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.     

Immunological relationships of OMC-1, an endogenous virus of owl monkeys, with mammalian and avian type C viruses

The relationships between OMC-1, an endogenous oncovirus of owl monkey, and representatives of the three oncoviral genera have been investigated by radioimmunological techniques. The major structural protein of OMC-1 was shown to share antigenic determinants with the corresponding proteins of certain type C viruses of rodent, feline, and cervine origin. It was not possible to demonstrate antigenic cross-reactivity between OMC-1 and endogenous type C viruses of baboons. These findings argue that OMC-1 and baboon endogenous viruses do not represent direct descendants of an ancestor virus that became integrated within primates prior to the divergence of New and Old World species. A close antigenic relationship was established between the major structural proteins of OMC-1, an endogenous virus of deer (deer kidney virus), and avian reticuloendotheliosis viruses. These findings establish OMC-1 and deer kidney virus in the evolutionary lineage that may have led to the generation of avian reticuloendotheliosis virus, a group of oncogenic viruses capable of crossing the interclass barrier between mammals and birds.     

Avian reticuloendotheliosis viruses: evolutionary linkage with mammalian type C retroviruses.

Reticuloendotheliosis viruses have been shown to be causative of tumors in a variety of avian species. The major structural protein of these non-genetically transmitted viruses is demonstrated to possess antigenic determinants common to those of all known mammalian type C viruses. These findings establish a mammalian origin for this oncogenic avian retrovirus group. None of the known mammalian type C virus groups demonstrated a closer immunological relationship to avian reticuloendotheliosis viruses. These results suggest that reticuloendotheliosis viruses have been non-genetically transmitted for a long period of evolution or that these viruses may have arisen by relatively recent infection of birds with an as yet undiscovered mammalian type C retrovirus.     

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.     

Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates.

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.     

Reticuloendotheliosis virus: detection of immunological relationship to mammalian type C retroviruses.

Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH2-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae.     

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.     

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.     

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.     

Genome of Reticuloendotheliosis Virus: Characterization by Use of Cloned Proviral DNA

Reticuloendotheliosis virus is an avian type C retrovirus that is capable of transforming fibroblasts and hematopoietic cells both in vivo and in vitro. This virus is highly related to the three other members of the reticuloendotheliosis virus group, including spleen necrosis virus, but it is apparently unrelated to the avian leukosis-sarcoma virus family. Previous studies have shown that it consists of a replication-competent helper virus (designated REV-A) and a defective component (designated REV) that is responsible for transformation. In this study we used restriction endonuclease mapping and heteroduplex analysis to characterize the proviral DNAs of REV-A and REV. Both producer and nonproducer transformed chicken spleen cells were used as sources of REV proviral DNA; this genome was mapped in detail, and fragments of it were cloned in lambdagtWES.lambdaB. The infected canine thymus line Cf2Th(REV-A) was used as a source of REV-A proviral DNA. The restriction maps and heteroduplexes of the REV and REV-A genomes showed that (proceeding from 5' to 3') (i) REV contains a large fraction of the REV-A gag gene (assuming a gene order of gag-pol-env and gene sizes similar to those of other type C viruses), for the two genomes are very similar over a distance of 2.1 kilobases beginning at their 5' termini; (ii) most or all of REV-A pol is deleted in REV; (iii) REV contains a 1.1 kilobase segment derived from the 3' end of REV-A pol or the 5' end of env or both; (iv) this env region in REV is followed by a 1.9-kilobase segment which is unrelated to REV-A; and (v) the helper-unrelated segment of REV extends essentially all of the way to the beginning of the 3' long terminal repeat. Therefore, like avian myeloblastosis virus but unlike the other avian acute leukemia viruses and most mammalian and avian sarcoma viruses, REV appears to be an env gene recombinant. We also found that the REV-specific segment is derived from avian DNA, for a cloned REV fragment was able to hybridize with the DNA from an uninfected chicken. Therefore, like the other acute transforming viruses, REV appears to be the product of recombination between a replication-competent virus and host DNA. Two other defective genomes in virus-producing chicken cells were also cloned and characterized. One was very similar to REV in its presumptive gag and env segments, but instead of a host-derived insertion it contained additional env sequences. The second was similar (but not identical) to the first in its gag and env regions and appeared to contain an additional 1-kilobase inversion of REV-A sequences.     

Immune response of the mouse to the major core protein (p30) of ecotropic leukemia viruses.

Sera from normal C57BL/6 mice contained low titers of antibodies against proteins of MuLV. Sera from C57BL/6 mice that were immunized with allogeneic leukemia cells sometimes contained high-titered antibodies against the p15 protein of MuLV; these antibodies detected group-specific antigenic determinants of the p15 protein, since reactions were observed with the p15 proteins of both AKR and Moloney viruses. In contrast, antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted strongly with the p30 protein of MuLV, as well as with p15. Antibodies in the C57BL/6 anti-AKR K36 sera detected group-specific antigenic determinants of the p30 protein; reactions were observed with the C57BL/6 anti-AKR K36 serum and the p30 proteins of both AKR and Moloney viruses. It was concluded that mice do have the capacity to respond immunologically to antigenic determinants of the MuLV p30 protein, although in most circumstances this is not observed.     

The avian retrovirus env gene family: molecular analysis of host range and antigenic variants.

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.     

Interstrain Variation of the Major Internal Structural Component (p30gag) of Two Murine Oncornaviruses: Comparative Immunochemical, Biochemical, and Biophysical Analysis

The major internal structural protein (p30(gag)) of the Moloney leukemia virus and the endogenous Y-1 murine oncornavirus was examined for biochemical and biophysical manifestations of interstrain antigenic variation. Although the two viral proteins share murine group-specific antigenic determinants, the Y-1 virus p30 appeared to have both a lower relative number of such determinants and a decreased affinity at the cross-reactive sites for Moloney virus p30 monospecific antibodies. Further, immunological analysis indicated the presence of unique antigenic sites on the Moloney virus p30 not shared by the analogous Y-1 virus molecule. The two polypeptides copurified and had similar isoelectric points (pH 6.2 to 6.3) and sedimentation coefficients (2.47S). However, equilibrium sedimentation yielded a significant mass difference between the two proteins, 28,300 +/- 600 and 31,000 +/- 300 daltons for the Moloney and Y-1 virus molecules, respectively. Amino acid analysis indicated a concomitant increase in total residues for the Y-1 virus p30, although a number of residues appeared to have been conserved between the two viral proteins. Conformational studies and hydrodynamic calculations demonstrated marked secondary and tertiary structural differences; with the Y-1 virus p30 being an asymmetric prolate ellipsoid containing 27 to 28% alpha-helix and Moloney virus p30 being somewhat more spherical and possessing an alpha-helical content of 50 to 55%. Two-dimensional mapping of (125)I-labeled tryptic peptides of each p30 suggested that considerable sequence heterogeneity is responsible for many of the biophysical, biochemical, and immunochemical differences in these two analogous structural proteins.     

Antigenic analysis of major structural proteins of avian leukoviruses.

The antigenic determinants of the avian leukovirus proteins p27, p19, p15, and p12 were examined by radioimmunoassay. There was no evidence of antigenic cross-reactivity among these polypeptides, indicating that they are four distinct components. The concentration of the proteins p27, p19, and p15 in the infected cell was measured and found greatly increased, indicating that synthesis of these proteins is induced after virus infection.     

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.     

Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24.

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.