RD-114 Virus Research Articles

Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.

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.

Human interferon-α- and -γ-mediated inhibition of retrovirus production in the absence of an inhibitory effect on vesicular stomatitis virus and encephalomyocarditis virus replication in RD-114 cells

RD-114 line is a human sarcoma cell line chronically infected with RD-114 retrovirus. Treatment of these cells with increasing doses of human interferon-α or -γ resulted in increasing inhibition of RD-114 virus production. Surprisingly, the replication of vesicular stomatitis virus and encephalomyocarditis virus, in these cells and in the parental RD cells which are not infected with the retrovirus, was insensitive to interferon treatment. Unlike reported differences in other properties of interferon-α and interferon-γ, there were no differences in their antiretroviral properties such as dose response, kinetics of establishment of the antiretroviral state, and kinetics of its dissipation upon removal of interferon.

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.

Isolation of Feline Leukemia Virus From a Leopard Cat Cell Line and Search for Retrovirus in Wild Felidae<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

A strain of feline leukemia virus (FeLV), subgroup A, was isolated in early subpassage of a testicular fibroblast culture obtained from a captive Asian leopard cat. Neither FeLV nor RD-114 virus was recovered from cultured tissues of 15 other animals, representing eight species of wild Felidae.

Relationship of the feline oncornavirus associated cell membrane antigen to a feline sarcoma virus encoded polyprotein.

The feline oncornavirus associated cell membrane antigen (FOCMA) is expressed on the membranes of cells transformed by infection with the feline leukemia virus (FOCMA-L) or feline sarcoma virus (FOCMA-S) (Hardy et al. 1977; Essex et al. 1977). Expression of FOCMA is transformation-specific and not dependent upon concomitant expression of antigens associated with feline leukemia virus (FeLV) or the endogenous virus RD114 (reviewed in Snyder et al., to be published a). In the natural environment FOCMA is the target for an effective immunosurveillance response: complement-dependent lytic antibodies directed to FOCMA reverse or prevent tumor development (Essex et al. 1975: Grant et al. 1977). FOCMA-L has been isolated from feline lymphosarcoma (LSA) cell membranes and has been shown to reside on a 70,000 dalton protein which is neither glycosylated nor phosphorylated (Snyder et al. 1978, to be published a). The nature of FOCMA-S is a subject of intense investigation at the present time.

Isolation and partial characterization of the reverse transcriptases of Rauscher murine leukemia virus, Simian sarcoma virus and RD-114 virus

Reverse transcriptases were purified from Rauscher murine leukemia virus, Simian sarcoma virus and RD-114 virus by affinity chromatography using poly(rC)-Sepharose. Molecular weight determinations of the viral enzymes gave values of 80 000 for all three enzyme preparations. Two-dimensional tryptic peptide maps of the purified enzymes showed only a few spots in similar positions.

Use of vesicular stomatitis virus pseudotypes to map viral receptor genes: Assignment of RD114 virus receptor gene to human chromosome 19.

Vesicular stomatitis virus pseudotypes bearing envelope glycoproteins of the endogenous feline type C retrovirus, RD114, were used to assay the expression of receptors specific to RD114 on the surfaces of mouse-human hybrid cells carrying different human chromosomes. These studies show that the gene encoding the RD114 receptor is located on human chromosome 19.

Feasibility studies of oncornavirus production in microcarrier cultures.

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

Search for viruses in rheumatoid macrophage-rich synovial cell populations.

The adherent cells remaining after short-term culture of synovial fluid and synovial membrane cells from rheumatoid and non-rheumatoid patients were examined for the presence of a productive virus infection and for various viral antigens. Labelling was carried out with 3H-thymidine and 3H-uridine followed by sucrose density gradient centrifugation of the culture supernatant. Only in 1 case was there incorporation of 3H-uridine into material of density 1 . 21 g/cm3. Viral antigens were tested for by indirect immunofluorescence with antisera to rubella virus, the retroviruses RD-114 and simian sarcoma associated virus, early adenovirus type 2 antigens, late adenovirus type 2 antigens, SV-40 T antigen, and in 1 case measles virus. No cell showed immunofluorescence with any antiserum except the early adenovirus type 2 antiserum, which stained the cytoplasm of about half the synovial cell cultures, some from rheumatoid and some from non-rheumatoid patients.

Evidence for type-C retrovirus production by Burkitt's lymphoma-derived cell line.

Burkitt's lymphoma cell line, P3HR-I, was found to secrete virions with properties of known type-C RNA tumor viruses. The viral particles had a buoyant density of 1.16 g/ml in sucrose gradients and contained a high-molecular-weight RNA and an RNA-instructed DNA polymerase. The viral polymerase was active in an endogenous reaction requiring the presence of the four deoxyriboside triphosphates and manganese ions, and was sensitive to RNase. The DNA product of the endogenous reaction specifically hybridized to P3HR-I viral 60 to 70S RNA. Electron microscopic examination of ultrathin sections of P3HR-I cells revealed immature, mature and budding virions typical of type-C retroviridae. Nucleic acid hybridization assays showed no sequence homoblastosis virus, murine oncornaviruses, simian sarcoma virus or RD114 virus.

Functionally conserved determinants on gp70s of endogenous primate retroviruses

Endogenous viruses of Old and New World monkeys are divided morphologically into type C isolates of baboons, macaques, and owl monkeys and type D isolates of squirrel monkeys and langurs. Mason-Pfizer monkey virus (MPMV), a type D virus, is horizontally transmitted in rhesus monkeys and is highly related to the langur isolate. Although these endogenous type C and D viruses possess little nucleic acid sequence homology and are antigenically distinct, they do possess cross-reactive determinants in their envelope gp70 antigen, as shown by sensitive interspecies radioimmunoassays. The ability of MPMV to induce syncytia in KC (Rous sarcoma virus-transformed human glioma) cells allowed for investigation of the role that these conserved determinants play in recognition of cellular receptors. The capacity of other viruses to bind to common cellular receptors and to inhibit MPMV-induced syncytia was measured in a quantitative assay using a high syncytia-inducing variant of MPMV. Pretreatment of KC cells with disrupted MPMV, baboon endogenous virus (BaEV), squirrel monkey retrovirus, and the BaEV-related endogenous cat virus RD114 blocked syncytia formation by MPMV. Partial competition was observed with endogenous macaque isolates MAC-1 and MMC-1. However, no inhibition was observed using feline leukemia, murine leukemia, equine infectious anemia, or murine mammary tumor viruses, none of which possess antigenic determinants which are cross-reactive with the endogenous primate retroviruses. Syncytia formation was also blocked by purified gp70s of MPMV and BaEV, demonstrating that inhibition was due to competition for viral gp70 cellular receptors.

Conserved Region of Mammalian Retrovirus RNA

The viral RNAs of various mammalian retroviruses contain highly conserved sequences close to their 3' ends. This was demonstrated by interviral molecular hybridization between fractionated viral complementary DNA (cDNA) and RNA. cDNA near the 3' end (cDNA(3')) from a rat virus (RPL strain) was fractionated by size and mixed with mouse virus RNA (Rauscher leukemia virus). No hybridization occurred with total cDNA (cDNA(total)), in agreement with previous results, but a cross-reacting sequence was found with the fractionated cDNA(3'). The sequences between 50 to 400 nucleotides from the 3' terminus of heteropolymeric RNA were most hybridizable. The rat viral cDNA(3') hybridized with mouse virus RNA more extensively than with RNA of remotely related retroviruses. The related viral sequence of the rodent viruses (mouse and rat) showed as much divergence in heteroduplex thermal denaturation profiles as did the unique sequence DNA of these two rodents. This suggests that over a period of time, rodent viruses have preserved a sequence with changes correlated to phylogenetic distance of hosts. The cross-reacting sequence of replication-competent retroviruses was conserved even in the genome of the replication-defective sarcoma virus and was also located in these genomes near the 3' end of 30S RNA. A fraction of RD114 cDNA(3'), corresponding to the conserved region, cross-hybridized extensively with RNA of a baboon endogenous virus (M7). Fractions of similar size prepared from cDNA(3') of MPMV, a primate type D virus, hybridized with M7 RNA to a lesser extent. Hybridization was not observed between Mason-Pfizer monkey virus and M7 if total cDNA's were incubated with viral RNAs. The degree of cross-reaction of the shared sequence appeared to be influenced by viral ancestral relatedness and host cell phylogenetic relationships. Thus, the strikingly high extent of cross-reaction at the conserved region between rodent viruses and simian sarcoma virus and between baboon virus and RD114 virus may reflect ancestral relatedness of the viruses. Slight cross-reaction at the site between type B and C viruses of rodents (mouse mammary tumor virus and RPL virus, 58-2T) or type C and D viruses of primates (M7, RD114, and Mason-Pfizer monkey virus) may have arisen at the conserved region through a mechanism that depends more on the phylogenetic relatedness of the host cells than on the viral type or origin. Determining the sequence of the conserved region may help elucidate this mechanism. The conserved sequences in retroviruses described here may be an important functional unit for the life cycle of many retroviruses.

Viral-specific antigen synthesis following de novo RD-114 virus infection of stationary cells

The cell cycle dependence of retrovirus replication was studied. Canine sarcoma (D-17) cells were infected de novo with the xenotropic feline retrovirus RD-114 under conditions previously reported to simultaneously inhibit virus replication and cell DNA synthesis and/or cell division. RD-114 viral antigen synthesis was observed under conditions previously reported to be inhibitory to avian and murine oncornavirus replication, including confluency and serum deprivation, X-irradiation, mitomycin C pretreatment, colchicine, and ethidium bromide treatments of cells. Several mechanisms that could account for viral antigen synthesis under the restrictive conditions used are discussed.

Suppressive Effects of Interferon on Syncytium Formation by RD-114 Virus in Human Transformed Cells

The RD-114 virus rapidly induced syncytia in the human transformed cell lines, RSa, RSb and IFr. Treatment of the virus with heat or ultrasonic vibration completely eliminated the syncytium-forming activity. Irradiation with u.v.-light or treatment with beta-propiolactone (BPL) reduced but did not completely destroy the activity. Pre-treatment of the cells for 16 h with 25 to 500 units/ml of human leucocyte interferon (Le-IF) or fibroblast interferon (F-IF) significantly reduced formation of syncytia by active virus or inactivated (u.v. or BPL) virus. This activity of interferon was inhibited by treatment of the cells with cycloheximide. Interferon did not increase the binding of 3H-uridine-labelled RD-114 virus to the cells. It is postulated that interferon treatment altered the plasma membrane of the cells and thus reduced their capacity to fuse.

Carnivores have sequences in their cellular DNA distantly related to the primate endogenous virus, MAC-1

Endogenous viral gene sequences, present in multiple copies and related to the primate type C virus, MAC-1, from Macaca arctoides, are found not only in related primates, as expected, but also in the cellular DNA of various carnivores (order Carnivora). These sequences are not related to the previously described endogenous viruses, RD-114 and feline leukemia virus, of certain Felis species.

Endogenous RD-114 Virus of Cats: Absence of Antibodies to RD-114 Envelope Antigens in Cats Naturally Exposed to Feline Leukemia Virus

Healthy cats, including those exposed to feline leukemia virus in leukemia cluster households, lack evidence of humoral immunity to virion surface antigens of the endogenous RD-114 virus.

A new virion precipitation test for oncovirus envelope antigens which detects common antigenic determinants in mammalian type-C viruses and Mason-Pfizer monkey virus.

A method for the study of oncovirus envelope antigens was developed, bases on the precipitation of intact virions by a double antibody technique. The amount of precipitated virus was then measured as reverse transcriptase activity. The method was designated the virion precipitation test (VPT). It has been used for titration of antibodies to envelope antigens of oncoviruses. The study of envelop antigens of 11 different oncoviruses permitted their differentiation into the following groups: (1) murine type-C viruses: (2) feline type-C viruses; (3) simian type-C viruses; (4) the RD-114/BEV group; (5) Mason-Pfizer monkey virus (M-PMV); (6) bovine leukemia virus; (7) avian type-C viruses; (8) mouse mammary tumor virus. No common antigenic determinants were detected in the last three groups. Mammalian type-C viruses (RD-114, NIH-MuLV, G-MuLV) had common antigenic determinants in the envelope, as demonstrated with an anti-RD-114 serum. Mammalian type-C viruses also shared antigenic determinants with M-PMV. The relationship of type-C viruses to M-PMV decreased in the following order: RD-114--NIH-MuLV--G-MuLV. It was also shown that the endogenous xenotropic feline RD-114 virus was more closely related to xenotropic NIH-MuLV than to ecotropic G-MuLV. The nature of the common antigenic determinants, as demonstrated by VPT on the surface of mammalian type-C viruses and M-PMV, and their significance for the concept of oncovirus evolution are discussed.

Characterization of a retrovirus isolated from normal mink cells co-cultivated with a dog mammary tumour.

A retrovirus antigenically distinct from known type C, B and D viruses was isolated from normal mink (Mustela vison) lung cells that had been co-cultivated with 5-iododeoxyuridine- and dexamethasone-treated dog mammary tumour cells. Cytogenetic studies of the virus-releasing co-culture showed mitotic figures identical to the normal mink cell line (MvlLu) with the exception of a low frequency of cells with extensive chromosomal breakage and uncoiling. The new virus bands at a buoyant density of 1.16 g/ml, contains 60S RNA and a reverse transcriptase which prefers Mn2+ over Mg2+ for the synthesis of DNA. This enzyme utilizes poly(rA).oligo(dT) more efficiently than poly(dA).oligo(dT) and is also able to synthesize DNA copies from the endogenous RNA. Morphologically, it is a typical type C virus. Filtered virus readily infects mink, dog and other mammalian cells indicating the amphotropic nature of its cell growth requirement. Hybridization studies showed that normal mink DNA contains multiple copies of proviral sequences of this newly isolated virus. Serological analyses indicate that the mink endogenous virus contains in its core protein, in addition to the interspecies type-C determinant, an antigenic component related to one of the determinants found in the feline leukaemia virus p30 protein. This determinant is not present in the Rauscher leukaemia virus, RD114 virus or simian sarcoma virus.

Nonconditional replication mutants of type C and type D retroviruses defective in gag gene-coded polyprotein post-translational processing

A rapid method for the isolation of nonconditional replication-defective mutants of type C retroviruses of primate and nonprimate origin and a representative type D retrovirus is described. By this means, six RD114, three M7-baboon, four Rauscher-murine leukemia virus (R-MuLV), and nine squirrel monkey retrovirus (SMRV) mutant clones were isolated. Although defective for virus production, all but two of the mutant clones isolated were characterized by relatively high levels of gag and env gene-coded protein expression. While expression of gag and env gene translational products was coordinate in most of the R-MuLV and SMRV mutant clones, certain clones nonproductively infected with either RD114 or M7 baboon virus showed noncoordinate synthesis. Further analysis of representative mutant clones indicated impaired gag gene-coded polyprotein post-translational processing. This latter observation made possible identification of the component structural proteins of both RD114 and SMRV gag gene-coded precursor polyproteins. The susceptibility of many of the clones to superinfection by wild-type virus indicated that the defect in virus production was due to mutation in the viral, rather than host, genome. Several nonproductively-infected clones, on the other hand, were resistant to superinfection. The demonstration of greater cross-interference to superinfection between M7, RD114, and SMRV than between any of these viruses and the amphotropic murine type C virus isolate, 4070-A, corroborates the presence of shared envelope glycoprotein determinants between primate-derived type C and type D viruses.