Analysis of antigenic determinants of structural polypeptides of avian type C tumor viruses.

Radioimmunoassays were developed for the 19,000, 15,000, and 12,000 molecular weight polypeptides of avian myeloblastosis virus and for the 19,000 and 12,000 polypeptides of RAV-0, a subgroup E avian tumor virus. Each polypeptide was shown to possess both group- and type-specific antigenic determinants, in contrast to the 27,000 mol wt polypeptide, which contained only group-specific determinants. The corresponding low-molecular-weight polypeptides of subgroup A, B, and E viruses were shown to be immunologically indistinguishable. The findings that low-molecular-weight polypeptides of subgroup C and D viruses reacted very differently in immunoassays for the respective polypeptides of avian myeloblastosis virus or RAV-0 suggest that subgroups C and D may have evolved differently form subgroups A, B, and E.     

Structural proteins of mammalian RNA tumor viruses: relatedness of the interspecies antigenic determinants of the major internal protein.

The relatedness of antigenic determinants of purified major core proteins of the murine, feline, RD 114/baboon, and woolly monkey/gibbon ape groups of RNA tumor viruses was examined by competition radioimmunoassay. In assay systems of a homologous antigen and antiserum, high affinity competition for binding to all of the antibodies was observed only with the homologous unlabeled protein; the core proteins of other groups of viruses showed only low affinity binding of a small fraction of antibodies, presumably those reactive with the interspecies determinants, at concentrations of competing protein 10- to 100-fold greater than that of the labeled antigen. The cross-reactive (interspecies) antigens of every two viruses were selectively examined by precipitating the purified 125-I-labeled protein with antiserum against each of the other proteins. The extent to which these shared determinants were common to the other viruses was then tested by the effectiveness of the proteins of each virus to compete for antibody binding. Several classes of interspecies determinants were distinguished: those common to two of the groups of viruses, others to three, and some to all four. Moreover, an even greater variety of interspecies determinants was indicated by differences in the affinity of the individual proteins for antibody binding, supporting the hypothesis that there are at least several, if not many, different interspecies determinants with a broad spectrum of antigenic cross-reactivity. These studies suggest that the murine and feline viruses are closely related as they contain cross-reactive antigenic determinants not shared with the other viruses, that the feline virus is more closely related to the woolly monkey virus than to RD 114, and that the RD 114 and woolly monkey viruses retain interspecies determinants shared relatively equally with each of the other viruses.     

Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.     

Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses

Immunoglobulin G directed against the DNA polymerase of Rauscher murine leukemia virus (R-MuLV) could bind to 125I-labeled DNA polymerase of spleen necrosis virus (SNV), a member of the reticuloendotheliosis virus (REV) species. Competition radioimmunoassays showed the specificity of this cross-reaction. The antigenic determinants common to SNV and R-MuLV DNA polymerases were shared completely by the DNA polymerases of Gross MuLV, Moloney MuLV, RD 114 virus, REV-T, and duck infectious anemia virus. Baboon endogenous virus and chicken syncytial virus competed partially for antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. DNA polymerases of avian leukosis viruses, pheasant viruses, and mammalian type B and D retroviruses and particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. We also present data about a factor in normal mammalian immunoglobulin G that specifically inhibits the DNA polymerases of REV and mammalian type C retrovirus DNA polymerases.     

Effect of chemical modification and fragmentation on antigenic determinants of internal protein p30 and surface glycoprotein gp70 of type C retroviruses

The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of α and ε amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH₂-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27.     

The genome-associated,specific RNA binding proteins of avian and mammalian type C viruses

A structural protein purified from the Rous sarcoma virus (RSV) can specifically bind in vitro to purified avian, but not mammalian, type C viral RNA. Following ultraviolet irradiation of viral particles under conditions which stabilize the polyploid 70S viral RNA, the same polypeptide can be directly purified from the RSV genome. Based on its electrophoretic mobility in polyacrylamide gels containing sodium dodecylsulfate, the RNA binding protein has been identified as the major phosphoprotein (p19) of avian type C viruses. Similar experiments show that the major phosphoproteins of mammalian type C viruses (p12 for murine viruses and p16 for endogenous primate viruses) are also the specific RNA binding proteins and, similarly, are found closely associated with the 70S RNA genomes in the intact viral particles.     

The occurrence of antigenic determinants of human blood proteins in mammalian plasmas

Summary In an attempt to follow up the persistence of single antigenic determinants of plasma proteins, the patterns of their determinants as revealed by cross-reactions with human plasma were tested in man, chimpanzee, the rhesus monkey, ox, sheep, pig, guinea pig and rat. The proteins investigated include ceruloplasmin, ₁-lipoproteins, fibrinogen, plasminogen, C-A- and ₁E-globulins. The results are presented in the form of pedigrees for the different plasma proteins: The phylogenetical pathways of the single determinants of human plasma proteins can be followed up; phylogenetically old determinants can be distinguished from others, which are possibly younger. The sensitivity of immunological techniques, which have been shown to be able to demonstrate single base changes in the DNA, for evolution research at the molecular level is stressed.     

Comparative studies on the structural phosphoproteins of mammalian type C viruses.

The major phosphoprotein common to woolly monkey sarcoma virus, gibbon ape lymphosarcoma virus, and type C viruses of the lower mammalian species (mouse, rat, cat), with the exception of the endogenous cat virus (RD-114), is the polypeptide of about 12,000 molecular weight. The protein-phosphate bond in this polypeptide of several viruses is of the phosphoserine variety excepting gibbon ape virus, which contains both phosphoserine and phosphothreonine. The primary phosphoprotein of RD-114 virus and the endogenous baboon type C virus, on the other hand, is the polypeptide of about 15,000 molecular weight which contains phosphothreonine as its phosphoamino acid. A second major phosphoprotein of molecular weight of 10,000 is detected only in viruses genetically related to rat species including those derived from the RPL cell line, from Sprague-Dawley rat embryo cells, and the Kirsten mouse sarcoma virus which was recovered from a mouse erythroblastosis virus after in vivo propagation through rat. These phosphorylated polypeptides of molecular weight 15,000, 12,000, or 10,000 are present in the virion structure in several different but nonrandom phosphorylated states.     

5''-terminal nucleotide sequences of mammalian type C helper viruses are conserved in the genomes of replication-defective mammalian transforming viruses.

The RNAs of replication-defective murine and primate type C transforming viruses were analyzed for the presence of nucleotide sequences homologous to the genomes of their respective helper type C viruses by using DNAs complementary (cDNA) to either the 5'-terminal (cDNA5') or total (cDNAtotal) nucleotide sequences of the helper virus RNA. The defective viruses examined have previously been shown to vary in their ability to express helper viral gag gene proteins. With cDNAtotal as a probe, these transforming viruses were shown to vary in their representation of helper sequences (15 to 60% hybridization of cDNAtotal). In striking contrast, 5'-terminal-specific sequences of the helper virus were conserved in the RNAs of every transforming virus tested (is greater than 80% hybridization of cDNA5'). These findings suggest a critical role for these sequences in the life cycle of the defective transforming virus.     

Interspecies immunologic cross-reactivity of mammalian sperm basic proteins

The species uniqueness of the sperm basic nuclear proteins (SBNPs) of mammals is in sharp contrast to the highly conserved function of those proteins, to mediate extreme condensation of the sperm chromatin. Despite their molecular uniqueness, however, the SBNPs of all eutherian mammals share certain characteristics: low molecular weight, high percentage of arginine and cysteine residues and, in the final step of chromatin condensation, extensive SS cross-linking. By radioimmunoassay, we have demonstrated that rabbit antiserum to mouse SBNP is reactive with purified mouse SBNP and mouse sperm and is cross-reactive with purified bull SBNP and sperm of bull, rat, rabbit, dog and man. The immunologically cross-reactive sites, therefore, may represent the homologous regions of the various species of SBNPs that serve the function of chromatin condensation. Immunologic reactivity was displayed only by sperm made to swell by treatment with 2-mercaptoethanol. Unswollen sperm were not reactive, confirming previous observations by immunofluorescence [1] indicating that reduction of SS bonds is necessary to render the antigenic sites of SBNP available for immunologic recognition, and suggesting that formation of SS bonds within that protein may be a molecular mechanism of antigen sequestration in vivo.