Mammalian Retroviruses Research Articles

Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein.

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.

Genes and gene products involved in growth regulation of tumor cells.

Protooncogenes, the cellular homologs of retroviral oncogenes, are cellular genes that control the growth and affect the differentiation of eukaryotic cells. Long before the discovery of viral oncogenes and their cellular homologs (Bishop 1983), it was clear that malignantly transformed cells were genetically altered with regard to their growth factor requirements for propogation in culture (Temin 1967, 1970). Moreover, the fact that cells transformed by oncogene transducing mammalian retroviruses were blocked for the binding of epidermal growth factor (DeLarco and Todaro 1978) and released transforming growth factors into the culture medium (Todaro et al. 1979, 1980; Marquardt et al. 1983) suggested early on that some viral oncogenes might be coding for or regulating the expression of such growth factors by infected cells. These observations have led to the proposal that transformed cells maintain autonomous growth by autocrine secretion (Sporn and Todaro 1980). Substance was added to such hypotheses by the recent finding that one oncogene, v-sis, was derived from a cellular growth factor gene, that for platelet-derived growth factor (PDGF) (Doolittle et al. 1983; Johnsson et al. 1984). Moreover, another viral oncogene, erb-B, was identified as a portion of the receptor gene for epidermal grwoth factor (EGF) (Downward et al. 1984; Ullrich et al. 1984). Evidence is accumulating that other oncogenes also code for portions of growth factor receptors or for proteins that affect the signal transmission from growth factor receptors to the nucleus. Analogous to the observation that certain combinations of peptide growth factors may have synergistic effects on cell growth properties, it was recently demonstrated that combinations of “complementary” oncogenes may act synergistically to transform a particular cell type (Land et al. 1983a, b).

Monoclonal antibody defining a human syncytiotrophoblastic polypeptide immunologically related to mammalian retrovirus structural protein p30

Antigenic material previously detected in human placental trophoblastic cells by immunoperoxidase staining using a goat antiserum against the feline RD114 retrovirus structural protein p30 was isolated by immunochromatography from normal syncytiotrophoblast. The antigen was used to immunize mice, and of the monoclonal antibodies produced by murine hybridomas an IgG1 was selected which reacted in enzyme immunoassay with the syncytiotrophoblast antigen and with purified RD114. This antibody, designated HPS-1, stained normal and neoplastic syncytiotrophoblasts in a manner similar to that of the goat antibodies, detected in immunoblotting a Mr = 130 000 polypeptide in cultured human choriocarcinoma cells and reacted in spot immunoblotting tests with purified preparations of mammalian retroviruses but not with an avian retrovirus. The polypeptide antigen may represent activation of human endogenous retroviral genes in syncytiotrophoblast.

Polypeptides of Mason-Pfizer monkey virus I. Synthesis and processing of the gag-gene products

Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, differs from the mammalian C-type retroviruses by preassembling core structures in the cytoplasm of infected cells during morphogenesis. Studies that define the protein composition of M-PMV virions and identify two gag-related polyprotein precursors in M-PMV infected cells are reported. The polyprotein precursor to the internal structural ( gag) proteins of M-PMV was identified by immunoprecipitation from lysates of pulse-labeled, virus-infected cells with an antiserum to the major structural protein, p27. Tryptic peptide-mapping experiments have shown that this precursor (Pr78) is cleaved to yield five virion structural polypeptides—p27, pp16, p14, p12, and p10. The pp16 polypeptide represents an additional gag-gene encoded polypeptide, not described previously; it is a phosphoprotein and present in virions in a number of forms. A second gag-related polyprotein precursor, P95, is also present in infected cells although in smaller amounts. This nonglycosylated polypeptide contains all of the leucine-containing tryptic peptides of Pr78 plus three others. Studies of the rate of synthesis and half-life of this protein argue against it being the major gag-gene precursor polypeptide. The possibility that it represents a precursor to the viral protease is discussed.

Purification of a specific inhibitor of reverse transcriptase from human placenta.

Human placental extracts contain a factor which specifically and reversibly inhibits the reverse transcriptase of mammalian retroviruses. This placental inhibitor has been partially purified and characterized. It elutes at 0.1-0.2 M phosphate on hydroxyapatite chromatography and can be further purified by phosphocellulose chromatography where it elutes at 0.4 M KCl. By these purification procedures, specific activities of 40-70,000 units of inhibitor per mg of protein were obtained. The size of the inhibitor is about 60-65,000 daltons as estimated by velocity sedimentation. The inhibitor purified by these techniques selectively inhibits the activity of purified reverse transcriptase from Rauscher murine leukemia virus and baboon endogenous virus. It is substantially less active against the reverse transcriptase of avian myeloblastosis virus. The specificity of this inhibitor for mammalian enzymes and particularly for the human placental reverse transcriptase suggests that it plays a role in the regulation of DNA synthesis in human placental development.

International Symposium on Retroviruses and Human Pathology

Introduction: The Role of Retroviruses.- Opening Remarks.- Retroviruses in Neoplasia Yesterday, Today, and Tomorrow.- The Role of Retroviruses in Nature.- W. P. Rowe Memorial Lecture.- Changing Dogmas in Retrovirology.- Section 1 Retroviruses and the Murine Model System.- The Biology of Endogenous Leukemia Viruses: A Study of the DBA/2 Mouse.- Murine Xenotropic Retroviruses in the Computer Age.- Viral and Nonviral Mammary Tumorigenesis.- The Proviral Genome of Radiation Leukemia Virus: Molecular Cloning, Nucleotide Sequence of Its Long Terminal Repeat, and Integration in Lymphoma Cell DNA.- Metastatic Potential of Friend Leukemia Cells: Fibronectin Induces Specific Arrest, Survival, and Growth of FLC in Liver of DBA/2 Mice.- The Envelope Gene of Mammalian Retroviruses: Analysis of Its Structure Reveals Specific Recombination Signals.- The Major Histocompatibility Complex (H-2) of the Mouse Influences the Phenotype of Murine Leukemia Virus-Induced Lymphomas.- Mouse Mammary Tumor Virus (MMTV) Morphology in the Gastrointestinal Lumen of Suckling Mice.- Murine Retroviruses: An Alternative Screening Approach for Antiviral Drug Research.- Section 2 Retroviruses and the Vertebrate Model System.- Retroviruses and Oncogenes in Rats.- Target Cells for Avian Leukemia Viruses Revisited.- Feline Retroviruses.- Bovine Leukemia Virus, A Distinguished Member of the Human TLymphotropic Viruses Family.- Association of Ultraviolet-Induced Retrovirus Expression with Anchorage-Independent Survival in Rat Embryo Cells.- Participation of 7S L RNA in Reverse Transcription by an Avian Retrovirus.- Different Retroviruses Show Different Susceptibilities to 2-Deoxy-D-glucose.- A Comparative Study of Bovine Leukemia Virus (BLV) Detection Methods.- Structural and Functional Heterogeneity among Type D Retroviruses.- Retroviruses and Oncogenes in the Rat Sarcoma Galliera: An old Tumor as a Unique Model System.- Section 3. Retroviruses and Human Pathology.- Molecular Biology of Viruses of the HTLV Family.- Activation of Retroviral Genes in Human Reproductive Tissues and Tumors.- Retrovirus and AIDS.- Human T-Cell Leukemia (Lymphotropic) Retroviruses: The HTLV Family and Their Role in Leukemias, Lymphomas, and AIDS.- The Organization of Mouse Mammary Tumor Virus-Related Sequences in Human Cellular DNA.- Close Structural Similarities Between the LTRs of Bovine Leukemia and Human T-Cell Leukemia Viruses.- Analysis and Immunological Properties of Lymphadenopathy- Associated Virus (LAV) Structural Proteins.- RNA Viruses and Lymphocyte Immune Functions.- Anti HTLV-III and Anti T-Cell Antibodies in AIDS and ARC Patients.- Seroepidemiological Study of Lymphadenopathy-Associated Virus, by ELISA, in Acquired Immunodeficiency Syndrome.- IgG Antibodies to SAIDS-D Virus Associated Antigens in Patients with AIDS and at Risk for AIDS.- Is a Retrovirus-Like Particle Expressed at a Specific Stage During Human Oocyte Maturation?.- Psoriasis - A Retrovirus Disease?.- Purification and Characterization of a High Molecular Weight Human Milk Ribonuclease: Its Potential use for Diagnosis and Prognosis of Human Mammary Neoplasia.- The Role of Retroviruses in AIDS and Slow Infections.- Section 4. Retroviruses and Oncogenes.- Retroviruses with Two Oncogenes.- Molecular Assays for Detection Of ras Oncogenes in Human and Animal Tumors.- The raf Oncogene.- Role of Retroviral Oncogenes in the Attachment of Cells to Natural Substrata.- Amplification of Cellular Oncogenes in Colon and Lung Cancer Cells.- The Nucleotide Sequence of the erbA Gene of Avian Erythroblastosis Virus Appears Unrelated to Other Known Oncogenes.- Characterization of a Human Osteosarcome Oncogene.- Coordinated Regulation of the c-myc Gene and a Tumor-related Multigenic Set in Human Neoplastic Cells Treated with Differentiation Inducers Including Tumor Promoters.- Transcripts of c-fes Oncogene are Present at Detectable Levels in Total Cellular RNA of Human Myeloid but not Lymphoid Normal and Neoplastic Cells.- Heterogeneity of Human Normal and Leukemic Cells of Lymphoid and Myeloid Lineage in Respect to the Expression Of c-myc and c-myb Mrna Transcripts.- Detection Of c-myc, c-myb and c-fes Oncogene Transcripts in Human Myeloid and Lymphoid Normal and Leukemic Cells by In Situ Hybridization.

Incorporation of lipids into variants of Moloney sarcoma virus which produce gag-mos fusion proteins

Several transforming gene products of mammalian retroviruses undergo post-translational addition of fatty acids. This report demonstrates that gag-mos fusion proteins from variant strains of murine sarcoma viruses incorporate both myristate and palmitate. However, the inefficiency of palmitate incorporation, as well as incorporation of that fatty acid into gag proteins, suggest that palmitate is metabolized to myristate before becoming bound to virus proteins. Furthermore, no detectable post-translational processing event is apparently required for addition of myristate, suggesting that such an event is unnecessary for association of gas-mos proteins with cell membranes.

Hamster endogenous retrovirus (HaER) ? Distinct properties of structural proteins and DNA polymerase

The structural proteins as well as some features of the RNA-dependent DNA polymerase of the hamster endogenous retrovirus (HaER) were examined. The polypeptide pattern of this virus is substantially different from that of other known retroviruses in containing major polypeptides with molecular weights of 68,000, 59,000, 27,000 and 24,000 daltons. Double antibody competitive radioimmunoassays showed that the HaER particles do not share any detectable antigenic relatedness with the murine viruses' p30, but manifest a considerable relatedness with the feline leukemia virus p27 and a slight cross-reactivity with the rat virus major protein. The RNA-dependent DNA polymerase of HaER virus has a molecular size of approximately 73,000 daltons and in contrast to other mammalian retroviruses shows no significant preference for Mn2+ over Mg2+. Apart from the lack of antigenic relatedness between the HaER virus proteins and the p30 protein of murine viruses, there is also no antigenic relatedness between HaER and murine viruses insofar as their DNA polymerase is concerned.

New mammalian transforming retrovirus: demonstration of a polyprotein gene product.

A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.

A common region for proviral DNA integration in MoMuLV-induced rat thymic lymphomas.

Similarly to other mammalian and avian retroviruses that lack a transforming gene, moloney murine leukaemia virus (MoMuLV) causes no morphological transformation in infected tissue culture cells. However, following injection in an appropriate animal host, MoMuLV induces mainly thymic lymphomas after a long latency period. A common characteristic of neoplasms induced by retroviruses lacking transforming genes is their clonal origin. Here we have generated MoMuLV-induced rat thymic lymphomas and confirmed their clonal nature. Furthermore, we took advantage of the clonality of these tumours to investigate the specificity of provirus integration in the tumour DNA. We reasoned that if several independently derived thymic lymphomas would contain the provirus integrated in the same region of cellular DNA, this would be a strong indication that this integration event is a contributing factor in oncogenesis. The results indicate that there is indeed a cellular DNA region (termed the MLVI-1 locus) that serves as the substrate for proviral DNA integration in 5 out of 16 tumours we examined.

Transforming genes of avian (v-ƒps) and mammalian (v-ƒes) retroviruses correspond to a common cellular locus

The Gardner (GA) and Snyder-Theilen (ST) isolates of feline sarcoma virus (FeSV) represent genetic recombinants between feline leukemia virus (FeLV) and transformation-specific sequences (v-ƒ es gene) of cat cellular origin. A related transforming gene (v-ƒ ps), common to the Fujinami, PRC II, and UR 1 strains of avian sarcoma virus has also been described. Translational products of each of these recombinant virus isolates are expressed in the form of polyproteins exhibiting protein kinase activities with specificity for tyrosine residues. In the present study, v-ƒ es and v-ƒ ps homologous sequences of GA-FeSV, ST-FeSV, and Fujinami sarcoma virus (FSV) are defined and these independently derived transforming genes are shown to correspond to a common cellular genetic locus which has remained highly conserved throughout vertebrate evolution.

Basolateral maturation of retroviruses in polarized epithelial cells.

We have investigated the maturation sites of avian and mammalian C-type retroviruses in polarized epithelial cells. Examination of thin sections of Madin Darby canine kidney cells infected with RD114 or avian reticuloendotheliosis virus revealed that these viruses mature from the basolateral membrane domains. Similar results were obtained with a continuous line of mouse mammary epithelial cells infected with Friend, Moloney, Rauscher, or Kirsten murine leukemia viruses, or Friend virus-related or Moloney virus-related mink cell focus-forming viruses. Immunofluorescence observations indicate that viral glycoproteins are inserted only at the basolateral membranes in these cells. Because of the availability of DNA and protein sequence data, and of molecularly cloned viruses, these virus systems offer advantages for molecular studies on directional transport of plasma membrane glycoproteins.

Detection of group and interspecies reactivities in mammalian C-type virus p30 proteins and corresponding human antigens.

We previously reported the presence of RNA tumor virus related antigens in human leukemic sera. The antigens were detected by the ELISA method in 30%–40% of patients with acute leukemia [1, 2]. Since these antigens from human sera that cross-react with primate C-type viral p30 protein had been observed with the ELISA method and since retroviral group and interspecies reactivities had been previously characterized mostly by competition radioimmunoassay, we examined the applicability and reliability of the ELISA method for the detection of group and interspecific determinants in p30 proteins of mammalian C-type retroviruses [4].KeywordsELISA MethodHuman SeronHuman AntigenHomologous AntigenCompetition ELISAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Myristyl amino-terminal acylation of murine retrovirus proteins: An unusual post-translational protein modification

The primary structure of the NH(2)-terminal region of the gag gene encoded internal membrane-associated protein p15 has been determined for both Rauscher and Moloney murine leukemia viruses. Peptides generated by endopeptidases and purified by HPLC were subjected to semi-automated Edman degradation. Dipeptides obtained with dipeptidyl carboxypeptidase were identified by gas chromatography-mass spectrometry. The amino acid sequence of the first 16-residue segment of Rauscher p15 is identical to the sequence of Moloney p15 except for a single amino acid substitution (Gly-->Asp) at position 13. Both proteins were found to have an acylated NH(2) terminus. By mass spectroscopy, myristic acid [CH(3)(CH(2))(12)COOH] was found to be bound through an amide linkage to the NH(2)-terminal glycyl residue in both p15s. The results of liquid chromatography show that the NH(2)-terminal myristyl group greatly contributes to the strong binding of these modified proteins and peptides to hydrophobic surfaces. Because p15 is known to be derived from the NH(2)-terminal region of a precursor polyprotein Pr65(gag) by proteolytic cleavage in the assembled virus, it is suggested that myristylation in vivo takes place during the biosynthesis of Pr65(gag). Preliminary data indicate that such modification of gag precursor polyproteins may be common to mammalian retroviruses. The role of NH(2)-terminal myristyl acylation of Pr65(gag) in virus assembly and the possibility of similar NH(2)-terminal modifications of gag-related fusion proteins of transforming viruses are discussed.

Role of endogenous cat retrovirus in cell differentiation.

Several long-term cultures were established from a spontaneous melanoma of a cat. Cells were rounded or spindle shaped and exhibited black/brown pigmentation in the cytoplasm. No virus was released from these cells spontaneously or after treatment with chemicals. However, exogenous infection of the cat melanoma cells with the endogenous cat virus RD114 resulted in remarkable morphological and functional changes. Most of the RD114 virus-infected cells exhibited multiple neuritic extensions and about 1-2% of the population showed characteristics of neuronal cells. Because human, mouse, and hamster melanoma cultures infected with various mammalian retroviruses, including the RD114 virus, did not display any morphological alteration, it is concluded that the neuronal cell differentiation in the cat melanoma cells is a consequence of its specific interaction with the endogenous cat retrovirus.

Synthesis of Retrovirus-Like Particles in Testicular Teratocarcinomas

Two lines of evidence led to the investigation of human teratocarcinoma cells in vitro for oncogenic retroviruses: the observation by electron microscopy of retrovirus-like particles budding from the syncytial trophoblasts of human placentas, and the demonstration that teratocarcinoma patients before treatment show a high serum antibody reactivity against envelope proteins of mammalian retroviruses. In all 5 teratocarcinoma cell lines studied so far, retrovirus-like particles have been detected by electron microscopy. The production of these human teratocarcinoma-derived (HTD)-particles is enhanced by induction procedures known to be effective in animal virus model systems. In parallel, virus induction also increases the level of chorionic gonadotropin in the culture supernatant, demonstrating syncytial trophoblast-like cells in the heterogeneous cell population of teratocarcinomas which could be responsible for HTD-particle production. The origin of these virus like particles—endogenous or exogenous—as well as their role in the pathogenesis of teratocarcinomas is not known so far. The potential value of serum antibody reactivity in teratocarcinoma patients as a diagnostic marker is discussed.

Demonstration of primer stimulated DNA synthesis by soluble reverse transcriptase.

An oligo (dT) primed terminal transferase-like activity was demonstrable with a number of disrupted mammalian retroviruses and the soluble reverse transcriptases therefrom. The characteristics of this activity were consistent with it being due to reverse transcriptase.

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses.

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.

Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy.

The secondary structural features in the 70S RNAs of the Prague strain of avian Rous sarcoma virus, subgroup A (PR-RSV-A), and Moloney murine leukemia virus (M-MuLV) were compared by electron microscopy. The PR-RSV-A genome contained two subunits joined by a linkage structure as in the genomes of M-MuLV and other mammalian retroviruses. In both viral genomes, a highly reproducible hairpin occurred at about 70 nucleotides from the 5' end of each subunit and contained 320 +/- 8 nucleotides. The stable point of linkage between the subunits in both viral genomes involved fewer than 50 nucleotides and occurred at 466 +/- 9 nucleotides from the 5' end. This places the linkage about 350 nucleotides further toward the 3' end of the subunit than the binding site of primer tRNA. Another structural feature common to both genomes was a loop in each subunit. In M-MuLV, the loop contained 3.9 +/- 0.10 kilobases (kb) and occurred at a distance of 2.2 +/- 0.05 kb from the 5' end. In PR-RSV-A, the loop was smaller (2.3 +/- 0.10 kb) and further (3.3 +/- 0.10 kb) from the 5' end. When M-MuLV RNA was heated to 70, 85, or 90 degrees C and cooled, the hairpin consistently reformed at the 5' end. No other structures typical of the native molecules reappeared. In RNA samples heated to 70 degrees C, a new loop reproducibly occurred near the 5' end of each subunit, but this loop was not found in samples heated to higher temperatures. Based on all of these findings, we conclude that the genome of PR-RSV-A shares several features with M-MuLV and other mammalian retroviruses and that the primer tRNA molecules are not involved in the linkage of the two subunits in either genome. We also conclude that the dimer linkage and the loops in subunits are typical of the native molecules and that their formation requires a special environment.

Antigenic determinants shared by the DNA polymerases of reticuloendotheliosis virus and mammalian type C retroviruses

Antiserum to the DNA polymerase of spleen necrosis virus, a reticuloendotheliosis (RE) group virus, inhibited the polymerase activity of reverse transcriptases from two RE group virus isolates and from mammalian type C retroviruses of murine, feline, and primate origin, but did not inhibit reverse transcriptases of avian myeloblastosis virus (AMV) or the type B or type D mammalian retroviruses tested. Conversely, antiserum to DNA polymerase of a mammalian type C retrovirus, Rauscher murine leukemia virus, inhibited the polymerases of mammalian type C viruses and a RE group virus but was ineffective against AMV polymerase.