DNA Polymerase Activities and Nucleic Acid Components of Virions Isolated from a Spontaneous Mammary Carcinoma from a Rhesus Monkey

Abstract

Virions isolated from a spontaneous mammary carcinoma of a rhesus monkey and propagated in human cells possess an RNA-instructed DNA polymerase. They also exhibit DNA polymerase activities that respond to either double-stranded DNA or synthetic RNA DNA hybrid complexes as templates. The virion has been shown to have a density of 1.16 g/ml and to contain a nucleic acid species of high molecular weight (sedimentation coefficient, 60-70 S), which bands as RNA at 1.670 in a Cs2 SO4 equilibrium density gradient. In addition, the virions contain species of low molecular weight (4-6 S) that consist of RNA as well as components banding at densities characteristic of DNA RNA complexes. The nucleoid of this virion has been isolated and shown to have a density of 1.23 g/ml; it also contains a 60-70S nucleic acid species.The presence of a virus, morphologically similar to that of the known RNA tumor viruses, has been recently reported in a spontaneous mammary tumor of a rhesus monkey (1-4).These particles were initially propagated by cocultivation of the mammary tumor tissue with monkey embryonic cells and they have since been grown in a variety of monkey, chimpanzee, and human cell lines (4,5). This virus has recently been designated the Mason-Pfizer monkey virus (M-PMIV) (6).We have previously shown (7) that these particles contain an RNA-instructed DNA polymerase. We report here on further characterization of the product synthesized and a demonstration that M-P]MV possesses many of the biochemical and biophysical features common to the oneogenic RNA viruses of other species. MATERIALS AND METHODS Virus and cellsThe virus, isolated from a spontaneous monkey mammary tumor, was propagated in suspension cultures of the cell line NC-37, which was established from normal human leukocytes.The medium employed was RPMII 1640, with 10% fetal calf serum and potassium penicillin G (50 units/ml) and streptomycin sulfate (50,4g/ml). All virus for polymerase studies was Glycerol gradient sedimentation analysis Nucleic acid to be examined was pelleted by centrifugation at 23,000 X g for 30 min at 0C, resuspended in 0.5 ml of TNE (0.01 Al Tris * HCl (pH 8.3)-0.1 M NaCl-0.002 M EDTA), and layered onto a previously formed density gradient of 10-30% (v/v) glycerol in TNE. Gradients were centrifuged at 201,000 X g for 2.5 hr at 40C in an SW41 swinging-bucket rotor. 24-drop fractions were collected from below; optical density at 260 nm was measured for each fraction when nucleic acid from unlabeled virions was analyzed; for radioactively labeled material, 50-Md portions were mixed with 0.5 ml of NCS reagent (Amersham/Searle) and radioactivity was counted after addition of toluene-BBOT. 18S and 28S [3H ]RNA extracted from rat embryo fibroblast cells were used as markers in sedimentation analysis. DNA polymerase reactionsThe RNA-instructed polymerase activity, DNA-instructed l)olyrmerase activity, and the polymerase activity employing synthetic RNA -DNA hybrids as templates were all assayed as previously described (7-9). RESULTS Density...

The 60 to 70S RNA and Reverse Transcriptase of Simian Sarcoma and Simian Sarcoma-Associated Viruses

“…Nonidet P-40 is approximately as effective as Sterox SL, despite the finding by Stromberg [20] that treatment with Sterox SL resulted in more endogenous polymerase activity than treatment with other non-ionic deter gents. The range of concentration of detergent required for maximum activity is between 0.05 and 0.5%, and is similar to the level noted by Schlom and Spiegelman for the Mason-Pfizer virus (M-PMV) [21].…”

Immunological Characterization of Type-D Retrovirus Isolates from Human Cells

“…The various type-D retrovirus isolates (ta ble I) were purified using equilibrium density gradient centrifugation [20], MPV and LV were iodinated and subjected to electropho resis in 10% polyacrylamide gels in the pres ence of SDS. The iodinated polypeptides in the p27 region and the p i0-12 region were eluted from the gel slices and used in the RIAs described (see 'Materials and Methods').…”

“…MPV and all other viruses used were purified from culture super natant fluids by equilibrium density gradient centri fugation and were concentrated as previously described [20], X-381 virus was purified from the supernatant fluid of X-381 cells established from a rhesus lactating mammary gland (3], LV was grown in bat lung cells (supplied by Dr. Todaro) [7], SMRV grown in canine Cf2Th cells cocultivated with squirrel monkey lung cells was supplied by Dr. R. Heberling and Dr.S.Kalter [6], Type-D retroviruses were purified from superna tant fluids of HeLa cells [10][11][12], from a cell line derived from a human amniotic cell, AO [13], and from two cell lines derived from human leukemic cells, T9 [14] and J96 [15]. Simian sarcoma virus I grown in NC-37 cells, baboon virus grown in BKCT (baboon kidney cocultivated with canine thymus) cells, and mouse mammary tumor virus grown in murine Mm5mt cells were obtained through the Biological Carcinogenesis Branch of the National Cancer Institute.…”

RNA‐dependent DNA polymerase associated with a simian lymphoid cell line derived from a Herpesvirus saimiri‐induced lymphoma

“…Morphologically, all of these resemble various murine oncogenic RNA tumor viruses. Among these, M-PMTV and SSV-I are known to possess a DNA polymerase that exhibits template preference and enzymatic characteristics of a reverse transcriptase (Gallo et al, 1972;Scolnick et al, 1972;Schlom and Spiegelman, 1971). M-PMTV also sediments at a density of 1.14-1.16 g/cm (Jensen et al, 1970).…”

“…Both are capable of replication in monkey embryonic cell culture and/or in established monkey cell lines, although M-PMTV replication is of a lower efficiency than that of murine RNA tumor viruses in murine cell culture (Jensen et al, 1970). M-PMTV also possesses RNA as an intrinsic nucleic acid (Schlom and Spiegelman, 1971). Both viruses are also capable of cell transformation and in vivo tumorigenesis (Chopra et al, 1971 ;WoIfe et al, 1971).…”