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Serological analysis of an oncornavirus (PMF virus) detected in malignant permanent human cell lines.

Immunodiffusion analysis of the PMF virus which was detected in malignant permanent human cell lines revealed positive reactions with antisera against the Mason-Pfizer monkey virus (MPMV). No cross-reactivity was demonstrated with murine leukemia virus (MuLV), rat leukemia virus (RaLV), hamster leukemia virus (HaLV), feline leukemia virus (FeLV), simian (woolly monkey) sarcoma virus (SSV-1) and mouse mammary tumor virus (MTV). The cross-reactive antigens of the PMF virus and the MPMV are considered as evidence for the human origin of the PMF virus.

Animals↗

Apparent allosterism by avian myeloblastosis virus reverse transcriptase and E. coli DNA polymerase I.

A recent report (1) presented evidence for allosterism in reverse transcription by Mason-Pfizer monkey virus reverse transcriptase and by E. coli DNA polymerase I. Our experiments also demonstrate these apparent cooperative effects when synthesis is catalyzed by either avian myeloblastosis virus DNA polymerase, feline sarcoma virus DNA polymerase, or E. coli DNA polymerase I (large fragment). We show that the apparent cooperativity depends on the use of oligo(dT)12-18 as primer. However, if the polymerase reaction products are isolated chromatographically, then the polymerases obey classical Michaelis-Menten kinetics with respect to substrate and enzyme concentrations. These results suggest that the cooperative effects are an acid precipitation artifact. The results are also consistent with the enzyme operating by a distributive mechanism with the oligo(dT)12-18 primer.

Allosteric Regulation↗

Oncornavirus-like particles in squirrel monkey (Saimiri sciureus) placenta and placenta culture.

Oncornavirus-like particles similar in morphology to type D particles were observed in 1 of 2 squirrel monkey (Saimiri sciureus) placentas. Intracytoplasmic type A particles, immature virus particles, and mature viruses with eccentric or occasionally centric nucleoids were associated with placental syncytiotrophoblasts. A spike layer typical of type B viruses was not detected in viral envelopes. Onvornaviruses, identical to those previously isolated from squirrel monkey tissues and similar to Mason-Pfizer monkey virus, were seen in cultures derived from the virus-positive squirrel monkey placenta cocultivated with a mink lung culture. The major morphologic difference between the in vivo and the in vitro squirrel monkey virus was in the nucleoid position of mature virus particles.

Animals↗

Serological definition of the lentivirus group of retroviruses.

The major polypeptides of visna viruses and other lentiviruses have been isolated and shown to be closely related if not identical in radioimmunoassays. By this criterion the lentiviruses form a distinct group of retroviruses unrelated to spuma viruses, mammalian and avian retroviruses that cause tumors, and unclassified retroviruses of cattle and horses. Two sera obtained from goats immunized with Mason-Pfizer monkey virus or squirrel monkey virus reacted with visna p30. Additional data suggest that this reaction represents infection of goats with a lentivirus or a new retrovirus closely related to the lentiviruses.

Animals↗

Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates.

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.

Animals↗

Bovine leukemia virus: an exogenous RNA oncogenic virus?

Short term cultures of bovine leukemic lymphocytes release virus particles with biochemical properties of RNA oncogenic viruses. These particles, tentatively called Bovine Leukemia Virus (BLV) have a high molecular weight-reverse transcriptase complex and a density averaging 1.155 g/ml in sucrose solutions. Molecular hybridizations between BLV-3H cDNA and several viral RNAs show that BLV is not related to Mason-Pfizer Monkey Virus (MPMV) Simian Sarcoma Associated Virus (SSV-1) Feline Leukemia Virus (FeLV) or Avian Myeloblastosis Virus (AMV). Rauscher Leukemia Virus (RLV) exhibits a slight but reproducible relatednesse to BLV. The high preference of BLV reverse transcriptase for Mg++ as the divalent cation suggests that BLV might be an atypical mammalian leukemogenic type C virus. Hybridization studies using BLV 3H cDNA as a probe suggest that the DNA of bovine leukemic cells contains viral sequences that cannot be detected in normal bovine DNA.

Animals↗

Bovine leukemia virus: an exogenous RNA oncogenic virus.

Short-term cultures of bovine leukemic lymphocytes release virus particles with biochemical properties of RNA oncogenic viruses. These particles, tentatively called bovine leukemia virus (BLV), have a high molecular weight RNA-reverse transcriptase complex and a density of 1.155 g/ml in sucrose solutions. Molecular hybridizations between BLV/[3H]cDNA and several viral RNAs show that BLV is not related to Mason-Pfizer monkey virus, simian sarcoma associated virus, feline leukemia virus, or avian myeloblastosis virus. These results were confirmed by hybridization between BLV 70S RNA and [3H]cDNA synthesized in the various viruses tested. The high preference of BLV reverse transciptase for Mg++ as the divalent cation suggests that BLV might be an atypical mammalian leukemogenic "type C" virus. DNA-DNA hybridization studies using BLV [3H]cDNA as a probe strongly suggest that the DNA of bovine leukemic cells contains viral sequences that cannot be detected in normal bovine DNA.

Animals↗

Identification of a generalised packaging sequence for D-type retroviruses and generation of a D-type retroviral vector.

In order to construct vectors based upon D-type, rather than C-type, retroviruses, we have identified a 624-bp fragment of Mason-Pfizer monkey virus (MPMV) which constitutes a packaging sequence for at least two D-type retroviruses. When this fragment was included in an extensively deleted D-type vector genome, the D-type viruses MPMV and SRV-5, but not the C-type viruses MLV-A or MLV-E, rescued the vector RNA from HeLa cells. The recombinant virus stocks have the host range of the rescuing D-type virus as shown by expression of an internal (SV40-puromycin) cassette replacing the retroviral structural genes. The recombinant MPMV was specifically neutralized by anti-MPMV serum and receptor interference was demonstrated when it was plated on cells productively infected with wild type MPMV. When the putative D-type packaging sequence was removed from the vector genome, even though the other sequence elements required for efficient reverse transcription remained, the vector was no longer rescued from HeLa cells. These results complement the recent demonstration of broad specificity of rescue of a C-type vector (carrying only the packaging sequence of Mo-MLV) by several different C-type, but not D-type, viruses. Replacement of the D-type packaging sequence by most of the extended packaging sequence of Mo-MLV prevented the otherwise D-type vector from being rescued by D-type viruses and did not allow it to be rescued by C-type viruses. This was probably because of the incompatibility of the D-type vector sequences with the C-type retroviral proteins involved in viral reverse transcription and integration. Hence, we have localized a packaging sequence that is recognized by D-type, but not by C-type, retroviruses and have constructed a D-type vector which may be useful in gene transfer experiments.

Betaretrovirus↗

Cell-mediated immune response to simian oncornavirus antigens in pregnant women.

Tritiated thymidine incorporation into lymphocytes of 118 adult women was studied in the presence of mitomycin C-treated cells prepared from cell lines continuously producing Mason-Pfizer monkey virus (MPMV), baboon C-particle virus, or simian sarcoma virus (SSV) and in the presence of control cell lines documented for the absence of oncornaviruses. At the end of pregnancy, women who had 5-9 pregnancies showed a high frequency (53%) of specific positive responses to cells with MPMV antigens. The frequencies were 15% for pregnant women with smaller numbers of pregnancies and 3% for nonpregnant women with similar numbers of previous pregnancies as in the pregnancy group. None of these nonpregnant women had lymphocytes responding to stimulation by baboon C-virus antigens, but positive responses were obtained in 20 and 16% of the pregnant groups, respectively. No correlation was found between responses to MPMV or baboon C-virus antigens. Of 48 women (35 pregnant, 13 nonpregnant) who were tested for lymphocyte responses to SSV antigens, only 2 showed a positive response. The results indicated that two distinct antigens, related to MPMV and to baboon C-virus, may be expressed during pregnancy and may then induce a transient cell-mediated response.

Adult↗

The use of trypan blue for counterstaining in the Sepharose bead immunofluorescence test. The application of the test for the demonstration of primate retrovirus-specific antibodies and antigens.

The Sepharose bead immunoflurorescence test was performed by counterstaining the beads with trypan blue. This results in a red staining of negative beads which allows an easy distinction from positive green-fluorescent beads. Sepharose beads conjugated with viral proteins or antiviral antibodies were used to demonstrate Mason-Pfizer monkey virus (MPMW)- and simian sarcoma virus (SSV) - specific antigens or antibodies. The test shows a high sensitivity and specificity and needs a small amount of material.

Antibodies, Viral↗

[Comparative antigenic analysis of J-96 cell oncornavirus and Mason-Pfizer virus from a spontaneous monkey mammary tumor].

A comparative study of the antigenic structure of oncornavirus of J-96 cells and Mason-Pfizer virus (M-PVM) isolated from cells of M. rhesus spontaneous mammary tumour was carried out using immunodiffusion in agar, immune autoradiography and indirect immunofluorescence procedures. Immune rabbit serum to disrupted J-96 virus detected by agar immunodiffusion in preparations or purified virus three soluble antigens one of which was identical to group-specific M-PVM antigen.

Animals↗

[Chronic infection caused by the simian Mason-Pfizer virus in primate cell cultures].

Cell cultures of monkey prepuce (Rhfs) and African green monkey kidney (BSC-1) were infected once with simian Mason-Pfizer virus (MPV) and virus expression in the course of establishment of chronic infection was studied. The productive infection was characterized by changes in the cell metabolism (DNA synthesis increased 2-3-fold as early as the "zero" passage), the appearance of gs-antigen, formation of virions of type D and high activity of RNA-dependent DNA-polymerase. Multinuclear giant cells appeared only in the infected Rhfs cell culture most sensitive to MPV. In human embryo kidney culture (HEK) productive infection was also established however, HEK cultures did not survive after 3-4 passages. No signs of transformation could be found in any of the cultures studied.

Animals↗

Molecular diversity among five different endogenous primate retroviruses.

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.

Animals↗

Use of molecular hybridization to detect type D retrovirus markers in rhesus placentas and other tissues.

We have shown previously that approximately 20% of the Mason-Pfizer virus (MPV) genome is present as endogenous provirus in rhesus monkeys. We report here that several full-term rhesus placentas examined contain additional MPV proviral sequences in their DNA. Competitive molecular hybridization experiments demonstrated that some of these placentas also contain RNA complementary to the entire MPV 60 to 70S RNA genome. Examination of internal organs of rhesus monkeys captured in the wild also revealed the presence of additional MPV proviral sequences and expression of MPV RNA in some tissues. These results provide further evidence that MPV is being transmitted via a non-germ line mechanism in the rhesus population and now demonstrate the placenta as a good source for the identification of retrovirus transcriptional products and proviral DNA.

Animals↗

The use of standard and relaxed hybridization conditions to detect two classes of sequences related to type-D retroviruses in the DNAs of primates.

Using standard hybridization conditions (68 degrees C and 0.4 M sodium phosphate) and assaying for RNAase-resistant RNA . DNA duplexes in the presence of 2 X SSC (1 X SSC is 0.15 M NaCl and 0.015 M sodium citrate), sequences representing approx. 20% of the Mason-Pfizer virus (MPV) genome have previously been shown to be endogenous in DNAs of all Old World monkeys examined (Drohan, W., Colcher, D., Schochetman, G. and Schlom, J. (1977) J. Virol. 23, 36--43). We now report that titration of both the temperature at which hybridizations are carried out and the Na+ concentration at which 125I-labeled MPV RNA . DNA hybrids are scored, reveals a second class of sequences related to the MPV genome in the DNAs of primates. These MPV-related sequences, which are similar to an additional 40% of the MPV genome, are detected when the temperature of hybridization is reduced to 54 degrees C and when the resulting 125I-labeled RNA . DNA duplexes are scored for RNAase resistance in 8 X SSC. These sequences are found in the DNAs of all Old World monkeys examined, and the Tm values of the hybrid duplexes are approx. 6--7 degrees C lower than those of the hybrid duplexes formed using standard conditions. These studies further demonstrate the wide distribution of type-D retrovirus sequences in primates.

Animals↗

Squirrel monkey retrovirus: an endogenous virus of a new world primate.

Squirrel monkey retrovirus (SMRV) was isolated by cocultivation of squirrel monkey lung cells with canine cells. 3H-labeled 60-70S SMRV RNA, isolated from virus grown in canine cells, hybridized to the same extent and to the same Cot1/2 value to the DNA of all tissues of all squirrel monkeys tested; Cot1/2 values show that SMRV proviral sequences are present in the low repetitive range. No SMRV proviral sequences were detected in tissues from a variety of other New World monkeys, Old World monkeys, or apes. Murine, feline, bovine, and canine cells also contain no detectable SMRV proviral sequences. Competitive molecular hybridization studies revealed no detectable sequence homology between the 60-70S RNAs of SMRV and Mason-Pfizer virus (MPV). The virion-associated DNA polymerase of SMRV is similar to that of MPV in that it has a molecular weight of approximately 80,000 and prefers magnesium as a divalent cation using oligo(dG)-poly(rC) as primer-template. The virion-associated DNA polymerase of SMRV can be clearly distinguished from those of MPV and the mouse mammary tumor viruses, however, by its preference for manganese as a divalent cation in the presence of high salt.

Animals↗

Sizes and concentrations of several type C oncornaviruses and bacteriophage T2 by the resistive-pulse technique.

Viruses above about 60 nm in diameter may be rapidly sized to a few nanometers in their natural hydrated state as they pass one by one through a single pore in a newly developed nanometer-particle analyzer based on the resistive-pulse technique of the Coulter Counter and the use of submicron diameter pores made by the Nuclepore process. Size measurements for several type C oncornaviruses are: Rauscher murine leukemia, 122.3 +/- 2 nm; simian sarcoma, 109.7 +/- 3 nm; Mason-Pfizer monkey, 140.0 +/- 2.5 nm; RD-114, 115 +/- 5 nm; and feline leukemia, 127.4 +/- 2 nm, relative to standard 109-nm latex spheres. The T2 bacteriophage has a volume of (5.10 +/- 0.15) X 10(-16) cm3. Concentrations of viruses near 10(9) to 10(11)/ml that are fairly clear of debris are routinely measurable in a few minutes to an accuracy near 15%. A lower practical count limit is near 5 X 10(7) viruses per ml.

Coliphages↗

[Detection of common antigenic determinants in the envelopes of mammalian type D (Mason--Pfizer) and type C virions by using the virion precipitation reaction].

Immune sera to Mason-Pfizer virus (M-PMV) are capable of precipitating type C viruses of mice (NIH-MuLV, G-MuLV, and to a lower degree R-MuLV), cats (FeLV, RD-114), and monkeys (SSV-1, GALV, BEV). The immune sera to RD-114, BEV, SSV-1 and GALV viruses can precipitate M-PMV virions. The adsorption analysis suggests that cross-reactions between M-PMV, RD-114, NIH-MuLV, and G-MuLV viruses depend on common virus-specific antigenic determinants in the envelopes of the virions. The relationship between M-PMV and type C viruses decreases in the series RD-114--NIG-MuLV--G-MuLV. Type C viruses of mammals have common envelope antigenic determinants lacking in M-PMV. No common antigenic determinants with M-PMV have been found in the envelope of virions of bovine leukemia virus, type B virus of mice (MMTV), or type C avian virus (Pr-RSV).

Animals↗