Reverse Transcriptase Activity In Supernatant Research Articles

Pharmacologic reactivation of latent feline immunodeficiency virus ex vivo in peripheral CD4+ T-lymphocytes

FIV establishes a latent infection in peripheral CD4+ T-cells, and the latent FIV promoter is associated with deacetylated, methylated histones, consistent with a restrictive chromatin structure. Here we explored the use of 5 histone-modifying agents – 4 histone deacetylase inhibitors (HDACi) and 1 histone methyltransferase inhibitor (HMTi) – to reactivate latent FIV ex vivo. All compounds tested were able to alter histone lysine residue modifications in feline cells, both globally and at the FIV promoter locally. When latently FIV-infected peripheral CD4+ T-cells were cultured ex vivo in the presence of these inhibitors, viral transcription was significantly activated relative to no treatment controls. Transcriptional reactivation of virus mediated by the HDACi suberoylanilide hydroxamic acid (SAHA) was dose-dependent, detected after as little as 1h of exposure, and resulted in virion formation as evidenced by supernatant reverse transcriptase activity. A synergistic effect was not found when SAHA was combined with HMTi under the conditions tested. At low therapeutically relevant concentrations in primary feline PBMC, SAHA was found to be minimally cytotoxic and non-immune activating. HDACi and HMTi can reactivate latent FIV ex vivo, and SAHA, also known as the anticancer drug Vorinostat, in particular is a promising candidate for in vivo use because of its efficacy, potency, and relative safety. Use of the FIV/cat model of lentiviral latency to explore in vivo treatment with SAHA and other anti-latency therapeutics will allow investigations that are either ethically or logistically not addressable in patients persistently infected with human immunodeficiency virus (HIV-1).

Isolation and Characterization of Griffithsin, a Novel HIV-inactivating Protein, from the Red Alga Griffithsia sp.

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM. GRFT also aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations. High concentrations (e.g. 783 nM) of GRFT were not lethal to any tested host cell types. GRFT blocked CD4-dependent glycoprotein (gp) 120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner. GRFT preferentially inhibited gp120 binding of the monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent motif, and the (mAb) 48d, which binds to CD4-induced epitope. In addition, GRFT moderately interfered with the binding of gp120 to sCD4. Further data showed that the binding of GRFT to soluble gp120 was inhibited by the monosaccharides glucose, mannose, and N-acetylglucosamine but not by galactose, xylose, fucose, N-acetylgalactosamine, or sialic acid-containing glycoproteins. Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner. GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.

Gag, vif, and nef genes contribute to the homologous viral interference induced by a nonproducer human immunodeficiency virus type 1 (HIV-1) variant: identification of novel HIV-1-inhibiting viral protein mutants.

We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr, pol, or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.

Infection of bone marrow macrophages by equine infectious anemia virus

SUMMARY Objective To characterize infection of bone marrow-derived macrophages (BMDM) with equine infectious anemia virus (EIAV) by determining virus production, effects on viability, and induction of cytokines. Sample Population BMDM obtained from bone marrow of 6 clinically normal adult horses. Procedure BMDM were infected with EIAV at a multiplicity of infection of 8. Cell viability, percentage of cells with detectable viral protein, reverse transcriptase activity, and concentrations of infective virus (focus-forming units/ml), interleukin 6, and tumor necrosis factor-α were measured in culture supernatant samples obtained at various days after infection. Results Cell viability was decreased on day 4 and was maximally decreased on day 8. The number of cells with detectable viral protein and supernatant reverse transcriptase activity increased significantly on day 4 and increased until day 6. Virus concentration (focus-forming units per milliliter) peaked on day 4 after infection and was constant thereafter. Infection with EIAV caused significant induction of interleukin 6 production by BMDM. The maximal difference was seen on day 4 after infection. Control and infected BMDM produced only negligible amounts of tumor necrosis factor-α. Conclusions BMDM are useful, as a cell population, to study the effects of infection with EIAV, including cell death and induction of interleukin 6 but not tumor necrosis factor-α production. (Am J Vet Res 1997;58:1402–1407)

Phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus

Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR sequences of U.S. field strains of EIAV to sequences of a laboratory adapted strain of the virus. All field strain sequences were more closely related to Wyoming strain than to the Malmquist laboratory adapted strain or a previously sequenced infectious molecular clone of EIAV. Primary equine monocyte-derived macrophage cultures were infected with virulent and avirulent strains of EIAV and the effects of macrophage stimulation on EIAV expression were determined. Stimulation of macrophages with phorbol ester activated the cells to secrete tumor necrosis factor α (TNFα). This activation signal also resulted in a significant downregulation of viral expression as determined by supernatant reverse transcriptase activity. This effect occurred independent of the virulence of the virus strain used or the nucleic acid sequence of the viral LTR. This may represent an adaptive response of EIAV to evade the host immune response and establish a persistent infection.

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.

Prevention of Activation of HIV-1 by Antiviral Agents in OM-10.1 Cells

The development of a reliable and simple system for evaluating compounds that could prevent activation of latent HIV would allow us to devise new therapeutic approaches. These compounds could eventually be used in combination with drugs that are effective against acute and chronic infections. The OM-10.1 cell line is a chronically infected clone which remains CD4+ until HIV-1 activation with tumour necrosis factor-α. A variety of compounds are known to have antiviral properties against either acutely or chronically infected cells were evaluated for their ability to inhibit HIV induced expression in these cells. We also examined the effect of several compounds that interact with biochemical pathways that may interfere with or enhance the reactivation process. These included nucleoside analogues, cytokines, steroidal and nonsteroidal anti-inflammatory agents, polyoxometalates, a TAT inhibitor, various natural products (including nerve growth factor, N-acetyl-L-cysteine, taxol, and interferons), TIBO, porphyrins, and various oligomers. CD4 cellular expression and supernatant reverse transcriptase activity were quantitated as markers of induced viral expression. Among the 58 compounds evaluated, 3′-fluoro-3′-deoxythymidine (FLT), interferon γ, Ro 5–3335 (a TAT inhibitor) and desferrioxamine were modest and selective inhibitors of HIV-1 activation.

Glutathione and N-acetylcysteine suppression of human immunodeficiency virus replication in human monocyte/macrophages in vitro.

Glutathione (GSH), its derivatives and N-acetylcysteine (NAC) inhibit the induction of HIV-1 expression in a chronically HIV-1-infected promonocytic cell line (U1/HIV) and peripheral blood mononuclear cells (PBMC). We have examined the effects of GSH and NAC on HIV-1 replication in human primary monocyte/macrophages cultured in vitro. Ficoll-gradient purified human monocytes were cultivated in vitro for 7-10 days and then infected with HIV-1 (Bal and Ada-M). Infection was blocked or substantially reduced by GSH or NAC (5-20 mM). Significant reduction (greater than or equal to 90%) in the amount of virus released, as determined by measuring supernatant reverse transcriptase activity and secreted p24 protein, was obtained when the cells were treated for 4 h with greater than or equal to 10 mM of GSH or NAC. The inhibitory effects of GSH and NAC were concentration dependent. This anti-HIV-1 effect persisted in these cultures for at least 35 days without evidence of significant increase in HIV-1 expression. Thus, a single pulse exposure of HIV-1-infected monocyte/macrophages with GSH or NAC led to a sustained, concentration-dependent decrease in HIV-1 p24 antigen levels, as well as, reverse transcriptase activity without producing detectable cellular toxicity in monocyte/macrophages.

Macrophage tropism of an acutely lethal simian immunodeficieny virus, SIVsmm-PBjl4

Simian immunodeficiency viruses (SIV) have many features of human immunodeficiency viruses and provide models for retroviral pathogenesis and vaccine studies. A SIV isolate from sooty mangabcy monkeys (SIVsmm-PBj14) is the most acutely pathogenic primate lentivirus described, causing rapidly fatal disease within 8 days in pig-tailed macaques. The parent virus (SIVsmm-9) normally causes an AIDS-like immunosuppressive illness in macaques. Both viruses have very similar genetic sequences, with the major difference being a duplicated enhancer region present in the LTR of SIVsmm-PBj14. We found that both viruses readily infected human macrophages and PHA-stimulated PBL (measured by culture supernatant reverse transcriptase activity and confirmed by in situ hybridisation and electron microscopy), but that the SIVsmm-PBj14 alone caused a marked cytopathic effect in macrophages. A previous report suggested that SIVsmm-PBj14 could activate and infect non-PHA stimulated PBL, but we found that this occurred only in PBL preparations that included macrophages. Non-PHA stimulated PBLs depleted of macrophages by adherence could neither be stimulated (measured by H 3 -thymidine uptake) or infected by either isolate. We have shown that SIVsmm-PBj14, in comparison to its parent virus, could cytopathically infect macrophages, and that these features are associated with acute lethality in pig-tailed macaques.

A semiautomated multiparameter approach for anti-HIV drug screening

We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3'-dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited.

Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4- and antibody-independent fashion.

Incubation of the human T cell lymphotropic virus (HTLV)-IIIB and HTLV-RF strains of human immunodeficiency virus type 1 (HIV-1) with normal seronegative human serum under conditions that allow complement activation resulted in enhancement of infection of the MT2 human T cell line cultured in the presence of low amounts of virus. Infection of MT2 cells was assessed by measuring reverse transcriptase activity in supernatants at day 9 of culture. Complement activation by viral suspensions occurred through the alternative pathway. Opsonization of HTLV-RF viral particles with complement was sufficient to allow a productive infection to occur in cells exposed to suboptimal amounts of virus. Infection of MT2 cells with suboptimal amounts of serum-opsonized HIV-1 was suppressed by blocking the C3dg receptor (CR2, CD21) on MT2 cells with monoclonal anti-CR2 antibody and rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Blocking of the gp120-binding site on CD4 under similar experimental conditions had no inhibitory effect on infection of MT2 cells with opsonized virus. Opsonization of HIV-1 with seronegative serum also resulted in a CR2-mediated enhancement of the infection of normal peripheral blood mononuclear cells and T lymphocytes. These results indicate that complement in the absence of antibody may enhance infection of C3 receptor-bearing T cells with HIV-1, and that the interaction of opsonized virus with the CR2 receptor may result by itself in the infection of target T cells in a CD4- and antibody-independent fashion.

5-azacytidine and 5-azadeoxycytidine inhibit human immunodeficiency virus type 1 replication in vitro

Chemotherapeutic agents which affect the integration, stability, or inducibility of the human immunodeficiency virus (HIV) provirus would have considerable value in treating acquired immunodeficiency syndrome. Two nucleoside analogs of cytosine, 5-azacytidine and 5-azadeoxycytidine, which seem to have such value because of their capabilities to affect both the stability and the methylation patterns of the nucleic acids into which they are incorporated, were tested for their ability to inhibit the replication of HIV type 1 (HIV-1) in human CEM T cells in vitro. 5-Azadeoxycytidine (1 microM) completely inhibited HIV replication in CEM cells, by the criteria of reduced viral antigen expression and decreased supernatant reverse transcriptase activity, with little toxicity for the treated cells. 5-azacytidine (1 microM) also inhibited HIV replication, but less effectively. When added 2 or more h after CEM cells were infected with HIV-1, both 5-azacytosine derivatives were less effective than they were when added at the time of infection. Even 2 h of exposure to 5-azadeoxycytidine was sufficient for inhibition of HIV replication. Although long exposure to either analog at concentrations of 1 microM would result in pronounced cellular cytotoxicity, the the fact that short exposures to the same dose of drug inhibit HIV replication but are not toxic for the cells implies that cellular toxicity itself is not an important mechanism of the antiviral action of the analogs.

Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone.

Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, was demonstrated to induce the expression of human immunodeficiency virus (HIV) in a chronically infected T-cell clone (ACH-2). Concentrations of recombinant TNF-alpha as low as 50 pg/ml induced a significant increase over background of HIV expression in the ACH-2 cells as determined by supernatant reverse transcriptase activity. The HIV-inducing effects of TNF-alpha could not be explained by toxic effects on the cells. In addition, both the uninfected parental cell line (A3.01) and the infected ACH-2 cells were shown to have high-affinity receptors for TNF-alpha. Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat. These studies provide evidence that TNF-alpha may play a role in the mechanisms of pathogenesis of HIV infection.

Molecular cloning of biologically active proviruses of bovine immunodeficiency-like virus

A series of independent proviral molecular clones of bovine immunodeficiency-like virus (BIV) obtained from a genomic library of BIV-infected bovine cell DNA were physically and biologically charactarized. Heteroduplex mapping shows that two of these BIV clones (106 and 127) contain uninterrupted proviral sequences approximately 9.0 kb in length, flanked by nonhomologous bovine cellular sequences. Microinjection of purified DNA from BIV clone 106 or 127 into susceptible bovine cells produces virus-specific cytopathic effects, including syncytium induction, supernatant reverse transcriptase activity, and infectious virus particle formation, similar to the effects produced by parental virus stock. Using restriction enzyme mapping, it was determined that the two infectious clones share 13 of 14 sites mapped within the provirus; thus, based on this criterion, the two clones are nearly identical, with the exception of a single polymorphic site recognized in the 3′ half of the genome. BIV appears to be an exogenous pathogenic virus, because Southern hybridization analyses detected no endogenous sequences related to BIV in DNA from a variety of uninfected bovine cells and tissues. Most of the BIV-related DNA found in cells 96 hr after infection is present as linear unintegrated viral DNA, although the presence of host flanking sequences in our proviral clones indicates that integration takes place. These biologically active clones of BIV will be of use in defining further the mechanisms of BIV pathogenesis and in engineering specific diagnostic reagents to determine the prevalence of BIV in cattle populations.

Infection and Replication of HIV-1 in Purified Progenitor Cells of Normal Human Bone Marrow

Myeloid progenitor cells were highly purified from normal human bone marrow by positive immunoselection with high-affinity monoclonal antibodies linked to magnetic beads and were successfully infected in vitro with the human immunodeficiency virus type 1 (HIV-1). From 99 to 100 percent pure bone marrow cells expressing the CD34 phenotypic marker were obtained. These cells were devoid of mature myeloid or T cell surface and intracellular markers as analyzed by immunohistochemical staining and flow cytometry. HIV-1 particles were detected by supernatant reverse transcriptase activity and transmission electron microscopy 40 to 60 days after infection. Viral particles were predominantly observed assembling and accumulating from within intracellular membranes, while phenotypically the cells were observed to have differentiated into CD4+ monocytes. These studies have important implications in understanding the pathogenesis of HIV-1 as well as the possible cause of certain of the observed hematologic abnormalities in HIV-1 infection. They also indicate that the bone marrow may serve as a potentially important reservoir of HIV-1 in the body.

In vitro infection of human monocytes with human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV).

We explored the possibility that normal human monocytes can be infected with the retrovirus human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). The T4 antigen, believed to be the receptor for HTLV-III/LAV binding to CD4 cells, is found on monocytes at low levels. Anti-T4A, which recognizes an epitope on the T4 molecule, inhibits viral binding to monocytes, and virus inhibits anti-T4A binding, although inhibition in both cases is not total. Virus particles were detected in HTLV-III/LAV-pulsed monocytes by electron microscopy as early as 10 min and for up to 3 days after inoculation, although budding virus was not observed. Monocytes were exposed to virus, were washed, and were cultured. Monocyte cultures were monitored by conventional assays for virus replication: immunofluorescence detection of cytoplasmic virus, supernatant reverse transcriptase activity, and supernatant virus antigen. These assays were either negative or at the lower limits of positivity. However, the amount of infectious virus was shown to increase over time in monocyte cultures by harvesting monocytes or their culture supernatants and titrating them into assay cultures containing stimulated T cells. Virus recovery from monocytes and virus recovery from T cells differed both quantitatively and qualitatively. Recovery from T cells and T cell supernatants peaked at 3 to 6 days and declined thereafter. Recovery from monocytes and monocyte supernatants increased over time in culture and never attained the levels of T cell cultures. Taken together, these studies indicate that HTLV-III/LAV binds to monocytes via the T4 molecule and enters the cells. Infectious virus is retained and increases with time in infected monocyte cultures. Both viral binding and infection are at low levels compared with levels in T cells. Unlike the usual infection of T cells characterized by high level virus replication with cell depletion, the infection appears to be persistent in monocytes.

Amphotropic Retrovirus Vector Transfer of the v-ras Oncogene to Human Hematopoietic and Stromal Cells in Continuous Bone Marrow Cultures

Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am-MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.

Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures

Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am- MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.

Role of natural prostaglandins in the control of murine mammary tumor virus expression.

The regulation of murine mammary tumor virus (MuMTV) production by mammotropic hormones, hormonomimetic substances, and cyclic nucleotides was investigated. The virus produced in control and treated mammary tumor cell cultures was quantitated by measuring the supernatant reverse transcriptase activity in exogenous reaction using poly(rC).oligo(dG) as template-primer. Two days after exposure, the synthetic glucocorticoid, dexamethasone (DXMT), increased spontaneous MuMTV production at optimal concentration (0.1 mumol) up to ten times. Dibutyryl derivative of cyclic AMP had no effect on spontaneous MuMTV production, whereas the drug potentiated suboptimal concentrations of the glucocorticoid. Natural prostaglandins, potent agonists of adenylate cyclase catalyzing intracellular synthesis of cyclic AMP, enhanced both basal (up to five times) and DXMT-stimulated (up to 1.6 times) MuMTV replication. The MuMTV-stimulating activity of prostaglandins decreased in the order of PGA1 greater than PGE1 greater than PGB1 greater than PGF2 alpha. Prostaglandins can be replaced partially by norepinephrine and isoproterenol by enhancing the DXMT-mediated MuMTV stimulation, whereas these drugs remained without effect on spontaneous MuMTV production. Theophylline, an antagonist of cAMP-phosphodiesterase converting cAMP to AMP, enhanced the virus-stimulating activity of DXMT as well as of prostaglandins. The enhancement of MuMTV production by adenylate cyclase agonists do not correlate absolutely with the estimates of intracellular cAMP levels, since the highest amounts of cAMP has been repeatedly observed in cells treated with PGE1 and norepinephrine. The results indicate that besides hormones, other hormone-like substances and cyclic nucleotides may be involved in the complex mechanism of hormone-regulated MuMTV genome expression.

Mouse mammary tumor virus production stimulated by hormones and polyamines in cells grown in semi‐synthetic in vitro conditions

Hormonal regulation of mouse mammary tumor virus (MMTV) synthesis was studied in the CCL-51-SF cell subline derived from the Sykes' mammary tumor cell line CCL-51 and adapted to grow in semi-synthetic in vitro conditions. The virus was quantitated by measuring the supernatant reverse transcriptase activity in exogenous reaction using poly (rA)-oligo (dT) and poly (rC)-oligo (dG) as template/primers. The cells produced a low but significant amount of virus in the absence of any hormones and serum proteins. The synthetic glucocorticoid dexamethasone increased production considerably, up to 100-fold. Pretreatment of CCL-51-SF cells with serum or 5-bromodeoxyuridine, BrdUrd, partly reduced the stimulation by dexamethasone of MMTV production. Insulin and prolactin alone or in combination had no stimulating effect on spontaneous MMTV synthesis and cell growth. Prolactin, and more efficiently the prolactin-insulin combination, enhanced the MMTV production stumulated with dexamethasone. Insulin alone remained without effect. The polyamine spermidine, but not spermine, increased the MMTV production over the control by a factor of 2. Polyamines did not influence cell replication at the concentrations used.