Type-C RNA Virus Gene Expression in Human Tissue

Partially purified fractions of human tissues have been analyzed by competition radioimmunoassay for the presence of two of the principle structural components of type-C RNA viruses, the major core protein (p27 to p30) and the major envelope glycopeptides (gp69/71). Screening of tissues was carried out by use of a heterologous assay system of (125)I-labeled Rauscher murine virus p30 antigen and anti-RD 114 virus serum which was found to detect a class of interspecies determinants common to murine, feline, and primate viruses. A competitor with the same apparent affinity for antibody binding as that of purified viral core proteins was found in relatively high concentration in tissues from patients with systemic lupus erythematosus, in some neoplastic tissues, and also in normal human tissues. This competitor from a lupus spleen chromatographed on phosphocellulose and showed size fractionation during gel filtration similar to known p27 to p30 viral proteins. An immunologically reactive protein was also demonstrated by immunodiffusion and by immunoprecipitation of (125)I-labeled human protein with anti-RD 114 p28 serum. Analysis of these human competitor proteins with homologous assay systems of viral core proteins and corresponding antisera showed that all, including the normal tissue extracts, appear similar to core proteins of known viruses, especially the RD 114 and woolly monkey species. A hypothesis suggested by these data is that many, if not all, humans harbor at least part of the genome of one or more type-C viruses, the properties of which are similar to those of viruses from other mammalian species, particularly primates.     

Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.     

Structural polypeptides of mammalian type C RNA viruses. Isolation and immunologic characterization of a low molecular weight polypeptide, p10.

Low molecular weight polypeptides of several mammalian type C RNA tumor viruses were purified by sequential ion exchange chromatography and molecular sizing techniques. These included a polypeptide with a molecular weight of 10,000 to 11,000, p 10, from two type C viruses of mouse origin. Rauscher- and Moloney-murine leukemia virus (MuL virus), and from an infectious type C virus isolate of the woolly monkey. The p12 structural polypeptides of these viruses as well as Rauscher-MuL virus p15 were also purified. By using radioimmunoassays developed for each polypeptide, it was possible to demonstrate that all three low molecular weight polypeptides, p15, p12, and p10, were immunologically unique. Among type C viral structural polypeptides, p10 has been least well characterized immunologically. The results of the present study indicate that p10 is virus-coded and possesses strong group-specific antigenic determinants. By use of appropriate immunoassays, broadly reactive interspecies determinants shared by mammalian type C virus isolates of murine, feline, and primate origin, were also demonstrated. The interspecies antigenic determinants of p10 were shown to be as broadly cross-reactive as those exhibited by the major type C virus structural polypeptide, p30.     

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.     

Species and interspecies radioimmunoassays for rat type C virus p30: interviral comparisons and assay of human tumor extracts.

The major internal protein, p30, of rat type C virus (RaLV) was purified and utilized to establish intra- and interspecies radioimmunoassays. Three rat viruses were compared in homologous and heterologous intraspecies assays with no evidence of type specificity. The only heterologous viruses to give inhibition in these species assays were the feline (FeLV) and hamster (HaLV) type C viruses; these reactions were incomplete and required high virus concentrations. An interspecies assay using a goat antiserum prepared after sequentially immunizing with FeLV, RD 114, and woolly monkey virus p30's and labeled RaLV p30 was inhibited by all mammalian type C viruses, although preferentially by RaLV, FeLV, and HaLV. Thus, as in a previously reported assay developed with HaLV p30, rat, hamster, and cat p30's seem more closely related to each other than to mouse type C virus p30. High levels of specific antigen were found in all cell lines producing rat virus, whereas embryonic tissues from several rat strains and cell lines considered virus-free based on other tests were negative for p30. Rats bearing tumors containing Moloney murine sarcoma virus (RaLV) did not contain free circulating antibody to RaLV p30. Fifty-one human tumor extracts (including two tumor cell lines) were tested for activity in the RaLV species and 47 in the interspecies assays after Sephadex gel filtration and pooling of material in the 15,000- to 40,000-molecular-weight range. At a sensitivity level of 7 ng/ml (0.7 ng/assay) in the interspecies assay, all human tissues, with one exception, were negative. The one positive result is considered nonspecific based on proteolysis of the labeled antigen. Input tissue protein of the purified tumor extracts averaged 1.9 mg/ml with a range of less than 0.025 to 22 mg/ml. Tissues from NIH Swiss mice processed in the same manner were positive in the interspecies assay but negative in the intraspecies RaLV assay.     

Species and Interspecies Radioimmunoassays for Rat Type C Virus p30: Interviral Comparisons and Assay of Human Tumor Extracts

The major internal protein, p30, of rat type C virus (RaLV) was purified and utilized to establish intra- and interspecies radioimmunoassays. Three rat viruses were compared in homologous and heterologous intraspecies assays with no evidence of type specificity. The only heterologous viruses to give inhibition in these species assays were the feline (FeLV) and hamster (HaLV) type C viruses; these reactions were incomplete and required high virus concentrations. An interspecies assay using a goat antiserum prepared after sequentially immunizing with FeLV, RD 114, and woolly monkey virus p30's and labeled RaLV p30 was inhibited by all mammalian type C viruses, although preferentially by RaLV, FeLV, and HaLV. Thus, as in a previously reported assay developed with HaLV p30, rat, hamster, and cat p30's seem more closely related to each other than to mouse type C virus p30. High levels of specific antigen were found in all cell lines producing rat virus, whereas embryonic tissues from several rat strains and cell lines considered virus-free based on other tests were negative for p30. Rats bearing tumors containing Moloney murine sarcoma virus (RaLV) did not contain free circulating antibody to RaLV p30. Fifty-one human tumor extracts (including two tumor cell lines) were tested for activity in the RaLV species and 47 in the interspecies assays after Sephadex gel filtration and pooling of material in the 15,000- to 40,000-molecular-weight range. At a sensitivity level of 7 ng/ml (0.7 ng/assay) in the interspecies assay, all human tissues, with one exception, were negative. The one positive result is considered nonspecific based on proteolysis of the labeled antigen. Input tissue protein of the purified tumor extracts averaged 1.9 mg/ml with a range of < 0.025 to 22 mg/ml. Tissues from NIH Swiss mice processed in the same manner were positive in the interspecies assay but negative in the intraspecies RaLV assay.     

Radioimmunoassay for the Major Structural Protein of Hamster Type C Viruses

A radioimmunoassay for the major, group-specific antigen (p30) of hamster type C viruses was developed. The test detected approximately 5 ng of viral protein per ml and was highly specific for hamster viruses when used with homologous antibody. Comparison of three hamster viruses, two being mouse-hamster pseudotypes, in homologous and heterologous intraspecies assays, showed no evidence of type specificity for these proteins. The pseudotype viruses showed no evidence of mouse virus p30 antigenic determinants. An interspecies antigen assay employing (125)I-labeled hamster p30 and anti-feline p30 was completely inhibited by cat (feline leukemia virus), hamster, and rat viruses, to a slightly lesser degree by mouse viruses, and only poorly by RD 114 and Gibbon ape viruses. The Mason-Pfizer virus did not inhibit this assay. Hamster p30 was detected by radioimmunoassay in individual embryos from two LSH hamsters and in several adult tissues, excluding muscle at levels below that required for detection in complement-fixation tests.     

Evolutionary relationships between gag gene-coded proteins of murine and primate endogenous type C RNA viruses.

Several low molecular weight proteins of endogenous type C viruses of the RD114/baboon group are compared with the gag gene translational products of endogenous type C viruses of murine origin. The p10 proteins of each virus group are shown to be immunologically and biochemically related, while the p12 proteins of RD114/baboon viruses are demonstrated to share antigenic determinants with murine viral p15. Moreover, highly type-specific phosphoproteins, p15 of RD114/baboon viruses and p12 of murine viruses, are shown to possess very similar biochemical properties. These findings, along with previous studies indicating immunologic cross-reactivity between their major internal antigens, p30, demonstrate that each of the gag gene-coded proteins of murine type C viruses has a analogue in viruses of the RD114/baboon group. The immunologic and biochemical relatedness of their gag gene translational products supports the concept of a common progenitor in the evolution of these endogenous viruses.     

Interspecies homology of RNA tumor virus proteins.

We report the application of a highly sensitive column chromatographic technique to the comparison of tryptic peptide maps of some RNA tumor virus proteins. By combining microbore ion-exchange chromatography with a sensitive fluorescent assay using o-phthalaldehyde, we obtained high-resolution peptide maps starting with only microgram amounts of protein. Our discovery of coincident peptides from the 15,000 and 30,000 molecular weight proteins from murine and feline leukemia viruses supports serological evidence for interspecies antigenic determinants; coincident peptides were also found for the 10,000 molecular weight proteins from these viruses, although immunochemical data did not reveal interspecies determinants. The relatively large number of coeluting peptides found in the 15,000 and 10,000 molecular weight proteins is strong evidence for the existence of homology.     

Chemical and Immunological characterization of the major structural protein (p28) of MMC-1, a rhesus monkey endogenous type C virus: homology with the major structural protein of avian reticuloendotheliosis virus.

The major core protein (p28) of MMC-1, an endogenous type C virus of the rhesus monkey (Macaca mulatta), was purified and subjected to structural and immunological analyses. The NH2-terminal amino acid sequence of MMC-1 p28 showed extensive homology to the sequences of the major structural proteins (p30) of known mammalian type C viruses. Similarly, interspecies antigenic determinants shared by all the above viral proteins were detected in MMC-1 p28. Competition radioimmunoassays together with the results of statistical analysis of the primary structure data provided evidence that MMC-1 p28 is not more closely related to primate type C viruses of the Papio genus than to those isolated from rodents, cats, or New World monkeys. MMC-1 p28 was found to be closely related structurally to the p30 protein of the avian reticuloendotheliosis virus (REV-A), a horizontally transmitted type C virus of putative mammalian origin. In addition, MMC-1 p28 and REV-A p30 shared a specific subset of antigenic determinants not present in any of the other avian or mammalian type C viruses studied. These findings suggest that MMC-1 and REV may have a common evolutionary origin.     

Structural Proteins of Mammalian Oncogenic RNA Viruses: Immunological Characterization of the p15 Polypeptide of Rauscher Murine Virus

The immunological properties of the purified 15,000-dalton protein of Rauscher murine type-C virus were analyzed by radioimmunoassay. The majority of the antigenic determinants of this protein were found to be remarkably specific to Rauscher and Friend virus and to a lesser extent to Moloney virus. Determinants reactive with other murine viruses (group-specific) or type-C viruses of other species (interspecies) were also demonstrated but were minor components of the total antigenic specificities of the protein. The results provide evidence that the antigenic properties of this protein specify the Friend-Moloney-Rauscher subgroup of type-C viruses.     

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.     

Feline Leukaemia Viral Antigens and Antisera to the Group Specific Antigens of the Murine Leukaemia Viruses

GEERING et al.¹ reported that feline leukaemia viruses shared one of the group specific antigens of the murine leukaemia viruses, gs-3, as detected by immunoprecipitation in agar gels with broadly reactive rat antisera to the group specific antigens of the murine leukaemia viruses (MuLV). Subsequently, they found that this shared group specific antigen was also present in the hamster and rat C-type viruses². Work by Schafer³ and our own immunodiffusion⁴ and complement fixation studies have confirmed the immunological reactivity between the feline leukaemia viral antigens and broad-reacting murine leukaemia group specific antisera. We have now applied this interspecies immunological reaction between the murine and feline C-type viruses to quantitative studies of the feline leukaemia viruses. Broad-reactive murine leukaemia-sarcoma group specific antisera prepared in rats by the induction of murine sarcoma virus (MSV) tumours^{5, 6} were found to be as useful and nearly as sensitive as a feline leukaemia-sarcoma specific, group specific antiserum for the in vitro detection and assay of the noncytopathogenic feline leukaemia virus (FeLV).     

Selective host range restriction of goat cells for recombinant murine leukemia virus and feline leukemia virus type A

We isolated a strain of normal goat fibroblasts which was uniquely selective in that it allowed the replication of xenotropic murine leukemia virus but not polytropic recombinant murine leukemia virus. In addition, feline leukemia virus type A replication was severely diminished in these goat cells, whereas feline leukemia virus type B and feline endogenous RD114-CCC viruses replicated efficiently. No other known cells exhibit this pattern of virus growth restriction. These goat cells allow the study of xenotropic murine leukemia virus in mixtures which also contain recombinant murine leukemia virus and may be helpful in eliminating feline leukemia virus type which often coexists in feline sarcoma or leukemia virus mixtures with other feline leukemia virus types.     

Serological analysis of an oncornavirus (PMF virus) detected in malignant permanent human cell lines.

Immunodiffusion analysis of the PMF virus which was detected in malignant permanent human cell lines revealed positive reactions with antisera against the Mason-Pfizer monkey virus (MPMV). No cross-reactivity was demonstrated with murine leukemia virus (MuLV), rat leukemia virus (RaLV), hamster leukemia virus (HaLV), feline leukemia virus (FeLV), simian (woolly monkey) sarcoma virus (SSV-1) and mouse mammary tumor virus (MTV). The cross-reactive antigens of the PMF virus and the MPMV are considered as evidence for the human origin of the PMF virus.