Hog cholera virus: molecular composition of virions from a pestivirus.

Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.     

Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity.

A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus. Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins. The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein. The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus. The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.     

Bovine viral diarrhea virus proteins: relatedness of p175 with p80 and p125 and evidence of glycoprotein processing.

Three monoclonal antibodies, which recognized two nonoverlapping antigenic domains and were reactive to the bovine viral diarrhea virus (BVDV) p80 protein, were found to cross react with the p125 protein of both cytopathic and noncytopathic BVDVs and a molecular weight 175,000 BVDV protein (p175). Results from limited proteolysis and chemical cleavage experiments confirmed the relatedness of these three proteins. In pulse-chase experiments it was apparent that p175 was a transient protein, as it was diminished during the chase, with a half-life of about 30 min. However, both p125 and p80 were also observed in short-pulsed lysates. Furthermore, during the chase, radiolabel was not found to accumulate into p125 or p80. Rather, these two proteins were stable with half-lives greater than 2 h. A fourth nonglycosylated protein, p37, increased during the chase. Processing of several glycoproteins was evident in these experiments. A glycoprotein of molecular weight 75,000 (gp75) diminished during the chase period, while glycoproteins gp62, gp48, and gp25 appeared or increased during the chase period. In contrast, the glycoprotein gp53 was a major protein in pulse-labeled cell lysates and remained constant throughout the chase period. In further experiments two stable forms of p80 differing in intramolecular disulphide bonding were observed.     

Envelope polypeptides of Friend leukemia virus: purification and structural analysis.

Roughly 10% of surface glycoproteins in the envelope of mature Friend murine leukemia virus are coupled to membrane polypeptides by disulfide bridges. The remaining 90% of these glycoproteins are associated noncovalently. However, they could also be linked to membrane polypeptides by the treatment of purified Friend murine leukemia virus with 2,2'dithiobis(m-nitropyridine). These amphiphilic heterodimer polypeptides, gp84/86, were recovered almost quantitatively in the form of aggregates, termed rosettes, when prepared by solubilization of the viral membrane with Triton X-100 and subsequent velocity sedimentation. gp69/71 and p12(E)/15(E) were purified from these protein micelles after reduction of the disulfide bonds by gel chromatography. Electron micrographs of rosettes, as well as of purified p12(E)/15(E), showed structures different from native viral knobs. Isolated gp84/86 could be reassociated and then displayed more similarity to these viral surface projections. As shown by peptide mapping, the primary structures of the glycoproteins gp69/71 are highly related as are those of the membrane polypeptides p12(E) and p15(E). Furthermore, it was shown by two-dimensional polyacrylamide gel electrophoresis and re-electrophoresis of purified gp84/86 that the larger component, gp86, was composed of gp71 associated with p15(E) and p12(E), whereas the smaller component, gp84, was formed by gp69 bound only to p12(E).     

Acid-resistant bovine pestivirus requires activation for pH-triggered fusion during entry

The route of internalization of the pestivirus bovine viral diarrhea virus (BVDV) was studied by using different chemical and biophysical inhibitors of endocytosis. Expression of the dominant-negative mutant Dyn(K44A) of the GTPase dynamin in MDBK cells, as well as the treatment of the cells with chlorpromazine and beta-methyl-cyclodextrin inhibited BVDV entry. BVDV infection was also abolished by potassium (K+) depletion, hyperosmolarity, and different inhibitors of endosomal acidification. We conclude that BVDV likely enters the cell by clathrin-dependent endocytosis and that acidification initiates fusion with the endosomal membrane. Further studies revealed that BVDV was unable to undergo "fusion from without" at low pH. The finding that low pH is not sufficient to force adsorbed BVDV into fusion with the plasma membrane is compatible with the remarkable resistance of pestiviruses to inactivation by low pH. The importance of the abundant intra- and intermolecular disulfide bonds in BVDV glycoproteins for virus stability was studied by the use of reducing agents. The combination of dithiothreitol and acidic pH led to partial inactivation of BVDV and allowed fusion from without at low efficiency. Evidence is provided here that acid-resistant BVDV is destabilized during endocytosis to become fusogenic at an endosomal acidic pH. We suggest that destabilization of the virion occurs by breakage of disulfide bonds in the glycoproteins by an unknown mechanism.     

Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gp1 and gp3

Two monoclonal antibodies were prepared against varicella-zoster virus proteins. One of the monoclonal antibodies (10.2) reacted only with the nuclei of infected cells and immunoprecipitated one nonglycosylated late viral protein (125,000 molecular weight). The other monoclonal antibody (19.1) with neutralizing activity, reacted with membrane antigens of infected cells and with the varicella-zoster virus envelope and immunoprecipitated two late major viral glycoproteins (gp1 and gp3). Synthesis of the 125,000-molecular-weight protein, gp1, and gp3 began at 20 to 22 h postinfection, 2 h after the peak of viral DNA synthesis, and continued until 29 h postinfection, when the first progeny virus appeared in infected cells. Pulse-chase experiments showed that during pulse-labeling, only gp1 was detected, whereas during the chase period, gp1 as well as gp3 was detected in infected cells. Under nonreducing conditions, gp3 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 130,000-molecular-weight protein as compared with the 62,000-molecular-weight species obtained when gels were resolved under reducing conditions. This finding indicates that gp3 is a dimer that is disulfide linked.     

Characterization of the envelope proteins of pseudorabies virus.

Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; gIIa, gIIb, and gIIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; gIII) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.     

Synthesis and processing of the envelope gp55-116 complex of human cytomegalovirus.

The envelope of human cytomegalovirus has been reported to contain between three and eight glycoproteins. Major constituents of the envelope include two abundant glycoproteins with estimated molecular weights of 55,000 (gp55) and 116,000 (gp116). These two glycoproteins have been shown to exist as a disulfide-linked complex (gp55-116) within the envelope of mature virions. Utilizing a panel of monoclonal antibodies reactive with the gp55-116 complex, we characterized the synthesis and processing of these two virion proteins. Infected cells were shown to contain two glycosylated proteins of 160,000 and 150,000 daltons as well as the mature gp55 and gp116. Pulse-chase analysis indicated that gp150 was a precursor protein of gp160. The mature gp55 and gp116 were generated, in turn, by cleavage of gp160. Antigenic and structural analysis revealed that gp55 and gp116 shared little structural homology and no detectable antigenic cross-reactivity. The results of this study are discussed in relation to the synthesis of envelope proteins of other herpesviruses.     

Immune response to vaccination with DNA encoding the bovine viral diarrhea virus major glycoprotein gp53 (E2)

Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle which has not been controlled by classical vaccination. The region encoding the BVDV major glycoprotein gp53 (E2) known to possess virus-neutralizing activity was cloned into a mammalian expression vector under the human cytomegalovirus (CMV) intermediate early promoter. Intramuscular and intradermal administration of the recombinant plasmid DNA into BALB/c mice induced BVDV gp53-specific antibody responses to both biotypes (cytopathic and noncytopathic) of BVDV genotype 1, and to cytopathic BVDV genotype 2. BVDV-neutralizing antibodies were generated against BVDV genotype 1 strains and they also persisted 6 months after the last injection.     

Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.     

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.     

Role of disulfide bond formation in the folding and assembly of the envelope glycoproteins of a pestivirus

Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, closely related to, and used as a surrogate model for the hepatitis C virus. Its envelope contains the E1 and E2 glycoproteins, disulfide linked into homo- and heterodimers. In this study, we investigate the role of disulfide bond formation in the folding, assembly, and stability of BVDV glycoproteins. We provide molecular evidence that intact disulfide bonds are critical for the acquirement of a stable conformation of E2 monomers. Forcing the E2 glycoproteins to adopt a reduced conformation either co- or post-translationally before assembly into dimers, determines their misfolding and degradation by proteasome. In contrast, dimerization of E2 glycoproteins results in a conformation resistant to reducing agents and degradation. Furthermore, inhibition of the ER-alpha-mannosidase activity leads to impairment of misfolded E2 degradation, demonstrating the involvement of this enzyme in targeting viral proteins towards proteasomal degradation.     

Determination of the protective epitopes on the glycoproteins of Venezuelan equine encephalomyelitis virus by passive transfer of monoclonal antibodies

We have previously characterized seven unique antigenic epitopes on the two envelope glycoproteins of the Venezuelan equine encephalomyelitis (VEE) virus vaccine strain, TC-83, by using monoclonal antibodies. The in vitro function of virus neutralization was primarily associated with one epitope on the gp56 (gp56c). To determine which epitopes were important in protecting animals from VEE infection, purified monoclonal antibodies were inoculated i.v. into 3-wk-old Swiss mice. Twenty-four hours later these animals were challenged i.p. with 100 IPLD50 of virulent VEE virus (Trinidad donkey). High-avidity anti-gp56c, anti-gp50b, anti-gp50c, and anti-gp50d monoclonal antibodies protected animals from virus challenge. Rabbit antisera to the gp56 and the gp50 glycoproteins were also effective in protecting mice from challenge with virulent VEE virus. Less antibody was needed to protect animals if the antibody was directed against the critical neutralization site. Less avid antibodies to the gp56c and gp50b epitopes demonstrated little or no protection in vivo. Protection, therefore, appeared to be a function of the passive antibody's specificity, avidity, and ability to bind to virion antigenic determinants topologically proximal to the critical neutralization site.     

Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor.

The leukemogenic glycoprotein (gp55) encoded by Friend spleen focus-forming virus is predominantly retained in the rough endoplasmic reticulum (RER). However, a small proportion (ca. 5%) is processed to form a derivative that occurs on plasma membranes and causes mitosis of infected erythroblasts. We have now found that gp55 folds heterogeneously in the RER to form components with different disulfide bonds and that this difference may determine their processing fates. RER gp55 consists predominantly of monomers with intrachain disulfide bonds. In contrast, the processed molecules are disulfide-bonded dimers. These dimers are extensively modified in transit to cell surfaces by conversion of four N-linked high-mannose oligosaccharides to complex derivatives and by attachment of a sialylated O-linked oligosaccharide. The plasma membrane dimers are then slowly shed into the medium by a mechanism that involves proteolytic cleavage of approximately 25 membrane-anchoring hydrophobic amino acids from the carboxyl termini of the glycoproteins. Consequently, shed molecules have shorter polypeptide chains than cell-associated gp55. We conclude that gp55 folds into different disulfide-bonded components that do not substantially isomerize, and that only one specific dimer is competent for export from the RER. Mitogenic activity of gp55 could be caused by the cell surface dimers, by the shed derivative, or by the carboxyl-terminal hydrophobic anchors that remain in the membranes after the shedding reaction.     

A unique glycoprotein containing GR-mouse mammary tumor virus peptides and additional peptides unrelated to viral structural proteins

A glycoprotein of molecular weight 130,000 (gP130) has been precipitated from the cytoplasm of GR-strain mouse mammary tumor (GR-MMT) cells by a rabbit antiserum (anti-MMTV) to GR-strain mouse mammary tumor virus (GR-MMTV). This protein was not precipitated by antisera specific for detergent-disrupted C3H-strain MMTV (C3H-MMTV); C3H-MMTV glycoproteins; C3H-MMTV nonglycosylated proteins; GR-MMTV p25 or p12; RIII strain (milk) MMTV proteins; or Rauscher murine leukemia virus (R-MuLV) proteins; nor was it precipitated by normal rabbit serum. Two-dimensional thin layer analysis of 35S-methionine-containing tryptic peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides are shared by gP130 and gPr76env. The envelope protein precursor, gPr76env, contains all of the gp33 peptides and six of seven gp55 peptides. One peptide in gPr76env, possibly a gp55-gp33 junction peptide, is also apparently present in gP130. Six of ten p25 peptides and four more gag-related peptides are shared by PR78gag and gP130. Protein gP130 also contains several tryptic peptides not found in gPr76env or in the core protein precursors Pr78gag, Pr110gag or Pr180gag-pol. Radioimmunoprecipitation experiments showed that gP130 could be precipitated from extracts of GR-MMTV cells with anti-MMTV serum even after antibodies to the known MMTV structural proteins had been removed from the serum by absorption. Both gP130 and a second protein, p30, were found in immunoprecipitates of detergent-disrupted isotopically labeled GR-MMTV treated with the absorbed anti-MMTV serum. These results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences unique to gP130; that is, those protein sequences which are not related to viral structural proteins. In light of these data and data published previously, gP130 is apparently a polyprotein containing juxtaposed components translated from the 5' and 3' end of the MMTV genome and protein components not previously identified as virus-specific.     

Hepatitis C Virus Infection Upregulates CD55 Expression on the Hepatocyte Surface and Promotes Association with Virus Particles

CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or full-length (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion.     

Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior and gp41 transmembrane envelope glycoproteins assemble into trimers on the virus surface that represent potential targets for antibodies. Potent neutralizing antibodies bind the monomeric gp120 glycoprotein with small changes in entropy, whereas unusually large decreases in entropy accompany gp120 binding by soluble CD4 and less potent neutralizing antibodies. The high degree of conformational flexibility in the free gp120 molecule implied by these observations has been suggested to contribute to masking the trimer from antibodies that recognize the gp120 receptor-binding regions. Here we use cross-linking and recognition by antibodies to investigate the conformational states of gp120 monomers and soluble and cell surface forms of the trimeric HIV-1 envelope glycoproteins. The fraction of monomeric and trimeric envelope glycoproteins able to be recognized after fixation was inversely related to the entropic changes associated with ligand binding. In addition, fixation apparently limited the access of antibodies to the V3 loop and gp41-interactive surface of gp120 only in the context of trimeric envelope glycoproteins. The results support a model in which the unliganded monomeric and trimeric HIV-1 envelope glycoproteins sample several different conformations. Depletion of particular fixed conformations by antibodies allowed characterization of the relationships among the conformational states. Potent neutralizing antibodies recognize the greatest number of conformations and therefore can bind the virion envelope glycoproteins more rapidly and completely than weakly neutralizing antibodies. Thus, the conformational flexibility of the HIV-1 envelope glycoproteins creates thermodynamic and kinetic barriers to neutralization by antibodies directed against the receptor-binding regions of gp120.     

Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins function as a membrane-anchored trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Previously, we reported three approaches to stabilize soluble trimers containing parts of the gp41 ectodomains: addition of GCN4 trimeric helices, disruption of the cleavage site between gp120 and gp41, and introduction of cysteines in the gp41 coiled coil to form intersubunit disulfide bonds. Here, we applied similar approaches to stabilize soluble gp140 trimers including the complete gp120 and gp41 ectodomains. A combination of fusion with the GCN4 trimeric sequences and disruption of the gp120-gp41 cleavage site resulted in relatively homogeneous gp140 trimers with exceptional stability. The gp120 epitopes recognized by neutralizing antibodies are intact and exposed on these gp140 trimers. By contrast, the nonneutralizing antibody epitopes on the gp120 subunits of the soluble trimers are relatively occluded compared with those on monomeric gp120 preparations. This antigenic similarity to the functional HIV-1 envelope glycoproteins and the presence of the complete gp41 ectodomain should make the soluble gp140 trimers useful tools for structural and immunologic studies.     

Identification with monoclonal antibodies of glycoproteins of Marek''s disease virus and herpesvirus of turkeys related to virus neutralization.

By use of monoclonal antibodies cross-reactive with Marek's disease virus and herpesvirus of turkeys, three glycoproteins (for Marek's disease virus, gp115/110, gp63, and gp50; for herpesvirus of turkeys, gp115, gp62 and gp52) related to virus neutralization were identified. Immunization of chickens or rabbits with these glycoproteins purified by affinity chromatography resulted in production of neutralizing antibodies.     

Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus.

Sera from virtually all individuals infected with human immunodeficiency virus contain antibodies against the viral envelope glycoproteins. By using a series of synthetic peptide antigens, we identified an immunodominant domain at amino acid position 598-609 of gp41. The minimal essential epitope is a 7-amino-acid sequence (amino acids 603-609) containing two cysteine residues. Both cysteine residues are required for the antigenic conformation of the sequence, possibly due to creation of a cyclic structure via disulfide bond formation.