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.     

Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III.

The human T-cell lymphotropic virus type III (HTLV-III/LAV) is a retrovirus associated with acquired immune deficiency syndrome. The region on the viral genome that is necessary for trans-activation of the HTLV-III/LAV long terminal repeat called tatIII has previously been determined to lie between nucleotides 5365 and 5607. Here we report that a bacterial fusion protein containing amino acid sequences specified by the first coding exon of the tatIII gene is recognized by some patient antisera. We also demonstrate that lymphoid and epithelial cells that express the trans activator function express a 14-kilodalton (kDa) protein recognized by a patient antiserum that reacts with the bacterial tatIII fusion protein. Cells transiently transfected with a deletion mutant of the trans activator protein produce a 12-kDa protein rather than the 14-kDa protein. These observations indicate that the tatIII region contains a functional gene and is capable of expressing a protein that migrates with an apparent molecular size of 14 kDa in some lymphoid and epithelial cells transfected with plasmids containing the tatIII region. We propose that the product of the trans activator gene be designated p14tat-III.     

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.     

Molecular cloning and characterization of murine leukemia virus-related DNA sequences from C3H/HeN mouse DNA.

Ten murine leukemia virus (MuLV)-related DNA sequences were isolated from C3H/HeN mouse genomic DNA by cloning of EcoRI fragments in a Charon 4A vector. Detailed restriction endonuclease maps of four of the clones were developed by using AKR MuLV [32P]cDNA as a probe. C3H clone 14-9 contains approximately 7 kilobase pairs of MuLV-related DNA, one copy of an MuLV long terminal repeat-like sequence, and a region of flanking mouse DNA. C3H clones 34.2 and 36.1 contain approximately 2 kilobase pairs of MuLV-related DNA, one copy of a MuLV LTR-like sequence, and differing lengths of flanking mouse DNA sequences. C3H clone 8.13 was found to contain an insert of 5.7 kilobase pairs of MuLV-related DNA with two long terminal repeat-like regions and sequences which are partially homologous to AKv-1. Comparison fo the restriction endonuclease cleavage maps of these C3H clones with maps recently developed for ecotropic and xenotropic MuLV DNAs indicates that C3H clone 14-9 corresponds to the 5'-terminal portion of a genomic DNA sequence related to xenotropic MuLVs, whereas C3H clones 34.2 and 36.1 correspond to the 3' terminal portions of genomic DNA sequences related to xenotropic MuLVs. Clone 8.13 represents a deleted, xenotropic MuLV-related provirus. C3H clones 14-9, 34.2, 36.1, and 8.13 provide defined DNA sequence probes with which to characterize the organization and expression of endogenous MuLV-related DNA sequences in the mouse genome.     

Multimeric forms of herpes simplex virus type 2 glycoproteins.

Molecular clones of closed circular DNA molecules of a mink cell focus-inducing murine leukemia virus (MCF-13 MuLV) were generated. Closed circular DNA molecules isolated from a Hirt extraction of recently infected NIH/3T3 cells were inserted at their unique EcoRI site into lambda gtWES.lambda B. Restriction endonuclease analysis of inserts of two clones indicated that they represented intact MCF-13 MuLV genomes. One viral insert contained two large terminal repeat sequences, and the other contained only one. A 300-base-pair DNA fragment located in the envelope region of the MCF-13 MuLV genome was determined to be related to xenotropic MuLV sequences.     

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.     

Comparison of endogenous murine leukemia virus proviral organization and RNA expression in 3-methylcholanthrene-induced and spontaneous thymic lymphomas in RF and AKR mice.

3-Methylcholanthrene-induced T-cell thymic lymphomas in RF mice were examined for involvement of murine leukemia virus (MuLV)-related sequences in leukemogenesis. Both the expression of MuLV-related RNA species and the organization of endogenous MuLV proviral DNA were analyzed. Of 27 primary tumors examined, only 5 exhibited elevated MuLV-related RNA species homologous to xenotropic specific env DNA. None of these RNA species hybridized with ecotropic p15E DNA sequences. Only two of these five tumors contained MuLV-like RNA species that hybridized with ecotropic MuLV long terminal repeat sequences, despite the probe's ability to detect both ecotropic MuLV and mink cell focus-inducing viral RNA. No muLV resembling mink cell focus-inducing virus whose expression could be correlated with lymphomagenesis was detected in either preleukemic thymocytes, primary 3-methylcholanthrene-induced thymic tumors, tumors passaged in vivo, or cell lines derived from tumors. Restriction endonuclease analysis of DNA from both primary tumors and cell lines failed to reveal either proviral DNA with recombinant env genes or rearrangement of endogenous MuLV proviruses. Therefore, chemically induced lymphomagenesis in RF mice appears different from the spontaneous lymphomagenic process in AKR mice with respect to the involvement of endogenous MuLV sequences.     

Viral proteins expressed on the surface of murine leukemia cells.

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.     

Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus Akv.

OBJECTIVE: Mouse strains carrying endogenous ecotropic murine leukemia viruses (MuLV) are capable of expressing infective virus throughout life. Risk of transplacental transmission of MuLV raises concerns of embryo infection and induction of pathogenic effects, and postnatal MuLV infection may lead to tumorigenesis. METHODS: Endogenous ecotropic MuLV-negative SWR/J embryos were implanted into Akv-infected viremic SWR/J mice, into spontaneously provirus-expressing AKR/J mice, and into noninfected SWR/J control mice; virus integration and virus expression were investigated at 14 days' gestation. Tumor development was monitored over 18 months. RESULTS: Of 111 embryos, 20 (18%) recovered from Akv-infected SWR/J mice, which had developed normally, were infected. New proviruses were detected in 10 of 111 (9%) embryos from Akv-infected SWR/J mice, and in 2 of 60 (3%) embryos from AKR/J mice; none expressed viral protein. Of 127 embryos recovered from Akv-infected SWR/J mice, 16 (13%) were dead; 4 of 5 (80%) were infected and expressed viral protein. Of 71 embryos from AKR/J mice, 11 (15%) were dead, and 2 of 2 had virus integration; virus expression was not detected. Numbers of dead embryos recovered from experimentally infected, viremic SWR/J mice and from spontaneously endogenous MuLV-expressing AKR/J mice were significantly higher, compared with numbers from nonviremic SWR/J control mice, and embryo lethality was significantly associated with prenatal provirus expression. Postnatal inoculation of Akv induced lymphoblastic lymphomas in 15 of 24 (61%) SWR/J mice within mean +/- SD latency of 14 +/- 2.4 months. Only 3 of 39 (8%) control mice developed lymphomas (P < 0.005). CONCLUSION: Embryos in MuLV-viremic dams are readily infected, and inappropriate prenatal expression of leukemogenic endogenous retroviruses may play a critical role in embryo lethality and decreased breeding performance in ecotropic provirus-positive mouse strains.