Evidence for the presence of repeating antigenic determinants in the major polypeptides derived from hepatitis B surface antigen

The immunogenicity of the four major and seven minor hepatitis B surface antigen (HBsAg)-derived polypeptides was evaluated in guinea pigs and compared to that of 22-nm HBsAg particles. Both humoral and cell-mediated immune responses were determined. All antisera reacted with homologous HBsAg/ayw and P22/ayw. However, the glycosylated polypeptides (P25, P27, P31, and P52) generally elicited the most intense immune responses. At least nine polypeptides induced antibodies which also reacted with heterologous HBsAg/adw particles and P22/adw. Whole HBsAg/ayw particles induced transformation in lymphocytes obtained from guinea pigs immunized with the major or minor polypeptides. These data tend to demonstrate that all HBsAg-derived polypeptides contain in their structure the group-specific and at least one of the subtype-specific antigenic determinants, and these determinants are also exposed on the surface of the HBsAg particle. It seems possible that a limited nucleotide sequence confers the same virus specificities to most if not all of the constitutive proteins of HBsAg.     

Evidence for antigenic variation in influenza A nucleoprotein.

Nucleoprotein (NP) antigens isolated from sodium sarcosyl detergent-disrupted influenza A virus particles by cellulose acetate electrophoresis were used to prepare specific immune sera. Antigenic analysis of the nucleoproteins by immuno-double-diffusion and antibody absorption tests revealed antigenic differences in the nucleoprotein antigens of human A/PR8/34 (HON1) virus and viruses of the Hong Kong (H3N2) subtype. The nucleoprotein antigens of avian A/duck/Ukraine/1/63 (Hav7Neg2) and A/swine/Iowa/15/30 (Hsw1Nl) viruses resembled more closely the NP of A/PR8/34 virus than the NP of human H3N2 strains. Antigenic analysis of recombinant strains prepared from A/PR8/34 and H3N2 parent viruses indicated that the parental origin of the NP antigens could be clearly identified. Identification of the nucleoproteins of parental and recombinant influenza A viruses by migration rate analysis of NP polypeptides and RNA genes by electrophoresis on polyacrylamide gels gave results which correlated with the antigenic characterization of their NP antigens. The findings suggested that antigenic “drift” occurs in the nucleoproteins of human influenza A viruses.     

Monoclonal antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic determinants

Twenty-one monoclonal antibodies that react with bluetongue virus (BTV) and have restricted or unrestricted serotype specificities were identified in culture supernatants of hybridomas derived from lymphocytes of mice immunized with BTV serotype 17. Hybridomas were screened and antibody specificities characterized in a solid-phase radioimmunoassay and by immunoprecipitation with radiolabeled, BTV 17-infected cell lysates. Three general serotype specificities were demonstrated by 13 antibodies that precipitated structural viral protein 9 (VP 9). One antibody precipitated VP 7, a 48,000 dalton nonstructural protein, and reacted in radioimmunoassay with 20 BTV serotypes and Epizootic Hemorrhagic Disease of Deer Virus serotype 1 (EHDV 1), EHDV 2, and Ibaraki virus, but not with uninfected cells. One serotype-specific antibody neutralized infectivity, inhibited hemagglutination by BTV 17, and precipitated VP 2 and VP 3. A second antibody, with restricted serotype specificity, precipitated VP 2 and VP 8. These results confirm those of others (Huismans and Erasmus, Onderstepoort J. Vet. Res. 48, 51-58, 1981) that BTV serotype-specific and neutralizing epitopes are associated with VP 2.     

Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: Evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition

A model showing the topological distribution, functions, and serological specifities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication [Heinz et al., 1983]. Virology 126, 525–537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and “Western blotting.” One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, α-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.     

A pH-dependent antigenic conversion of empty capsids of poliovirus studied with the aid of monoclonal antibodies to N and H antigen

Evidence is presented that the body temperature of chickens at 41° selects virus recombinants with a genome constellation necessary for pathogenicity in chickens. It could be shown that recombinants of influenza A viruses pathogenic for chicken grow equally well at 37 and 41° whereas the nonpathogenic recombinants have a significantly lower growth rate at 41°. Hybridization studies with recombinants derived from fowl plague virus and A/turkey/England/63 revealed that at 41° such isolates are selected which possess all the genes coding for the polymerase complex from one wild type parent virus. Furthermore, only pathogenic recombinants showed an increase in plaque size when the incubation temperature was shifted from 37 to 41°. The behavior of virus recombinants at elevated temperature therefore is a useful marker for rapid in vitro selection of pathogenic or nonpathogenic recombinants.     

Structure of the mengo virion. VI. Spatial relationships of the capsid polypeptides as determined by chemical cross-linking analyses.

The asymmetric structure unit of the Mengo virus capsid comprises one molecule each of the polypeptides α, β, and γ. Structure units are clustered in pentamers which are centered at each of the 12 vertices of a T = 1 icosahedral lattice (Mak et al., 1974). The location of the ～-60 δ polypeptides in the capsid has not been established. In order to obtain information about the arrangement of α, β, and γ in the structure unit and to determine which polypeptides participate in the noncovalent interactions responsible for pentamer formation and capsid stabilization, virions were reacted with bifunctional crosslinking reagents and the polypeptide complexes produced were identified by gel electrophoresis. Using the reversible crosslinkers dimethylsuberimidate (DMS) and dithiobis(succinimidyl propionate) (DSP), positive identification of βγ, αγ, αβγ, α₂, α₃, and α₄ complexes was made. Complexes involving S were not detected, nor were β_n or γ_n. The latter observation indicated that the hydrophobic interactions among αβψ structure units in a pentamer involve α-α contacts. When virions crosslinked with DMS, DSP, or dimethyladipimate (DMA) were subsequently dissociated by 0.1 M NaCl at pH 6 and examined in the electron microscope, it was shown that only treatment with DSP prevented complete capsid dissociation. Since DSP crosslinking alone produced αβ complexes, it was concluded that the interactions between adjacent pentamers probably result from α-β contacts. Treatment of Mengo virions with formaldehyde produced crosslinks between β and γ polypeptides and the RNA. Based upon these data, a model for the organization of individual polypeptide species within the Mengo virus capsid is presented.     

Structural domains of endogenous murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies

Eight distinct antigenic determinants, or epitopes (labeled a-h) were identified on endogenous ecotropic murine leukemia virus (MuLV) gp70s using a series of murine and rat monoclonal antibodies. These epitopes were characterized by their distribution patterns in a panel of cloned MuLVs, and by their localization in fragments of gp70 generated either by spontaneous breakdown of purified gp70, or by controlled proteolysis of gp70 in solubilized virions. A major 32K carboxy terminal fragment was formed which contained epitopes b, c, and f. This fragment also possessed the p15(E) disulfide linkage site, and contained approximately four complex (type 1) carbohydrate chains. An amino terminal 35K fragment contained epitopes a, d, g, and h, and possessed two glycosylated sites, including a site which occasionally retained an endoglycosidase H-sensitive oligosaccharide chain. A related 49K fragment was also obtained which included the entire 35K region and contained an additional sequence bearing epitope e. In a series of dual-tropic MCF-type viruses studied, only those epitopes located in the 32K fragment were ever retained, indicating that for these recombinant viruses at least a portion of that domain was derived from the ecotropic parent. A model is presented indicating the likely orientation of these fragments and their structural characteristics.     

Biological characterization of a herpes simplex virus intertypic recombinant which is completely and specifically non-neurovirulent

In this study, a pre-existing "library" of Herpes Simplex Virus (HSV) intertypic recombinants was found to be not useful for mapping HSV genes controlling viral neurovirulence in mice. Most of these agents were significantly less virulent than either parental type following intracranial inoculation, and in the general case this lessened virulence could be attributed to inefficient replication in any cell type at 38.5 degrees (the normal temperature of the mouse). One agent tested (recombinant RE6) was completely non-neurovirulent following intracranial inoculation of as much as 3.2 X 10(7) PFU. Since about 10 PFU of either 17 Syn+ or HG52 (the "parental" strains of this recombinant) were lethal for mice, RE6 is at least 10 million-fold less neurovirulent than the wild-type strains from which it was produced. The function of the defective gene(s) in RE6 is not yet known, but it is not required for the expression of viral thymidine kinase, efficient replication in cultured cells at 38.5 degrees, or replication in non-neural mouse tissue in vivo. Therefore, the defect in RE6 is in an HSV gene function(s) which is absolutely required for neurovirulence but not for general viral replication. Several possibilities for the molecular nature of the defect in RE6 are discussed.     

Monoclonal antibodies derived during graft-versus-host reaction. II. Antibodies detect unique determinants common to many MCF viruses

Hybridoma cell lines were recovered from the spleens of 6-week-old (B6 X D2)F1 mice undergoing graft-versus-host reaction induced by the transfer of 5-week-old B6 parental spleen cells. These cell lines produced antibodies reactive with envelope polypeptides of a variety of MuLV. The viral specificity assessed by membrane immunofluorescence and virus-binding radioimmunoassay indicated that the reactivity of these antibodies was distinctly different from monoclonal antibodies recovered from (B6 X D2)F1 recipients of D2 spleen cells reported previously (Portis et al., Virology 118, 181-190, 1982). Ten out of 17 monoclonal antibodies in the current study reacted exclusively with MCF viruses and three of these antibodies detected envelope determinants which were shared by a broad panel of MCF viruses of diverse origin. These common MCF determinants were expressed by the gp70 molecule as determined by Western blot analysis. The production of these antibodies by young mice in the absence of exogenous virus inoculation suggests that these antigens may be encoded by endogenous MCF-like sequences.     

Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins.

Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.     

Characterization of vaccinia polypeptides

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.     

Identification of poliovirus precursor proteins by immunoprecipitation

Precursors of poliovirus capsid polypeptides were detected using immunoprecipitation followed by SDS-PAGE. Two common VPO-3-1 precursors were found, and the occurrence of alternative cleavage pathways was confirmed.     

Effect of arildone on modifications of poliovirus in vitro

The effect of arildone, a new antiviral agent, on the biological and physical alterations of poliovirus type 2 induced by heat or alkaline treatment in vitro are described. Arildone prevents inactivation of poliovirus infectivity and physical alterations of the virion at 47°. Although arildone protects the biological activity of poliovirus during treatment at pH 10.5, the drug did not fully prevent the physical alterations induced at alkaline pH. Our results indicate differences in the mechanism of destabilization of poliovirus by heat and alkaline treatment in vitro. Furthermore, these results show that arildone interacts directly with the viral capsid. The stabilization of poliovirus in vitro correlates well with prevention of uncoating in the cell by arildone.     

Structural polypeptides of parvoviruses

The structural polypeptides of X14 virus and adenovirus-associated viruses (AAV) types 1 and 4 were analyzed by SDS-polyacrylamide gel electrophoresis. The capsid of each defective parvovirus consists of two minor and one major polypeptide, but the capsid of X14 virus consists of only two polypeptides. The smallest polypeptide of each virus is the most abundant. The naturally occurring empty capsids of AAV type 4 and X14 have proteins indistinguishable in size and concentration from those of complete virions. The isolated structural polypeptides of the two hemagglutinating viruses, AAV4 and X14, did not show any hemagglutinating activity.     

Inhibition of uncoating of poliovirus by arildone, a new antiviral drug.

The antiviral effects of a new drug, arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl]-3,5-heptanedione, on poliovirus type 2 replication and host cell functions are described. Arildone inhibits poliovirus replication at a minimal inhibitory concentration (MIC) of 0.2 μM, while transport of radioactively labeled precursors and synthesis of DNA, RNA, and protein in uninfected HeLa cells are not inhibited. This drug is not virucidal and does not interfere with adsorption or penetration. Arildone inhibits uncoating of poliovirus and thereby prevents virus-induced shutoff of host cell protein synthesis. The possible mechanisms by which arildone interacts with the poliovirus icosahedral capsid to prevent uncoating are discussed.     

Structural polypeptides of Choristoneura biennis entomopoxvirus

The structural polypeptides of Choristoneura biennis (Free) entomopoxvirus (EPV) were analyzed. The virion contained at least 40 polypeptides ranging from 12,000 to 250,000 in molecular weight. Twelve major proteins constitute 95% of the total virus protein. Core particles generated by treatment with Nonidet P-40, under conditions during which endogenous alkaline protease is active, display a smooth exterior similar to those obtained for orthopoxviruses. The major component of these cores is a polypeptide with a molecular weight of 59,000.     

Similar frequencies of antigenic variants in Sendai, vesicular stomatitis, and influenza A viruses

The effects of three inhibitors of glycosylation (2-deoxy-d-glucose, d-glucosamine, and tunicamycin) on the formation of infectious extracellular snowshoe hare virus and the synthesis of intracellular virus-induced polypeptides have been examined. The inhibitor 2-deoxy-d-glucose, used at concentrations of up to 10 mM, did not significantly affect either infectious virus release or viral-induced polypeptide synthesis and maturation. By contrast, both d-glucosamine (40 mM) and tunicamycin (1 μg/ml) prevented the release of infectious virus and inhibited the maturation of the two viral glycoproteins (Gl and G2), but not the viral nucleoprotein (N). Two new polypeptides, Gl₀ and G2₀, of molecular weights 105 × 103 and 33 × 103, respectively, were found in infected cells treated with tunicamycin or D-glucosamine. These two polypeptides were identified as virus specific since they could be precipitated by virus immune serum. Tunicamycin was found to inhibit the release of extracellular virus particles at 33 and 38°, which correlated with the observation that no intracellular virus particles were detected in electron micrographs of cells treated with the drug.     

Early DNA-binding polypeptides of Epstein-Barr virus

The kinetics of Epstein-Barr virus (EBV)-specific protein synthesis has been studied after induction of P3HR-1 cells with n-butyrate. The majority of the EBV polypeptides became detectable on SDS-polyacrylamide gel electrophoresis between 15 and 18 hr after addition of the drug. Overall viral protein synthesis was at maximum after about 24 hr. Some polypeptides appeared significantly earlier (152K, 55K, 51K) while others were synthesized during a limited period of the cycle only (65K). At least four of the polypeptides detected (152K, 134K, 55K, 51K) bound to DNA cellulose columns. The EBV specificity of the polypeptides was confirmed by immunoprecipitation and by two-dimensional gel analysis. The 152K polypeptide had a pI around 8 whereas the other polypeptides varied between basic and acidic pI. The DNA binding proteins isolated by DNA cellulose chromatography represent a subclass of the virus-specific early proteins that can be immunoprecipitated from the total cell extract.     

Immunoprecipitable polypeptides specified by varicella-zoster virus

Polypeptides encoded by varicella-zoster virus (VZV) in infected cell cultures have been identified by radioimmune precipitation techniques. Detergent-solubilized extracts of VZV-infected cells were reacted with highly specific VZV antisera raised in strain-2 guinea pigs immunized with sonicates of syngeneic virus-infected cells. Fractionation of the immunoprecipitates in acrylamide slab gels demonstrated an average of 16 polypeptides, which ranged in molecular weight from 32,000 to å200,000. These included the three major immunogenic glycoproteins (gp 62, gp 98, and gp 118) and a prominent higher molecular weight nonglycosylated polypeptide at 155,000. One of the [³⁵S]methionine-labeled polypeptides comigrated with purified actin. Not all polypeptides were visible in any one particular fluorogram, but comparative analysis of polypeptide profiles derived from electrophoreses performed with different gel concentrations and different crosslinkers (methylene-bisacrylamide and N,N-diallyltartardiamide) clearly established a consistent and reproducible pattern of radioactive bands. A low background of radio-activity was nonspecifically precipitated by the antigen-antibody-protein A complexes; however, with the exception of a common band comigrating with actin, the electrophoretic profiles representing virus-specific and nonspecific immunoprecipitates were easily distinguished.     

Influenza virus RNA segment 7 has the coding capacity for two polypeptides

The genes of a recombinant human influenza virus A/Japan/305/57:A/Bel/42:A/PR/8/34 have been cloned in bacterial plasmids, and the nucleotide sequence of RNA segment 7 has been determined. The DNA sequence predict two open reading frames, which overlap by 68 bases and have a coding capacity for polypeptides of 27,000 and 11,000 daltons. Segment 7 has been previously assigned as the gene coding for the viral matrix protein of apparent molecular weight 25,000. Therefore gene 7 has the potential to code for a previously unsuspected protein of approximately 11,000 daltons in addition to matrix.