Induction and characterization of a replication competent cervid endogenous gammaretrovirus (CrERV) from mule deer cells.

Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference.

Human endogenous retroviral expression by leukocytes.

Budding endogenous retroviral particles were identified on the plasma membrane of a high percentage of neurotrophils in four widely varied pathological specimens.

Constitutive production of a murine retrovirus in the human B-lymphoblastoid cell line, DG-75.

During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission studies, the Epstein-Barr virus (EBV)-negative, B-lymphoblastoid cell line DG-75 was found to be chronically infected with a heretofore unrecognized retrovirus. Two DG-75 sublines obtained from different sources (designated UW and KAR) were found to produce constitutively particles identified as retroviral by electron microscopy and reverse transcriptase activity. The ultrastructure, morphogenesis, and density in sucrose of the particles were typical of C-type retroviruses. Immunoblot analysis of the DG-75(UW) retrovirus proteins showed antigenic similarity to Moloney murine leukemia virus. A third DG-75 subline in early passage, designated HAD, was free of retrovirus. The DG-75(UW) retrovirus was infectious and produced progeny virions that could be passaged to uninfected cells. We have thus demonstrated that DG-75 cells, which have been used extensively in studies of the biological effects of EBV-encoded genes and their promoters, may be chronically infected with a murine retrovirus and that an early passage subline is retrovirus free and available for such studies.

Ecotropic C-type retrovirus of B16 melanoma and malignant transformation of normal melanocytes.

We reported previously that B16,JB/RH and JB/MS melanomas of C57BL/6 mice express the common melanoma-associated antigen (MAA) recognized by MM2-9B6 monoclonal antibody (MAb). This MAA is encoded by the env gene of an ecotropic MuLV-type retrovirus that somatically emerged in melanomas of C57BL/6 mice. The potential role of this melanoma-associated retrovirus (MelARV) in melanoma formation remains unknown and has not been previously investigated. To test this, normal melanocyte lines (melan-a and C57M) of C57BL/6 mice were infected with the MelARV produced by B16BL6 melanoma. Infection of these melanocytes with the MelARV was associated with the appearance of the MAA recognized by MM2-9B6 MAb. Most of the infected melanocyte sublines were able to grow only in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Two infected melanocyte sublines showed morphological changes, were able to grow in the absence of TPA and, after inoculation into C57BL/6 mice, produced rapidly growing, highly pigmented tumors. These new melanomas, derived from the MelARV-infected melan-a and C57M melanocytes, were termed Meli-A1 and Meli-BL, respectively. Southern blot analysis of EcoRI- and HindIII-digested DNAs from these melanomas showed several retroviral insertion sites. One copy of MeIARV was found to be inserted at the end of the 6th leucine domain of the c-maf proto-oncogene, which encodes a basic region/leucine zipper transcription factor related to the AP-1 family that is able to form homodimers or heterodimers with Fos and Jun transcription factors. Our data indicate that c-maf is a common insertion site of MelARV in BL6, Meli-A1 and Meli-BL melanomas, whereas no such insertion site was found in the melanocytes infected with MelARV but not malignantly transformed. Thus, our data imply that the ecotropic MelARV that somatically emerged in B16 and other melanomas of C57BL/6 mice may play a role in malignant transformation.

Microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles.

The observation of murine retrovirus infection of microglial cells in brain regions expressing spongiform neurodegenerative changes suggests that these cells may play an important role in pathogenesis. To evaluate this potential in vitro, murine microglial cells were infected in mixed glial cultures with the highly neurovirulent murine retrovirus, FrCasE. The microglia were then isolated from the mixed cultures on the basis of their differential adherence and shown to be approximately 98% pure. The infected microglia expressed viral envelope protein at the plasma membrane, while viral budding was primarily intracellular. Evaluation of the viral envelope protein by immunoblotting indicated that the immunoreactive species produced was exclusively a 90-kDa precursor protein. Very little of the envelope protein was associated with particles released from these cells, and viral titers in the culture supernatant were low. Interestingly, these cells were still capable of infecting permissive target cells when seeded as infectious centers. This partially defective infection of microglial cells suggests a potential cellular means by which a neurovirulent retrovirus could disrupt normal microglia and in turn central nervous system motor system functioning.

Ultrastructural and biochemical evidence that the L929 cell retrovirus lacks the env gene translation product.

LCV, a murine retrovirus released by L929 mouse cell fibroblasts, is non-infectious when inoculated into SC-1, mink, D-17 or Vero cells. Ultrastructural examination by thin sectioning, freeze-etching or negative staining revealed the absence, on the viral envelope, of the radially disposed spikes. Polyacrylamide gel electrophoresis of radiolabelled viral components showed the absence of the glycosylated protein gp70 as well as of the p15E cleavage product of the polyprotein precursor gPr90env. The premature loss of the gp70 molecule from LCV to the culture medium was ruled out since no peak of D-[14C]glucosamine-labelled glycoprotein was detected by affinity chromatography or immunoprecipitation of concentrated medium. The ultrastructural and biochemical results all supported the hypothesis that the absence of infectivity was due to the lack of gp70 glycoprotein in the envelope of LCV. A possible block at a translational or post-translational level was also investigated by immunofluorescence studies with antisera directed against ecotropic or xenotropic gp70; Moloney murine leukaemia virus-infected or NZB cells were used as positive controls for eco- or xenotropic viruses respectively. The absence of fluorescent stain in L929 cells further supported these results and suggested that LCV and the L929 parental cell line lack the uncleaved precursor and the final product of the env gene translation process.

Mammary tumor virus DNA as a marker for genotypic variance within hormone-responsive GR mouse mammary tumors.

Mammary tumors of GR mice acquire extra mammary tumor virus (MMTV) DNA information within their DNA during tumor growth and development. These extra MMTV genes have been used by us as genotypic markers to investigate the heterogeneity of GR mammary tumors and their loss of hormone dependence during serial transplantation. Our studies reveal that the various subpopulations of cells within individual GR mammary tumors are characterized by differences in number and location of acquired extra MMTV DNA fragments. Losses of certain of these extra MMTV DNA fragments occur when mammary tumors become hormone independent, indicating a loss of hormone-dependent cells. The study of MMTV DNA markers also reveals that low levels of autonomous cells are already present in some hormone-dependent mammary tumors at an early stage of their development. The genotypic analysis strongly indicates that mammary tumor progression is not due to phenotypic adaptation but to clonal selection of the more aggressive sublines.

[Aspects of megakaryocytic viral production in relation to lymphocytic leukemia in the rat]. / Quelques aspects de la production virale mégacaryocytaire en rapport avec la leucémie lymphoïde chez le rat.

Two viral populations BL/F (EL) and BL/F (SL) were derived from RadLV-Rs by propagation in rats where they induced respectively a generalized lymphoma in 5-6 weeks or a thymic lymphoma killing the animals in 5-6 months. In both cases, 10 days after inoculation of viral extract, numerous viral particles are present in the megakaryocytes (MKC) of the bone marrow and the spleen. Our results suggested a production rather than a passive accumulation of those particles by the MKC. The kinetics of blood platelet level for both leukemias showed a thrombocytopenia corresponding with the macroscopic development of the tumor. Therefore the evolution of the blood platelet level is not related to the MKC viremia. This suggests a lack of direct effect of virus BL/F on the MKC metabolism.

Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase.

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.

Aberrant viruses in cells infected with murine sarcoma virus-feline leukemia virus.

This study describes an unusual type of virus seen when a feline leukemia virus (FeLV) pseudotype of murine sarcoma virus (MuSV) obtained by cocentrifugation procedures infected feline embryo cells (FEF) and two Crandell cat cell lines (CrFK1, CrFK2). When all three cell cultures were infected with MuSV-FeLV, only FEF and CrFK2 were transformed and only these showed normal and aberrant virus. The CrFK1 infected with MuSV-FeLV did not transform but did replicate normal type-C virus with a 50-A intermediate coat. The virus replicated in the two transformed lines showed three particles; a normal particle with a 50-A intermediate coat, a normal particle with a 100-A intermediate coat, and an aberrant particle with a 100-A intermediate coat.

The morphology of murine oncornaviruses following different methods of preparation for electron microscopy.

The effect of different preparative procedures for electron microscopy on the size and shape of murine oncornaviruses has been studied. With conventional negative staining procedures using neutral sodium phosphotungstate, both murine mammary tumor virus and murine leukemia virus appeared in head-and-tail forms, with a peak head diameter of 122 and 130 nm, respectively. Negative staining with uranyl accetate gave round virions with peak diameters of 148 and 130 nm. Prefixed virus was round with peak diameters of 141 and 130 nm, respectively, in phosphotungstate, and 148 and 117 nm, respectively, in uranyl acetate. With thin sections, the peak diameters were 143 and 123 nm. The preservation of the spherical shape of the virus was obtained by glutaraldehyde fixation dehydration in alcholic solutions of uranyl acetate, and critical point drying. Under these conditions the viruses had peak diameters of 99 and 82 nm, respectively. The size of murine mammary tumor virus has always been found to be larger than murine leukemia virus in all preparations except for negative staining with neutral sodium phosphotungstate. Shadowing of the virion preparations revealed considerable flattening of the particles in all cases except for critical point drying. Negatively stained preparations did not cast any shadow, and thus thethickness of the particles could not be evaluated. Virus can be reversibly converted from spherical to head-and-tail forms by altering osmotic strength. Under most of the conditions used, murine mammary tumor virus gave a bimodal size distribution with significant numbers of particles that were smaller than the major virus size.

Induction of endogenous murine C-type virus in spleen cell cultures treated with mitogens and 5-bromo-2'-deoxyuridine.

In short-term cultures of BALB/c spleen cells, treatment with a combination of 5-bromo-2'-deoxyuridine (BrdU) and either lipopolysaccharide W. Escherichia coli or concanavalin A resulted in release of C-type virus into the medium. Only lipopolysaccharide induced virus release when given alone. This could be potentiated by a combined treatment with BrdU. In contrast, phytohemagglutinin at mitogenic concentration had no effect with or without BrdU, suggesting that inducibility may vary between various mitogen-responsive spleen cell populations. In AKR mice, spontaneous virus release was detectable in nonstimulated spleen cell cultures. This could be potentiated by lipopolysaccharide, whereas no further increase occurred upon additional BrdU treatment. The induced viruses had C-type characteristics in that they contained reverse transcriptase that could be distinguished from cellular enzymes by template-primer preference experiments. Furthermore, the enzyme activities were particle-associated, banding in isopycnic sucrose gradients at 1.15-1.17 g/cm-3. The presence of C-type viruses was confirmed by electron microscopy.