Immunological Cross-Reactions Between Two Low-Molecular-Weight Polypeptides from A Murine Type C Virus

Two low-molecular-weight type-specific virion polypeptides from the Kirsten strain of Murine leukemia virus, polypeptides p10 and p12, are immunologically related by radioimmunoassay competition techniques.     

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.     

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.     

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses IV. Biochemical Typing of Murine Viral Proteins

Tryptic peptide maps were prepared for four purified structural proteins derived from several murine leukemia viruses (MuLV's). Analyses of these peptide maps reveal that the p30 proteins of Rauscher, Moloney, and Gross MuLV's are very similar to each other, as are the p10's obtained from these three viruses. In contrast, the peptide maps of the individual p15's and p12's from the same viruses establish that each of these polypeptides is highly strain specific. For all four polypeptides studied, unique peptides appear in the Rauscher MuLV and Moloney MuLV tryptic profiles that are not present in the corresponding Gross MuLV profile. By this method of analysis it was possible to distinguish the p30's of N-tropic and B-tropic MuLV's derived from the same BALB/c mouse.     

Antigenic characterization of type C RNA virus isolates of gibbon apes.

Type C RNA viruses initially isolated from a lymphosarcoma of a gibbon ape and from a fibrosarcoma of a woolly monkey are very closely related immunologically. However, recent studies have shown that these viruses are distinguishable in a radioimmunoassay for the 12,000-molecular-weight polypeptide (p12) of the woolly monkey virus. In the present report, an immunoassay has been developed for the p12 polypeptide of the gibbon ape type C virus. This assay is shown to further distinguish the woolly monkey and gibbon ape viruses. In type-specific assays for the p12 polypeptides of these viruses, two new type C viruses isolated from gibbons in a second colony, characterized by high incidence of hemopoietic neoplasia, are immunologically distinguishable from the original gibbon ape virus. The p12 type-specific immunoassays described in the present report may be of importance in studying the natural history of these viruses and their relationship to tumors of primates.     

In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane.

To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells.     

Characterization of the type and group specificities of the immune response in mice to murine leukemia viruses.

Normal sera from a variety of strains of inbred mice have precipitating antibodies to murine type C viruses that are detected by radioimmune precipitation assays. The results demonostrate that this humoral immune response is primarily directed against the AKR strain of murine leukemia virus (MuLV) proteins gp71, gp43, and p15(E). These sera also react with Friend- or Rauscher-MuLV in radioimmune precipitation assays. This reaction is not due to a separate immune response, but rather is primarily a consequence of the cross-reactivity of antibodies to the AKR strain of MuLV p15(E) with the p15(E) of these viruses. These data, using autogenous immune sera, emphasize the serological differences of the virion glycoproteins and the serological similarity of the p15(E) virion component of the viruses. Furthermore, based on the serological reactivities to the glycoproteins, the results suggest that the AKR strain of MuLV is endogenous to and expressed in mice, but that the Friend-Moloney-Rauscher virus group is not.     

Mouse strain resistant to N-, B-, and NB-tropic murine leukemia viruses.

Mouse strain G was studied for its susceptibility to various strains of murine leukemia and sarcoma viruses. Both N- and NB-tropic Friend leukemia viruses neither induced splenomegaly nor grew efficiently in strain G mice. Using the XC test, cultured embryo cells were found to be resistant, but not absolutely, to all the tested viruses, N-tropic AKR virus, N- and NB-tropic Friend leukemia viruses, NB-tropic Rauscher leukemia virus, B-tropic WN1802B virus, NB-tropic Moloney leukemia and sarcoma viruses, and N-tropic Kirsten sarcoma virus, although the resistance to Moloney leukemia and sarcoma viruses is sometimes not as strong as that for other viruses. Thus, the strain G mice are unique among mouse strains because they show resistance that is not related to the N-B tropism of murine leukemia viruses.     

Cells transformed by certain strains of Moloney sarcoma virus contain murine p60.

It was previously demonstrated that the 60,000 dalton (p60) precursor-like polyprotein containing murine p30 was a constituent of the feline leukemia virus pseudotype of Moloney sarcoma virus [m1MSV(FeLV)]. It is now shown that p60 is detected in cells of five mammalian species transformed by m1MSV, indicating that p60 is specified by this genome. Moreover, little or no murine p30 is detected in the m1MSV-transformed cells, suggesting that the murine group p30 antigenic reactivity of S + L- cells is ude to p60. Pulse-chase studies in cells producing m1MSV(FeLV) show that p60 is the largest polypeptide detectable during the pulse, and that intracellular p60 is not cleaved into smaller (for example, p30) polypeptides during chase periods of up to 10 hr. The lack of cleavage of p60 is in contrast to the properties of p30 precursors detected in cells containing replicating avian or mammalian RNA tumor viruses. The inefficient cleavage of intracellular p60 and the kinetics of appearance of murine p30 in extracellular m1MSV(FeLV) suggest that p60 cleavage to p30 occurs in cells shortly before virus release. While only p60 was detected in the m1MSV-transformed cells, p60 and p70 were detected in m3MSV-transformed cells, and no immunoprecipitable polypeptides were detected in HT-1 MSV-transformed cells. The observed differences in the intracellular polypeptide expression by each of the strains of MSV suggests differences in genetic content.     

Translation of murine leukemia virus RNA in cell-free systems from animal cells.

The virion RNA of Moloney murine leukemia virus (MuLV) has been translated in eukaryotic cell-free systems derived from mouse L- and human HeLa cells. In both systems at least three polypeptides, approximately 60,000, 70,000, and 180,000 in apparent molecular weight, were formed in response to the added 35S MuLV RNA. All three polypeptides were precipitable with antiserum to detergent-disrupted MuLV. Fingerprint analysis of tryptic digests indicated that all three contain anino acid sequences in common with each other and with the major methionine-containing structural proteins of the virion.     

Biochemical characterization of the amphotropic group of murine leukemia viruses.

The recently described amphotropic group of murine leukemia viruses constitutes a distinct biological group, differing from the ecotropic and xenotropic groups in host range, cross interference, and serological reactivity. Viruses of this group have been detected only in wild mice from certain areas in California. By using a [3H]DNA probe synthesized in an endogenous reaction from detergent-lysed amphotropic virus (strain 1504-A), it was demonstrated that the amphotropic murine leukemia viruses are distinct biochemically, in that 20% of the viral genome sequences are not shared by AKR-type ecotropic or nay of three types of xenotropic murine leukemia virus tested. A subset of these amphotropic unique sequences, comprising one half of them, is present in the genome of wild mouse ecotropic viruses and in Moloney and Rauscher viruses as well. Sequences homologous to the entire genome of 1504-A amphotropic virus are present in the cellular DNA of all eight inbred mouse strains tested, as well as in wild Mus in Asia, in amounts varying from three to six complete viral genomes per haploid cell genome. Evidence is presented that at least 20% of the DNA sequences in both mouse- and mink-grown murine leukemia virus probes are of host-cell origin.     

Determination of the proteolytic processing sites in the polyprotein encoded by the bottom-component RNA of cowpea mosaic virus

We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components.     

Feline leukemia virus: biochemical and immunological characterization of gag gene-coded structural proteins.

The major non-glycosylated structural proteins of feline leukemia virus have been isolated, and competition immunoassays have been developed for each. These proteins include the 27,000- to 30,000-molecular-weight major internal antigen designated p30, a 15,000-molecular-weight protein (p15), an acidic protein of 12,000 molecular weight (p12), and a highly basic 10,000-molecular-weight protein (p10). Immunologically and biochemically corresponding proteins of feline and murine leukemia viruses have been identified. and, on the basis of analogy to the known sequence of a prototype type C virus of mouse origin, the map order of the gag region of the feline type C viral genome has been tentatively deduced as NH2-p15-p12-p10-COOH. The demonstration of two feline leukemia virus gag gene-coded proteins, p15 and p12, expressed in the form of an uncleaved precursor in a mink cell line nonproductively transformed by feline sarcoma virus provides indirect support for the proposed sequence.     

Sequence analysis of Gardner-Arnstein feline leukaemia virus envelope gene reveals common structural properties of mammalian retroviral envelope genes

We have sequenced the envelope (env) gene and most of the adjacent 3' long terminal repeat (LTR) of Gardner-Arnstein feline leukaemia virus subtype B. The LTR of this virus contains, at corresponding positions, all signal sequence elements known from other retroviral LTRs. The deduced amino acid sequence of the longest open reading frame was compared with env polypeptide sequences of several murine leukaemia viruses. This allowed us to predict the positions of both p12/15env and gp70 polypeptides as well as a hydrophobic leader polypeptide. The env polypeptides of the different viruses show long stretches of homology and similar hydrophilicity profiles in the p12env region and in the carboxy-terminal half of gp70 (constant region). The most extensive variations are confined to certain parts of the amino-terminal half of gp70 (differential region). In this region, however, feline leukaemia virus and murine mink cell focus forming viruses are still closely related. A correspondingly spaced pattern of identical, short amino acid sequences appears in three different parts of the env polyprotein, suggesting its evolution from a primordial env-related precursor by tandem duplications.     

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.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.