Isolation of a unique retrovirus, MNV-1, from Macaca nemestrina.

Cell cultures established from the spleen of a Macaca nemestrina with enzootic retroperitoneal fibromatosis (ERF) spontaneously released a unique retrovirus. Throughout 14 serial passages, the spleen cell cultures remained fibroblastic and no cytopathic effect was evident. The virus incorporates [3H]uridine, contains an RNA-dependent DNA polymerase (RDDP), has a buoyant density of 1.15 g/cm3 in sucrose, and was designated MNV-1. Virion-associated reverse transcriptase showed no preference for either Mg2+ or Mn2+ in standard RDDP assays. Complementary DNA (cDNA) transcribed from polyadenylated MNV-1 RNA hybridized to genomic DNA and RNA extracted from diseased tissues but not to nucleic acids from normal tissues of a healthy Macaca nemestrina or a Macaca mulatta. MNV-1 is therefore exogenous to these species. MNV-1 had no detectable homology to the endogenous macaque virus isolates MAC-1 and MMC-1. Liquid hybridization of MNV-1 cDNA to viral RNA derived from exogenous and endogenous subhuman primate retroviruses (SiSV(SSAV), GALV-SF, BaEV-M7, and BILN) did not reveal any significant sequence homologies. In addition, MNV-1 does not share homology with bovine leukemia virus or Mason-Pfizer monkey virus as determined by Southern blot hybridization. We conclude that MNV-1 is a unique retrovirus which has not previously been described. As the ultrastructure of virions in in vitro cell cultures, as well as disease involved tissue, show some particles with type C morphology and others with type D morphology, MNV-1 may be comprised of more than one component.

Restricted HEL-12 virus infections in de novo infected human and canine cells.

Model systems to study restricted primate retrovirus expression were established by de novo infection of canine foetal thymus cells (CF-2Th) and superinfection of HEL-12 cells with HEL-12 virus. In the resulting CF-2Th/HEL-12V cells and HEL-12/HEL-12V cells, four sequential stages of virus infection were defined by the production of reverse transcriptase (RT)-containing particles and expression of virus antigens as detected by radioimmunoassays. Stage 1 cells did not synthesize virus antigens or produce RT-containing particles. Stage 2 cells synthesized virus antigen but not RT-containing particles. Stage 3 cells synthesized antigen and produced RT-containing particles, and stage 4 cells synthesized virus antigens but no longer produced RT-containing particles. The duration of the four stage infection is 2 to 3 weeks in both cell types. Monospecific competition radioimmunoassays to detect HEL-12V p30 or gp70 antigen showed high levels of virus antigen throughout stages 2 to 4 of infection. Analysis of immunoprecipitates formed under conditions to detect either p30- or or gp70-containing proteins in cells pulsed and pulsed--chased with [3H]leucine showed the same spectrum of virus precursor polyproteins, intermediates and mature virion components in stage 2 to 4 cells in canine and human infections. Spent culture fluids collected from stage 3 and stage 4 CF-2Th/HEL-12V cells failed to reveal inhibitors of RT activity. Stage 4 CF-2Th/HEL-12V or HEL-12/HEL-12V cells labelled with [3H]uridine produced virions which incorporated [3H]uridine but did not have RT activity, suggesting that restricted infection is characterized by production of HEL-12V defective in RT activity.

Comparative oligonucleotide analysis of exogenous and endogenous primate type C viruses.

RNAs of representative viruses of the exogenous simian sarcoma virus-gibbon ape lymphosarcoma virus (SiSV/GALV) and endogenous baboon virus (BaEV) classes of subhuman primate type C viruses were compared and related to HEL-12 virus, an isolate derived from human embryonic lung cells. The extent of sequence identity between different viral RNA preparations was determined by comparison of fingerprint patterns obtained after electrophoretic separation of RNase T1-resistant oligonucleotides. The studies presented indicate that HEL-12 viral RNA and simian sarcoma-simian associated virus [SiSV(SSAV)] RNA share 90 to 95% of the large oligonucleotides. From 5 to 10% of virus-specific oligonucleotides were detected in each of several virus preparations examined and their occurrence was independent of the cell line on which the virus ws propagated. HEL-12 virus and GALV-SF have 50% unique oligonucleotides in common. These are the same oligonucleotides that are shared between GALV-SF and SisV(SSAV) RNA. Two BaEV isolates, M7 and BILN, and RD114, and BaEV-related endogenous virus of cats, each easily displayed distinguishable oligonucleotides patterns. Large oligonucleotides characteristic for these three endogenous virus isolates were not detected in the fingerprints of HEL-12 virus, SiSV(SSAV) and GALV-SF.

Biological and antigenic characteristics of HEL-12 virus.

The biological and antigenic properties of HEL-12 virus have been compared with gibbon ape lymphosarcoma virus (GALV) and simian sarcoma and simian sarcoma-associated viruses, SiSV and SSAV, respectively, HEL-12 virus did not transform human or marmoset fibroblasts but rescued SiSV focus-forming activity from non-productively transformed marmoset cells (HF/SiSV-NP). Like SSAV and GALV, HEL-12 virus induced syncytia with XC cells. In addition, HEL-12 cells which did not produce virus but which contained HEL-12 proviral DNA, rescued SiSV from HF/SiSV-NP cells in co-cultivation experiments. Results of neutralization and serum cytotoxicity tests utilizing SiSV rescued by HEL-12 [SiSV-(HEL-12)] indicated that HEL-12 virus envelope proteins are very closely related to those of SSAV but readily distinguished from those of GALV. Antigenic diversity of SiSV(SSAV), SiSV(GALV) and SiSV(HEL-12) envelope glycoproteins (gp70) was shown in competition radioimmunoassays (RIA) designed to detect minor antigenic differences using antiserum monospecific for SiSV(SSAV) gp70 or Friend murine leukaemia virus gp70. Antigenic differences between these gp70s were demonstrated in RIA using purified SSAV gp70 or HEL-12 gp70. These data indicate that HEL-12 virus has biological properties similar to those of SSAV and GALV, is distinguished from GALV in neutralization tests and has both distinct and SSAV-related gp70 antigenic determinants.

The nucleotide sequence of a cloned human leukocyte interferon cDNA.

We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.

Retrovirus expression in normal and pathogenic processes of man.

C-type viruses have been isolated from primates only on rare occasions. To date, an endogenous C-type virus of man has not been isolated. Nevertheless, the evidence for C-type viral expression in human tissue increases. Evidence for viral antigen expression during human gestation and the pathogenesis of systemic lupus erythematosus (SLE) is summarized. The data are discussed in connection with the current hypotheses that retroviruses may participate in both normal and abnormal biologic processes.

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

We report that a cloned population of tumor cells can rapidly produce variants that differ in their quantitative expression of surface proteins and in their rate of immunoglobulin secretion. A fresh clonal isolate of S107 myeloma cells possessing large amounts of surface IgA was continuously passaged in vitro for 2 years. During this period, fluorescence-activated cell sorter analysis indicated the development of subpopulations possessing decreased amounts of surface IgA. Cells from these variant subpopulations were isolated by first using the cell sorter to enrich for cells with decreased amounts of surface IgA and then cloning the selected population in soft agar. The 50 sublines that were isolated showed heritable differences in their levels of surface IgA and H-2 antigens and in their rates of myeloma protein secretion. Sublines having either large amounts, intermediate amounts, or absence of surface IgA also had corresponding large amounts, intermediate amounts, or absence of myeloma protein secretion. In contrast, a decrease or loss of surface Ig did not correlate with a decrease or loss of viral envelope glycoprotein gp71 and H-2 antigens. The variants did not resemble the phenotypes of less-differentiated normal lymphocyte populations of the B-cell lineage. The isolation and characterization of these variants allows us to explore the mechanisms and pathways of tumor cell differentiation as well as to study the regulation and function of cell surface proteins.

Type C virus induced by iododeoxyuridine in the human embryonic cell strain HEL-12.

The induction of a type C virus from a strain of human embryonic lung cells (HEL-12) by iododeoxyuridine (IdUrd) was examined at various times during in vitro propagation. IdUrd did not elicit type C virus production immediately following initiation of cultures from frozen primary HEL-12 cells. After overnight treatment with 30 microgram/ml IdUrd the cells expressed viral antigens and produced a type C virus between the 25th and 80th day of in vitro growth. Production of the induced type C virus was transient. Single-cell clones of the parental HEL-12 strain were likewise susceptible to type C virus production by IdUrd. The ability of IdUrd to induce virus terminated with the onset of spontaneous type C virus production from HEL-12 cells at between 80 and 120 days of in vitro growth.

Time-dependent resistance or susceptibility of tumor cells to cytotoxic antibody after exposure to a chemotherapeutic agent.

We report that a chemotherapeutic agent (melphalan) can affect the sensitivity of tumor cells to cytotoxic antibody. Depending on the time interval between drug treatment and subsequent exposure to antibody and complement, the tumor cells can be either more resistant or more susceptible to antibody when compared to control cells. The number of tumor cells surviving the combined treatment was determined by a colony inhibition assay. The two antisera used in this study were directed against either virus-specific or myeloma protein-specific antigens on the surface of S107 murine myeloma cells; identical results were obtained with both sera. Twenty-four hours after exposure to the drug, the number of tumor cells surviving the antibody treatment increased. During this period of increased resistance, the tumor cells were temporarily arrested in the G(2) phase of the cell cycle. After this period of maximal resistance, the effect of cytotoxic antibody on the cells changed such that 4 days after melphalan treatment the cells were significantly more susceptible to the antibody than were the sham-treated control cells. The period of increased susceptibility correlated with an increased density of S107 myeloma protein and viral antigens on the surface of the tumor cells. Eight days after the drug treatment, the susceptibility of the tumor cells and the density of surface antigens both returned to normal levels. This study shows that the correct time interval between exposure to a drug and subsequent treatment with antibody is critical for maximal killing of the tumor cells. The basis for the differential sensitivity of the tumor cells to anti-body may be related to the drug-induced changes in the cell cycle and in antigen expression on the cell surface.

Cell generation and type C virus expression in the human embryonic cell strain HEL-12.

The spontaneous expression of a type C virus in a diploid strain of human embryonic lung fibroblast-like cells (HEL-12) was examined during serial culture. Virus antigen expression was determined by indirect immunofluorescenc with antisera to disrupted simian sarcoma virus and the 28000 mol. wt. internal antigen of the endogenous cat virus RD-114. Virus production was examined by reverse transcriptase assays of culture fluids. Virus antigens were not detected for 25 days after frozen, primary HEL-12 cells were reinstated in culture. The cells expressed virus antigens but did not release virus particles between 25 and 80 days. Spontaneous virus release and maximal antigen expression occurred in cells grown for 80 to 120 days. Virus particles were not detected after 120 days although virus antigens persisted until the experiment was terminated. The HEL-12 virus was infectious for cell cultures of human, rhesus monkey, dog and rabbit cells. The proportion of SiSV-like and RD-114-like antigenic components of HEL-12 virus were altered by passage through heterologous cells suggesting heterogeneity of the HEL-12 virus population.