Mammalian RNA Tumor Viruses Research Articles

Efficient production of mammalian RNA tumor viruses in serum-free culture medium allows rapid RNA subunit purification

A wide variety of infected mammalian cell cultures were observed to produce high levels of RNA tumor virus particles in the absence of serum for at least 12 h. Virus production was measured by yields of 50–70 S viral RNAs isolated directly from serum-free culture media by chromatography on oligo(dT)-cellulose. Yields of RNAs from viruses produced in serum-free medium were comparable to yields obtained from purified viruses produced in serum-containing medium. Subunits of viral RNAs were thermally dissociated and separated by a new sedimentation system using sucrose gradients with resolving power (in the relevant size range) equivalent to that obtained with electrophoresis in polyacrylamide gels. RNA subunits isolated directly from serum-free medium appeared to be intact as judged by poly(A) content and resedimentation. The overall approach developed here represents dramatic savings in time and effort over previous ways of producing purified RNA subunits from RNA tumor viruses.

Biochemical studies on RNA tumor virus information and its transmission in B16 murine melanoma.

The mouse melanoma B16 contains particles encapsulating a high molecular weight RNA of 60--70S size associated with a reverse transcriptase. The particles possess a density of 1.14--1.18 g/cm3. The RNA shares sequences with the 70S RNAs of several mammalian C-type RNA tumor viruses. The nuclear DNA of the mouse melanoma B16 possesses particle-related sequences not present in the genome of normal C57BL mice.

A competition immunoassay for characterizing the reverse transcriptase of mammalian RNA tumor viruses

A competition enzyme immunoassay for reverse transcriptase has been developed. This assay is based on competition between the enzymatically active reverse transcriptase probe and a sample tested for the presence of reverse transcriptase antigen on a limited amount of monospecific antibody. The extent of competition is determined from the residual DNA polymerase activity left in the probe. Any reverse transcriptase activity in the test sample that may interfere with the enzymatic assay is irreversibly inhibited by pretreatment with N-ethylmaleimide. Thus, increasing amounts of test antigen neutralize more antibodies and as a result restore more of the reverse transcriptase probe activity. By means of the competition immunoassay we were able to show type, group, and interspecies antigenic determinants in the reverse transcriptase of mammalian RNA tumor viruses. The competition immunoassay has also proved useful for quantitating the amount of reverse transcriptase present in virions. The enzyme was found to comprise 2.5% of the total proteins in Moloney murine leukemia virus.

Phenotypic mixing between avian and mammalian RNA Tumor viruses: I. envelope pseudotypes of rous sarcoma virus

Pseudotypes of Rous sarcoma virus (RSV) with the envelope properties of six strains of mammalian leukemia viruses (MaLV) were produced after mixed infection in avian cells permissive for the replication of both types of virus. RSV pseudotypes with the envelope antigens of xenotropic and ecotropic murine leukemia virus were also obtained by fusion of mammalian cells producing leukemia virus with avian cells producing RSV, but RSV(MaLV) pseudotypes were not obtained by MaLV superinfection of RSV-transformed mammalian nonproducer cells. Functional RSV(MaLV) pseudotypes were obtained with both nondefective RSV and RSV defective in envelope antigens, but were not obtained with RSV which is defective in RNA-directed DNA polymerase.

Cells transformed by certain strains of moloney sarcoma virus contain murine p60

It was previously demonstrated that the 60,000 dalton (p60) precursor-like polyprotein containing murine p30 was a constituent of the feline leukemia virus pseudotype of Moloney sarcoma virus [m1MSV(FeLV)]. It is now shown that p60 is detected in cells of five mammalian species transformed by m1MSV, indicating that p60 is specified by this genome. Moreover, little or no murine p30 is detected in the m1MSV-transformed cells, suggesting that the murine group p30 antigenic reactivity of S+L− cells is due to p60. Pulse-chase studies in cells producing m1MSV(FeLV) show that p60 is the largest polypeptide detectable during the pulse, and that intracellular p60 is not cleaved into smaller (for example, p30) polypeptides during chase periods of up to 10 hr. The lack of cleavage of p60 is in contrast to the properties of p30 precursors detected in cells containing replicating avian or mammalian RNA tumor viruses. The inefficient cleavage of intracellular p60 and the kinetics of appearance of murine p30 in extracellular m1MSV(FeLV) suggest that p60 cleavage to p30 occurs in cells shortly before virus release. While only p60 was detected in the m1MSV-transfromed cells, p60 and p70 were detected in m3MSV-transformed cells, and no immunoprecipitable polypeptides were detected in HT-1 MSV-transformed cells. The observed differences in the intracellular polypeptide expression by each of the strains of MSV suggests differences in genetic content.

Relationships between membrane antigens of human leukemic cells and oncogenic RNA virus structural components.

Leukemic cells from all human chronic granulocytic leukemia (CGL) and some acute myelomonocytic leukemia (AMML) donors are lysed by rabbit antisera to a purified glycoprotein of Friend murine leukemia virus (FLV gp71) in a microcytotoxicity assay. These antisera are not cytotoxic to cells from patients with acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or to peripheral blood lymphocytes from normal donors. A goat antiserum to gradient purified FLV in addition to reacting with cells from CGL and AMML donors also reacted with cells from AML patients and some ALL donors. However, this antiserum failed to react with cells from CLL patients. Peripheral blood and bone marrow leukocytes prepared from leukemic patients in clinical remission failed to react with antisera to FLV and FLV gp71. Absorption experiments demonstrated that the antigen on CGL cells which is reacting with the antiserum to FLV gp71 is also present on normal human platelets and neutrophils. Similar absorption studies showed that the antigen on AML cells detected by the FLV antiserum is not present on normal leukocytes and platelets and appears to be related to the major internal p30 antigens of mammalian RNA tumor viruses. Another antigenic relationship between oncornaviruses and membrane antigens of human leukemia cells was shown by the ability of FLV antigens to absorb the cytotoxic reactivity of nonhuman primate antisera detecting human leukemia-associated antigens. FLV and FLV gp71 antigens were able to absorb all cytotoxic activity of monkey and chimpanzee antisera to human myeloid leukemia antigens when these antisera were tested with CGL cells. These two approaches to an analysis of cross-reactivity indicate that the antigenic determinant(s) detected by the cytotoxic reactions of the FLV gp71 antiserum with human CGL cells is different from the determinant on FLV gp71 which is responsible for the inhibition of the reactivity of simian antisera with CGL cells. Since the goat and rabbit antisera to FLV and FLV gp71 are able to distinguish AML from CGL cells by direct cytotoxicity testing and absorption, they may be valuable reagents for the serological diagnosis of myeloid leukemia. In addition, since peripheral blood cells from AML and CGL patients in clinical remission were seronegative, the antisera may be valuable as management aids. The data in this report indicates that whatever the mechanism of leukemogenesis is in man, cells from CGL and AML patients possess certain membrane antigens which cross-react with FLV structural components such as p30 and gp71.

Polyriboadenylate sequences at the 3'-termini of ribonucleic acid obtained from mammalian leukemia and sarcoma viruses.

The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T(1) digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosine-labeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CCl(3)COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.

An Immunoelectron Microscopic Study of Type C Virus Particles in Feline Sarcoma Virus-Induced Rat Tumors

Many type C RNA tumor viruses can infect cells of different species. Following cross-species infection, these viruses are known to exhibit some altered properties. In an attempt to investigate host influence on properties of mammalian RNA tumor viruses, neonatal Wistar rats were inoculated with feline sarcoma virus of Snyder-Theilen strain. A transplantable tumor line designated as RT-FeSV was established from one of these induced tumors. Some syngeneic rats inoculated with RT-FeSV tumors which were passaged in rats less than 3 times developed precipitable serum antibodies to feline leukemia virus (FeLV).

Isolation of proteins by gel filtration in 6 m guanidinium chloride: Application to RNA tumor viruses

Separation of RNA tumor virus proteins by gel filtration in 6 m guanidinium chloride indicates that this method will effectively separate polypeptides in milligram amounts whose molecular weights differ by as little as 15 percent. Such separations are achieved because proteins in 6 m guanidinium chloride have a random coil conformation with diffusion coefficients considerably smaller than the corresponding native proteins. Consequently, protein bands that emerge from a gel filtration column in 6 m guanidinium chloride are remarkably sharp. A 60–70% yield of renatured RNA tumor virus proteins could be obtained following dialysis against a dilute solution of mercaptoethanol. The major proteins of avian RNA tumor viruses were obtained as pure components by gel filtration in 6 m guanidinium chloride. Mammalian RNA tumor viruses appear to have a more complex protein structure, since, in some cases, additional purification was required to obtain the pure proteins. This method of protein separation might be used as the initial stop in the isolation of components from other biological macrostructures.

Detection and isolation of a new DNA polymerase from human breast tumor cell line HBT-3 by (dT)12-18-cellulose chromatography.

Column chromatography on (dT)(12-18)-cellulose was used to detect and isolate a novel DNA polymerase activity from human breast tumor cell line HBT-3. The new enzyme is similar to those of mammalian RNA tumor viruses in its molecular size, preference for poly(A).(dT)(12-18) as compared to either poly(dA).(dT)(12-18) or poly(A).poly(dT), and ability to copy poly(C).(dG)(12-18). A similar DNA polymerase activity, defined by its binding to (dT)(12-18)-cellulose, is not detectable in several other cell lines of human tumors.

Molecular relatedness of mammalian RNA tumor viruses as determined by DNA hybridization.

Each of six mammalian C-type viruses-including two feline leukemia viruses, three murine leukemia viruses, and the human "candidate" virus RD-114-can be distinguished from each other by hybridizing DNA synthesized by viral reverse transcriptase with viral RNA. Characterization of the DNA . RNA hybrids by hydroxylapatite chromatography revealed nucleotide sequence diversity among the viruses, detectable both by the amount of cross-hybridization and by the decreased thermal stability of heterologous hybrids.

Antibody to the RNA-dependent DNA polymerase of mammalian C-type RNA tumor viruses.

Sera from rats bearing transplantable tumors induced by murine C-type tumor viruses contain an inhibitor of the activity of the viral RNA-dependent DNA polymerase. The inhibitor is shown to be an immunoglobulin (IgG) directed against the enzyme. Antiserum made in rabbits against partially purified murine leukemia virus polymerase also inhibits the polymerases of other mammalian C-type RNA-containing tumor viruses. Thus, the polymerases from different mammalian tumor viruses are antigenically related.