W-delta B5R, whereas at comparable doses the WR parent virus caused significant mortalities. Finally, an ORF with 93.4% amino acid identity to vaccinia WR B5R is present in variola major virus strain Harvey and the B5R protein was shown by Western blotting to be expressed by all orthopoxviruses tested.
We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.
The structural polypeptides of the isolate VR-2332 of porcine reproductive and respiratory syndrome virus were analyzed in sucrose gradient-purified virions. The virus had an average density of 1.15 g/cm3 and contained, by SDS-PAGE, three major polypeptides with apparent molecular weights of 15, 19 and 26-30 kDa, which were designated as nucleocapsid (N), matrix (M) and envelope (E), respectively. The predominant structural protein was N. N-glycosidase F digestion only affected E whereas O-glycosidase or endoglycosidase H digestion had no effect, suggesting that the viral glycoproteins contain only complex N-linked carbohydrates.
Computer-assisted alignment of known palmitylproteins was used to identify a potential peptide motif, TMDX1-12AAC(C)A (TMD, transmembrane domain; X, any amino acid; C, cysteine acceptor residues; A, aliphatic residue) responsible for directing internal palmitylation of the vaccinia virus 37-kDa major envelope antigen, p37. Site-directed mutagenesis was used to confirm this motif as the site of modification and to produce a nonpalmitylated version of the p37 protein. Comparative phenotypic analysis of the wild-type and mutant p37 alleles confirmed that the p37 protein is involved in viral envelopment and egress, and suggested that attachment of the palmitate moiety was essential for correct intracellular targeting and protein function.
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.
The rabbitpox virus (RPV) B5R protein, synthesized late in infection, is found as a 45-kDa membrane-associated protein of the envelope of infectious extracellular enveloped virus (EEV) and as a 38-kDa protein secreted from the cell by a process independent of morphogenesis. The protein is not found associated with intracellular mature virus (IMV). Deletion of the gene attenuates the virus (RPV delta B5R) in animals (mice and rabbits), has relatively little effect on formation of IMV, prevents EEV formation in some but not all cells, and leads to a reduced host range. Analysis of the sequence of the protein suggests relatedness to factor H of the complement cascade. Collectively, these observations suggest that attenuation of the virus in vivo could be linked to an inhibition of the inflammatory response, a deficiency in growth, or both. In this report we have analyzed the behavior of RPV delta B5R in infected mice and rabbits and conclude that attenuation of the mutant virus likely results from simple failure to grow within the infected animal and that the inflammatory response probably contributes little to the observed attenuation.
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.
The vaccinia virus hemagglutinin (HA) is a glycoprotein found on the plasma membrane of infected cells and the envelope of extracellular virus. Two forms of HA (85 and 68 kDa) are detected by immunoblot analysis. Although hemagglutination activity is only readily detectable late in infection, the 85-kDa HA appears early and accumulates throughout infection, whereas the 68-kDa form appears only late in the cycle. Production of the 68-kDa HA but not the 85-kDa HA was inhibited by either cytosine arabinoside or rifampin. Analysis of HA gene expression reveals a complex pattern of expression. The HA gene is transcribed early to yield a 1.65-kb dicistronic early transcript, consisting of the 945-bp HA open reading frame (ORF) fused to a 453-bp downstream ORF. Transcription from this site initiates 7 bases upstream of the AUG initiating codon of the HA ORF. Due to the discrepancy between the calculated size of the HA protein (33 kDa) and that reported for the unglycosylated HA protein derived from in vitro translation (58 kDa), we placed an early transcription termination signal (TTTTTAT) directly downstream of the 945-bp HA ORF. This led to a reduction in size of the early HA mRNA to 1.2 kb, as expected, but had no effect on the formation of either the 85- or 68-kDa protein. Transcripts originating from the early promoter are found throughout the infection cycle. However, after DNA replication, transcription from a second, late promoter ensues. The transcriptional start site of the late promoter is within a consensus TAAATG sequence located 135 bases upstream of the transcriptional start site of the first promoter. The late transcriptional start site is also found within an upstream ORF.
Systematic characterization in some depth of vaccinia virus-induced hemagglutinin activity (HA) was undertaken using a series of solubilization and chromatographic steps. HA labeled with either [35S]methionine or [3H]glucosamine or [3H]fucose was extracted with nonionic detergent from isolated plasma membranes (PM), then purified sequentially on DEAE-cellulose, WGA-Sepharose, and Bio-Gel A-5m columns. Following reconstitution of the final fraction with lecithin the HA was shown to reside exclusively in a 85K glycoprotein. Nature of the glycosidic bonds linking the oligosaccharide chains to the protein backbone was determined by means of tunicamycin, an inhibitor of glycosylation, and following alkali hydrolysis. In cells treated with tunicamycin the HA glycopeptide isolated from PM was reduced in MW from 85 to 65K, its carbohydrate content was diminished about 75% but its biological activity was unaffected, indicating that approximately three-fourths of the sugar chains attached via N-glycosidic bridges are not involved in hemagglutination. Peptide mapping indicates no evidence of alteration in the underglycosylated protein backbone. About 25% of the oligosaccharide within intact HA and virutally all of the carbohydrate remaining in the 65K product from tunicamycin-treated cells was susceptible to alkali-borohydride hydrolysis. Partial (5–50%) hydrolysis of the oligosaccharide using varying concentrations of a protease-free mixture of glycosidases abolishes the HA from both the 85K glycoprotein and a 65K underglycosylated product. This result implies that about one-fourth of the oligosaccharide chains which are linked O-glycosidically are responsible for the biological activity of the molecule. Thus vaccinia-induced HA is an 85K glycoprotein in which N-linked oligosaccharides predominate but the O-linked chains participate in expression of the biological activity.
The hemagglutinin negative (HA−) strain IHD-W of vaccinia virus was shown to induce a defective glycopeptide. The serorelatedness between HA and the altered molecule could be demonstrated by use of specific anti-HA antisera. The HA− antigen isolated from immunoprecipitates was analyzed by means of “Western” blots, peptide mapping, using liquid chromatography and antigen competition, in conjunction with radioimmuno assay. Despite the reduced molecular weight (MW) of the polypeptide in the HA− 41K glycoprotein as compared with that of the polypeptide in the normal 85K HA, antigenicity was conserved. Absence of HA activity from the 41K defective molecule was related to the presence of only the N-type glycosidic links between the carbohydrate and asparagine residues in the polypeptide. Following synthesis, the active HA molecule was transfered to the cell surface but the defective glycoprotein accumulated intracellularly, probably in association with the smooth membrane reticulum. This implies that vectorial transfer to the cell surface was defective in the case of the mutant molecule. In vitro translation produced the unglycosylated form of the HA polypeptide, 57K in MW and the HA− polypeptide, 30K in MW. With the latter product there was a 2K difference in MW between the protein synthesized in vitro and the 28K one, formed during in vivo infection in the presence of tunicamycin. This result suggests that reduction in MW may involve cleavage of a peptide, perhaps a “signal” peptide, during formation of the glycoprotein. When considered altogether, these observations indicate that mutation, in the gene encoding the HA resulted in profound changes in molecular structure and cytopathology. The mutation caused changes in both the protein and carbohydrate moieties of the molecule, failure of the nascent glycoproteins to move vectorially to the cell surface or produce hemagglutination and finally changed the cytopathology from that of cell rounding to syncytium formation.
The relationship between vaccinia virus-specified hemagglutinin (HA) and cell fusion (F) was studied using strain IHD-J which is HA+ and F− and strain IHD-W which is HA− and F+. Studies with specific antisera and inhibitors of virus biogenesis indicated that the substance which induces fusion is carried to the cell surface from within host cells by mature progeny particles. A variety of experiments involving double infection or the interaction of cells mixed in culture after single infection with the 2 vaccinia strains led to the conclusion that a relationship exists between acquisition of HA at the cell surface and inhibition of fusion. Perhaps presence of HA modified the plasma membrane in a way that inhibited the expression of F. Sendai virus inactivated by ultraviolet-induced polykaryocytosis of cells infected by IHD-J vaccinia, suggesting that the mechanism for inducing fusion is different in these two agents.
SUMMARY Extracellular rabbitpox virus released naturally from infected cells differed antigenically from intracellular virus released by artificial disruption of cells. Intracellular virus was neutralized by antiserum prepared against live rabbitpox virus and by antiserum against inactivated vaccinia virus. In contrast, extracellular virus was neutralized only by rabbitpox antiserum. The antibodies responsible for the neutralization of intracellular and extracellular virus could be absorbed separately from rabbitpox antiserum. Morphologically, extracellular virus differed from intracellular virus in possessing an outer envelope. This envelope was probably the site of the virus antigen characteristic of extracellular virus, and fluorescent antibody staining of infected cells suggested that it was derived from the modified host cell membrane. Antibody directed against extracellular virus was responsible for the ability of rabbitpox antiserum to control the spread of rabbitpox virus in tissue culture and probably for its ability to protect rabbits from rabbitpox infection. Extracellular virus should therefore be used as the test virus in titrations of neutral- izing antibody if these are to assess the protective activity of an antiserum.
The polypeptides of vaccinia were separated and analyzed by two-dimensional (2-D) gel electrophoresis patterned after that of O'Farrell (1975). Following labeling with [35S]-methionine, [33P]phosphate, or [3H]glucosamine, pure virions were dissociated and subjected to electrophoresis using either isoelectric focussing or nonequilibrium pH gradient conditions in the first dimension and SDS-polyacrylamide slab gels in the second dimension. By this means at least 111 spots, of which 7 or more were basic proteins, were resolved in the autoradiograms. Authenticity of several single spots was established. This included a glycoprotein of molecular weight 34,000 (34 K) labeled with [3H]glucosamine; a phosphorylated basic protein of about 11 K marked with 33PO4; isolated purified surface tubular elements of 58 K; two major core polypeptides of 60 and 62 K derived from larger precursors after proteolytic cleavage; a precursor polypeptide of 25 K known from previous studies with a ts mutant 1085 to undergo cleavage; and an 18 K polypeptide which appears in wild type and ts 1085 infections under circumstances permissive for cleavage. The 2-D analysis therefore reveals that poxviruses are in terms of their polypeptides, even more complex than had been anticipated previously.
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.
The relationship of vaccinia haemagglutinin (HA) to extracellular enveloped virus (EEV) was examined. EEV banded in caesium chloride gradients at a density of 1.23 to 1.24 g/ml coincident with a peak of HA activity. EEV of an HA+ vaccinia strain showed greater than 90% adsorption to rooster red blood cells (RBCs) as detected by infectivity and 3H-thymidine labelling whereas intracellular naked virus (INV) of the HA+ strain and EEV of an HA- strain failed to show significant adsorption. The adsorption was specifically inhibited by antiserum to vaccinia. Adsorption kinetic experiments demonstrated a lack of temperature dependence on the total amount of EEV adsorbed. No elution of EEV from RBCs could be detected. The capacity of EEV to adsorb to RBCs was found to be stable at 56 degrees C for 30 min.
Vaccinia virus IHD-J strain induces hemagglutinin (HA) on the surface membrane of infected cells and does not elicit cell-cell fusion (F-). We isolated 21 independent hemadsorption-negative (HAD-) mutant viruses from IHD-J and five HAD+ revertants from one of these mutants. Of the 21 mutants, 19 that synthesized either no or little HA at the cell surface caused cell-cell fusion (F+), whereas none of the five revertants that synthesized HA at the cell surface induced cell-cell fusion. Furthermore, anti-HA monoclonal antibody B2D10 induced extensive polykaryocytosis of IHD-J-infected cells and suppressed the ability of the IHD-J-infected cell extract to inhibit the polykaryocytosis induced by IHD-W. The other 2 of the 21 HAD- mutants, B1 and A2, which induced HAs at the cell surface, showed F- and F+ phenotype, respectively. The HA molecule of mutant B1 had a single amino acid substitution of Lys for Glu-121 in its extracellular domain, whereas that of mutant A2 had a single substitution mutation of Tyr for Cys-103. We conclude that the vaccinia HA is a fusion inhibition protein, that the active sites for the two activities reside separately in its extracellular domain, and that cysteine-103 is important in forming the proper tertiary structure of the protein to exert both activities.
The protein and glycoprotein composition of a sucrose gradient fraction from vaccinia infected cells treated with rifampicin was studied. This particulate fraction contained cytoplasmic membranes and pleomorphic membranous structures. The glycoproteins (89, 42 and 20-23 kDa, respectively) were identified as the same glycoproteins that are found in plasma membranes of infected cells and the envelope of extracellular enveloped vaccinia (EEV). These glycoproteins could be solubilized by 0.1% NP-40. The Golgi membrane associated 41K acylated vaccinia protein was also NP-40 soluble. In contrast, most particulate fraction proteins (125, 100, 86, 65, 41, 39, 31, 27, 25, 14 and 12.5 kDa) with the exception of the 33 and 29 kDa proteins remained essentially insoluble after NP-40 treatment. The 86 and 65 kDa proteins are the rifampicin inhibited precursors to INV core proteins while the 33 and 29 kDa proteins are INV surface proteins. Twelve proteins behaved like their respective comigrating INV proteins when extracted with NP-40 and 2ME. Electron microscopy showed that a centrifuged sediment from NP-40 treated cells contained pleomorphic protein containing membranous structures that we have called rifampicin bodies. We conclude that (1) the major glycoproteins found in the particulate fraction from sucrose gradients are vaccinia glycoproteins residing in cytoplasmic membranes while (2) the major non-glycosylated proteins are components of the rifampicin bodies and that (3) the rifampicin bodies represent an intermediate in the morphogenetic process leading to mature INV.
The vaccinia virus hemagglutinin (HA) has specific affinity for the structural protein, VP37K. The nature of this affinity and its relationship to the function of the HA were analyzed using HA mutants. The VP37K reactive site of the HA molecule is located in its transmembrane region, and the vaccinia virus HA associates with the viral particle via the VP37K-HA affinity. The viruses possessing an HA with fusion inhibitor activity were largely of the low infectivity form, whereas the viruses that associated mutant HAs defective in the activity were of the high infectivity form. D1 mutant virus does not produce HA. When it was incubated with the HA of the IHD-J strain, the HA associated with the virus particle. The HA-loaded D1 mutant virus acquired a high affinity not only for chick erythrocytes but also for KB and Vero cells. At the same time, the infectivity for Vero cells was decreased. The original high infectivity was recovered by treatment with trypsin. The virion-associated vaccinia HA has two functions; the HA protects the infectivity of the virus by the fusion inhibitor activity and exhibits affinity against host cells. Vaccinia virus first adsorbs to the cell via HA, and then proteolysis of the HA activates the second adsorption site which seems to be the fusogenic site of the virus. Proteolytic activation represents removal of the fusion inhibitor activity of the HA.
Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.
Vaccinia virus hemagglutinin (HA) is expressed at late time of infection cycle, and it is nonessential for virus growth. Location of the HA structural gene was determined by hybrid-arrested and hybrid-selected translation methods at the right terminus of the HindIII A fragment. The position of the HA gene was confirmed by the production of the complete HA protein in the cells transfected with the plasmid containing that region. Examination of this nucleotide sequence revealed the positions of cleavage sites for a number of restriction endonucleases. The deduced amino acid sequence revealed that the HA protein is a member of typical surface membrane glycoproteins. Comparison of the nucleotide sequence upstream of the HA coding region with corresponding region of other late genes suggested the existence of the consensus decanucleotides TTCATTTa/tGT between 34 to 18 bp upstream to the initiation codon followed by a cluster of A or T, a unique feature of the late genes of vaccinia virus. These results in conjunction with the ease of isolating HA- mutants provide a basis for a new site suitable for inserting foreign genes.
The release of vaccinia virus from mouse nasal epithelial cells infected in vivo was studied by electron microscopy. Intracellular naked vaccinia virus was enwrapped by Golgi membranes to form a double membrane intermediate. The outer membrane of the intermediate presumably fused with the plasma membrane, releasing extracellular enveloped virus. No signs of simple naked virus budding at the plasma membrane were observed. The majority of extracellular virus was enveloped and not naked.
A 37,000-dalton polypeptide (p37K) present on purified extracellular vaccinia virus but absent from intracellular virus particles of classical morphology (G. Hiller et al., J. Virol. 39:903-913, 1981; L. G. Payne, J. Virol. 27:28-37, 1978) was further characterized. The polypeptide was only expressed in infected cells after onset of viral DNA replication. Phase partition experiments showed that it is relatively hydrophobic. Although p37K apparently is not a glycoprotein, in vivo radioisotope labeling detected tightly associated palmitic acid. Antibodies to p37K were used to monitor its distribution within infected cells at the light and electron microscopic levels. After synthesis p37K first accumulated in the Golgi region due to a tight membrane association. During progressing infection p37K-carrying membranes were used to form double-walled envelopes around brick-shaped vaccinia particles. Within these specialized vesicles vaccinia particles were moved through the cytoplasm toward the cell's surface, presumably along cellular routes for certain secretory products. Finally, single enveloped viruses were released into the extracellular space by an exocytotic process.
Endo-beta-N-acetylglucosaminidase F (Endo F) and peptide:N-glycosidase F (PNGase F) were purified from cultures of Flavobacterium meningosepticum by ammonium sulfate precipitation followed by gel filtration on TSK HW-55(S). This system separated the two enzymes and provided PNGase F in a high state of purity, but the basis for the resolution appeared to be hydrophobic interaction and not molecular size. Studies using purified Endo F and PNGase F with defined glycopeptides demonstrated that Endo F was somewhat similar to Endo H in that it hydrolyzed many, but not all, high-mannose and hybrid oligosaccharides, as well as complex biantennary oligosaccharides. PNGase F, in contrast, hydrolyzed all classes of asparagine-linked glycans examined, provided both the alpha-amino and carboxyl groups of the asparagine residue were in peptide linkage. Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.
Vaccinia virus-induced antigens and polypeptides labelled late in infection with [14C]-leucine were examined in gel precipitin tests and polyacrylamide gel electrophoresis followed by autoradiography. Pulse-chase experiments, investigations on the effects of rifampicin and direct analysis of precipitin lines by gel electrophoresis allowed the identification of two major precipitin lines in terms of the polypeptides specific for the infected cell. One of these was non-structural. A small proportion of the other antigen was incorporated into sedimentable structures during a chase; this was prevented by rifampicin. Evidence suggesting that three virus-specified polypeptides are formed by cleavage of larger precursors is also presented and discussed.
The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn & Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.
A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week.
When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
A vaccinia virus core polypeptide, with a molecular weight of 76,000 and a relative deficiency in tryptophan, was shown by pulse-chase experiments to form from a precursor. The latter may be a rapidly labeled, 125,000-molecular weight, tryptophan-deficient, virus-induced polypeptide, which diminished in quantity during the chase period and was barely detectable after two to three hours.
Rifampicin completely prevented the formation of the core polypeptide without inhibiting the synthesis of the precursor. A rifampicin-resistant vaccinia mutant was used to demonstrate the specificity of this effect. The sequence of events after the removal of the drug suggested that cleavage of the precursor occurs during the formation of the virus core. Rifampicin appears to act by interrupting earlier maturational events which precede the formation of the core polypeptide.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Sendai virus injected intracerebrally into 3-week-old mice caused infection of ependymal and choroid plexus epithelial cells. Budding of mature viruses occurred only from the apical surfaces of these cells. The viral peplomere proteins, haemagglutinin-neuraminidase and fusion, were concentrated on the apical portion of the ependymal cells, while the nucleocapsid-associated polymerase protein was dispersed throughout the cytoplasm. This indicates that intracellular routing of the virus envelope glycoproteins to the cell surface may be one of the factors that determines the site of virus budding. Vesicular stomatitis and vaccinia viruses, on the other hand, budded or egressed predominantly from the baso-lateral cell surfaces.
A myeloma line has been developed which produces no globulin chains of its own, has a duplication of 8.7 h, fuses effectively with B-lymphoblasts and produces stable hybrids. An enhancing effect of macrophages on hybridoma yields has been observed. Among the fusing agents tested, PEG of mol.wt. 4000 gave the best results, 20 degrees C being the optimum working temperature. The maintenance medium of choice has been found to be Iscove's with 10% FCS. Direct exposure of fusion cultures to a selective medium with hypoxanthine, aminopterine and thymidine reduced the labor involved and increased the yield. A mechanical device for changing the medium has been designed. The replacement of standard trays by microtrays resulted in a higher frequency of surviving hybrids. By using a feeder layer, the spleen cell input can be reduced 50-fold. At such low multiplicities the positive cultures arise predominantly from single hybrids, eliminating the need for subsequent cloning. The hybrids can be labelled and will yield in serum-free medium. Since at least a third of them inherit the fast growth rate of their myeloma parent and keep producing over 2000 antibody molecules per second, readaptation to ascitic growth is also superfluous. A simplified technique of producing monoclonal antibodies is given in detail, together with the experimental evidence prompting modifications of the classical method of Köhler and Milstein (1975, 1976).
Structural proteins of vaccinia virus (IHD-J strain) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. Glycoproteins and phosphoproteins were identified. Correlations between the bands revealed by the continuous buffer method and those developed by the discontinuous buffer method were confirmed by two-dimensional SDS-PAGE, and the spots in the two-dimensional electrophoresis (first, isoelectrofocusing; second, SDS-PAGE) were identified by parallel electrophoresis of selectively solubilized polypeptides. Double labeling of the proteins with [3H]fucose and [14C]glucosamine showed that the proteins of virus grown in KB cells were glycosylated. When the virus was grown in L929 cells, the carbohydrate moieties lacked fucose, while glucosamine was detected in the same 14 polypeptides as in virus grown in KB cells. All glycopolypeptides migrated as clusters of spots upon two-dimensional electrophoresis. 32P incorporation was found in nine bands, five of which coincided with [35Szmethionine-labeled proteins. The phosphorylated polypeptides also migrated as multiple spots upon two-dimensional electrophoresis. The results indicated that the structural polypeptides of vaccinia virus detected as 39 bands in one-dimensional gels and 84 spots in two-dimensional profiles could be assigned to 52 polypeptide species, of which about 50% were basic.
The significance of extracellular enveloped vaccinia (EEV) for the in vitro and in vivo dissemination of vaccinia virus was investigated. The quantity of in vitro released extracellular virus correlated very closely with the ability of 13 vaccinia strains to cause long-range spread of infection (comet formation) in cell cultures infected at low m.o.i. but was not correlated with plaque size. The kinetics of virus spread after low m.o.i. was related to the amount of virus released from primary infected cells but not to their content of intracellular naked vaccinia (INV). Most extracellular vaccinia virus from IHD-J-infected RK-13 cells banded in CsCl density gradients as EEV (88%) while very little banded as INV (2%). Antisera to the enveloped prevented comet formation while antisera to INV did not. CsCl centrifugation of blood-borne extracellular virus from rabbits infected intravenously with vaccinia virus after cyclophosphamide treatment revealed that 64% of the virus banded as EEV but only 11% as INV. High in vitro EEV-yielding vaccinia strains were able to spread from the respiratory tract to the brains of mice and cause death. Low in vitro EEV-yielding vaccinia strains were generally not able to disseminate in vivo or cause mouse mortality. The notable exception to this trend was strain WR, which, although releasing small amounts of virus in vitro, could nevertheless very effectively disseminate in vivo, causing a high rate of mouse mortality. Treatment with anti-envelope serum protected mice from a lethal vaccinia infection whereas antiserum to inactivated INV did not. These results indicate that the in vitro dissemination of vaccinia infection is mediated by EEV and implicate EEV as having a role in the in vivo dissemination.
Vaccinia virus has been examined with the electron microscope by several techniques in conjunction with experimental modifications of structure. Negative staining revealed that over 80% of the virus in highly purified preparations were particles which appeared to have a beaded surface like a mulberry and were termed M forms. The beading was formed by loops of thread-like structures 90 A wide which were themselves double helices formed from two 30 A strands coiled to a 120 A pitch. Twenty per cent or less of the particles appeared as larger more electron-dense bodies with a capsule of complex structure; these have been termed C forms. Experimental interconversion of the two forms showed that both were structurally mature virus. The relationship of structures seen in C forms to those revealed by thin sectioning is considered. Experimental findings and the results of shadowing and replica examinations indicate the presence of a superficial protein layer of antigenic material not revealed by negative staining. The relationship of this layer to the structure of the virus and the nature of the ribbon structure of the M form are discussed.
6X-05214-1 1A from the Swedish Medical Research Council An antigenic difference between intracellular and extracellular rabbitpox virus A film detection method for tritium labelled proteins and nucleic acids in polyactylamide gels
- Lendon G Payne Grant References Appleyard
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Deglycosylation of asparagine-linked gly-case by peptide: N-glycosidase F Studies on the structure of vaccinia virus
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Vaccinia haemagglutinin Production of mono-clonal antibodies: Strategy and tactics
- F M Burnet
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