Optimized method for isolation of immature intracytoplasmic retroviral particles from mammalian cells.

To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles. The published methods for ICAPs isolation often involved several time-consuming centrifugation steps yielding damaged particles. Other papers describe the ICAPs production in non-natural host cells. Here, we optimized the method for purification of unstable Mason-Pfizer monkey virus (M-PMV) ICAPs from non-human primate derived cells, commonly used to study MPMV replication i.e. African green monkey kidney fibroblast cell line (COS-1). Our simple and rapid procedure involved separation of the intracytoplasmic particles from the cell debris and organelles by differential, low-speed centrifugation, their purification using sucrose velocity gradient and final concentrating by low-speed centrifugation. Importantly, the method was established for unstable and fragile M-PMV intracytoplasmic particles. Therefore, it may be suitable for isolation of ICAPs of other viruses.

Macaque homologs of EBV and KSHV show uniquely different associations with simian AIDS-related lymphomas.

Two gammaherpesviruses, Epstein-Barr virus (EBV) (Lymphocryptovirus genus) and Kaposi's sarcoma-associated herpesvirus (KSHV) (Rhadinovirus genus) have been implicated in the etiology of AIDS-associated lymphomas. Homologs of these viruses have been identified in macaques and other non-human primates. In order to assess the association of these viruses with non-human primate disease, archived lymphoma samples were screened for the presence of macaque lymphocryptovirus (LCV) homologs of EBV, and macaque rhadinoviruses belonging to the RV1 lineage of KSHV homologs or the more distant RV2 lineage of Old World primate rhadinoviruses. Viral loads were determined by QPCR and infected cells were identified by immunolabeling for different viral proteins. The lymphomas segregated into three groups. The first group (n = 6) was associated with SIV/SHIV infections, contained high levels of LCV (1-25 genomes/cell) and expressed the B-cell antigens CD20 or BLA.36. A strong EBNA-2 signal was detected in the nuclei of the neoplastic cells in one of the LCV-high lymphomas, indicative of a type III latency stage. None of the lymphomas in this group stained for the LCV viral capsid antigen (VCA) lytic marker. The second group (n = 5) was associated with D-type simian retrovirus-2 (SRV-2) infections, contained high levels of RV2 rhadinovirus (9-790 genomes/cell) and expressed the CD3 T-cell marker. The third group (n = 3) was associated with SIV/SHIV infections, contained high levels of RV2 rhadinovirus (2-260 genomes/cell) and was negative for both CD20 and CD3. In both the CD3-positive and CD3/CD20-negative lymphomas, the neoplastic cells stained strongly for markers of RV2 lytic replication. None of the lymphomas had detectable levels of retroperitoneal fibromatosis herpesvirus (RFHV), the macaque RV1 homolog of KSHV. Our data suggest etiological roles for both lymphocryptoviruses and RV2 rhadinoviruses in the development of simian AIDS-associated lymphomas and indicate that the virus-infected neoplastic lymphoid cells are derived from different lymphocyte lineages and differentiation stages.

Early immunopathology events in simian retrovirus, type 2 infections prior to the onset of disease.

Immunopathology during early simian retrovirus type 2 (SRV-2) infection is poorly characterized. Here, viral dynamics, immune response and disease progression in transiently- or persistently-infected cynomolgus macaques are assessed. Viral nucleic acids were detected in selected lymphoid tissues of both persistently- and transiently-infected macaques, even after viral clearance from the periphery. Immunohistochemical staining of lymphoid tissues revealed alterations in a number of immune cell populations in both transiently- and persistently-infected macaques. The precise pattern depended upon the infection status of the macaque and the marker studied. Gross immunopathological changes in lymphoid tissues were similar between SRV infection and those observed for other simian retroviruses SIV and STLV, suggesting a common immunopathological response to infection with these agents.

Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins.

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.

Type D retrovirus markers in healthy Africans from Guinea.

Sixteen matching sera and DNA samples from healthy African blood donors living in rural areas of Guinea were analysed for the presence of type D retrovirus markers. Screening for the antibodies against structural proteins of Mason-Pfizer monkey virus (M-PMV) was carried out by Western blot with a purified M-PMV as an antigen. Eight out of 16 sera samples were found to contain antibodies against at least two gag gene-coded proteins, and three of these were weakly positive against env gene-coded protein. Using PCR amplification and Southern hybridization, we detected M-PMV-like gag sequences in 11 out of 16 samples and env-related sequences in 8 out of 16 samples. Six DNAs were found to contain both M-PMV gag- and env-related sequences. Restriction endonuclease analysis of the PCR-amplified gag sequences from two individuals and direct DNA sequencing analysis of the amplimers confirmed their M-PMV-like origin. Detection of antibodies and M-PMV-related sequences in blood donors from Guinea, but not in French or Algerian blood donors, indicated exogenous SRV infection in humans from certain geographic areas of Western Africa.

Markers of type D retroviruses in children with Burkitt's-type lymphoma.

Antibodies to gag-coded proteins of type D retroviruses have been detected in children with lymphadenopathy [1]. We tested 41 HIV noninfected children with lymphoproliferative diseases (27 cases of Burkitt's-type lymphoma, six cases of Hodgkin's disease, four cases of T-cell lymphoma, three cases of lymphoblastic lymphoma and one case of large-cell anaplastic lymphoma) for the presence of type D retroviral serological and genetical markers. Twenty-five healthy donors were tested as a control. DNA samples from peripheral blood lymphocytes were analyzed by the polymerase chain reaction (PCR) and Southern blotting for the presence of type D retroviral related sequences. MPMV pro-pol specific sequences have been detected in 18 out of 27 children with Burkitt's-type lymphoma. By means of Western blotting, six patients positive in PCR/Southern blotting analysis were also found to contain Mason-Pfizer monkey virus (MPMV) specific antibodies, in their sera. All children with other lymphoproliferative diseases as well as healthy donors were negative in PCR/Southern blotting and Western blotting analysis. These data suggest the possible association of type D retroviral markers with Burkitt's-type lymphoma of children.

Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase.

We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.

Radioimmunoassay for infectious primate retrovirus reverse transcriptase: characterization, comparison with conventional immunologic assays and applicability to cellular extracts.

SSV reverse transcriptase (RT) was purified to homogeneity and used in a radioimmunoassay. Following iodination, the homogeneity of the protein and its identity with RT were confirmed by several criteria: (1) its molecular weight on an SDS-polyacrylamide gel; (2) its precipitation by anti-SSV RT but not by antisera to other SSV proteins; (3) its cross-reactivity in RIA with antisera to other retroviral polymerases; (4) its competition in RIA by active homogeneous SSV RT but not by other purified SSV proteins; and (5) its competition in RIA by only those fractions from a poly(U)-Sepharose column possessing SSV RT activity. Competition of the labelled probe with disrupted retroviruses of the infectious primate group showed that, while a homologous RIA detected only type-specific enzyme determinants, it did not distinguish the various woolly-gibbon retroviral DNA polymerases. A more broadly reactive heterologous assay utilizing an antiserum to R-MuLV RT detected group- but not interspecies-specific enzyme determinants. A comparison of immunologic assays for RT showed that: (1) highly purified RT is not essential for reliable results in enzyme neutralization or enzyme binding assays; (2) the greater sensitivity of enzyme binding compared to enzyme neutralization assays is a function of the antibody, not of the antigen. Competition RIAs using extracts of virus-infected cells showed that infectious primate retrovirus RT could be measured in a crude system and that cellular DNA polymerases alpha, beta and gamma did not compete with the labelled probe.