Rauscher Murine Leukemia Virus Research Articles

Chemoprevention of retroviral infection: success is determined by virus inoculum strength and cellular immunity.

We demonstrated earlier that post-exposure prophylaxis with 3'-azido-3'-deoxythymidine (AZT, zidovudine) or with AZT + interferon-alpha (IFN-alpha) prevented viremia and disease in BALB/c mice inoculated with Rauscher murine leukemia virus (RLV). After the 20-day treatment course, most animals were resistant to rechallenge with live virus. Adoptive transfer of T cells from such resistant but not from normal mice into naive recipients provided full protection against virus challenge. From these experiments, we concluded that post-exposure chemoprophylaxis restricted virus replication and allowed the animals to form protective, long-lasting cellular immune responses. Here, the role for cellular immunity during antiviral chemoprophylaxis was tested by comparing treatment success in normal BALB/c mice and in their nude, athymic counterparts. Both were inoculated with equal doses of RLV (10(4) plaque-forming units, pfu). Single-agent AZT or combination therapy with AZT + IFN-alpha, started before or after RLV inoculation, prevented viremia in all normal but not in most nude mice. A significant number of nude mice were completely protected by chemoprevention only when given a 10 times lower virus dose. When normal mice were injected with a 10 times higher virus dose (10(5) pfu), complete protection by chemoprevention was lost. These results demonstrate that the success of chemoprevention depends critically on the virus inoculum. The differential success of chemoprevention in normal and T-cell-deficient mice implies that effective cellular immunity plays an important role in protecting virus-exposed animals against viremia and disease.

Size Exclusion Removal of Model Mammalian Viruses Using a Unique Membrane System, Part I: Membrane Qualification

Abstract. We have previously described a new class of composite membrane that has the capability to efficiently remove particles, including viruses, from protein solution. The qualification of this membrane requires that it reproducibly and predictably remove model mammalian viruses. Using the Viresolve/70™ membrane, the mammalian viruses polio, Simian virus-40, Sindbis, reovirus type 3 and Rauscher murine leukemia virus are shown to be reproducibly removed via a sieving mechanism. Mammalian virus retention increases constantly with virus diameter independent of virus class or type, increasing from 3·5 logs with polio virus to greater than 6·8 logs with murine leukemia virus. Consistent with a sieving mechanism, mammalian virus retention with the Viresolve/70™ membrane is independent of virus concentration. These results are shown both in the presence and absence of protein in solution. The presence of protein in solution is shown to increase the virus retention coefficient of each virus above that measured in phosphate buffered saline. The model virus retention is shown to be well predicted by hard particle retention reported previously for this membrane. In addition, the hard particle retention is shown to predict the worse case performance expected of the membrane in the presence of protein.

TAP2-defective RMA-S cells present Sendai virus antigen to cytotoxic T lymphocytes.

The murine antigen-processing-defective mutant cell line RMA-S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)-defective RMA-S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA-S cells required 2-3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA-S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild-type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA-S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings confirmed leakiness of antigen presentation in RMA-S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I-mediated antigen presentation.

8 Two new (newborn) mouse models - harvey murine sarcoma virus (MSV-Har) and rauscher murine leukaemia virus (RLV), for anti retroviral drug testing: J.J. Harvey, M. Botcherby. Antigen Presentation Research Group, MRC Clinical Research Centre, Northwick Park, Harrow, U.K.

A structural protein with a molecular weight of approximately 12,000 has been purified from Gross murine oncornavirus by phosphocellulose column chromatography. The protein was distinct from all other of the principle Gross virus components and did not react with antisera prepared against other purified Gross virus proteins with molecular weights of about 30,000, 17,000, 14,000 and 10,000. The immunologic properties of the protein were analyzed by competition radioimmunoassay. The antigenic determinants were chiefly subgroup-specific with a close homology to the analogous protein of AKR, Kirsten, or BALB/c xenotropic viruses but not to proteins of Friend, Rauscher, Moloney, NIH Swiss xenotropic, or NZB xenotropic viruses. Group-specific determinants cross-reactive with the analogous protein of these latter viruses were present but were minor components of the total antigenic specificities of the protein. Weak interspecies cross-reactivity was demonstrated with the Theilen feline virus, but very little, if any, with different primate oncornaviruses. The identity of the related, cross-reactive proteins of other murine viruses was analyzed both by competition radioimmunoassay and immunoprecipitation. It was identified as the 15,000 MW component of Rauscher and Friend viruses, and a 12,000 MW component of Moloney and BALB xenotropic viruses. The close similarity in the properties of the p15 of Rauscher murine virus and this p12 of Gross murine virus establish these as the homologous subgroup-specific proteins of the FMR and Gross subgroups of viruses.

75 Activity of BRL47923 and its oral prodrug, SB203657A against a rauscher murine leukaemia virus infection in mice: R.M.Perkins 1, S.Barney 1, R.Wittrock 1, P.H.Clark 1, R.Levin 1, D.M.Lambert 1, S.R. Petteway 1, H.Serafinowska 2, S.Bailey 2, S.M.Jackson 2, M.R.Harnden 2, R.J.Ashton 2, D.Sutton 2, J.J.Harvey 3, A.G.Brown 2. SmithKline Beecham Pharmaceuticals, King of Prussia, USA 1, and Epsom, UK

75 Activity of BRL47923 and its oral prodrug, SB203657A against a rauscher murine leukaemia virus infection in mice: R.M.Perkins 1, S.Barney 1, R.Wittrock 1, P.H.Clark 1, R.Levin 1, D.M.Lambert 1, S.R. Petteway 1, H.Serafinowska 2, S.Bailey 2, S.M.Jackson 2, M.R.Harnden 2, R.J.Ashton 2, D.Sutton 2, J.J.Harvey 3, A.G.Brown 2. SmithKline Beecham Pharmaceuticals, King of Prussia, USA 1, and Epsom, UK

Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors.

Sulfonic Acid Dyes

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.

An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus

A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Comparison of rauscher and LP-BM5 MuLV for assessing anti-AIDS drugs In Vitro: M. G. Hollingshead, L. B. Allen, R. W. Buckheit, Jr. and L. Westbrook, Southern Research Institute, Birmingham, AL, USA

Comparison of rauscher and LP-BM5 MuLV for assessing anti-AIDS drugs In Vitro: M. G. Hollingshead, L. B. Allen, R. W. Buckheit, Jr. and L. Westbrook, Southern Research Institute, Birmingham, AL, USA

Antiviral Activity and Pharmacokinetics of Liposome-Encapsulated Phosphonoformate in Rauscher Murine Leukemia Virus-Infected Mice

AbstractLiposomal delivery of phosphonoformate (PF), an antiviral agent with poor membrane permeability, was evaluated using a passive targeting strategy to treat Rauscher murine leukemia virus (RMLV) infection. Pharmacokinetic studies in RMLV infected mice using the nonmetabolized [14C]PF reveal that nonencapsulated PF is rapidly excreted with an approximate clearance of 18.8 g blood/hour, while liposome (egg phos-phatidylcholine/egg phosphatidylglycerol/cholesterol: 9:1:8)-encapsulated PF has an approximate clearance of 0.61–0.96 g blood/hr. When administered in the liposome encapsulated form, 50–80% of the dose is found in the liver, spleen, and blood at 3 hr postinjection. The area under the drug level-time curve (AUC) in the liver and spleen following a single dose of encapsulated PF is 1.3–1.7 times higher than the AUC of a 25-fold larger dose of nonencapsulated PF. Biodistribution studies of 125I-labeled liposomes or encapsulated [14C]PF in vivo show that the specific uptake of liposomes and entrap...

Cellular metabolism and enzymatic phosphorylation of 9-(2-phosphonylmethoxyethyl) guanine (PMEG), a potent antiviral agent.

PMEG (9-(2-Phosphonylmethoxyethyl)guanine) is a potent, broad spectrum antiviral agent in the nucleoside phosphonate class (Figure 1). In vitro, PMEG is active against HSV-1, HSV-2, VZV, HCMV and Rauscher murine leukemia virus with IC50s (the concentrations of drug required to reduce the plaque formation by 50%) less than 1 ug/ml (1,2,3). Similar antiviral activity was demonstrated with TK-minus mutants of HSV-1. However, the toxic effect exerted by PMEG on CEM and Vero cells in culture (TC50 5-10 μg/ml, the concentrations of drug affecting the viability of uninfected cells by 50%) limits the utility of PMEG as an antiviral agent. On the other hand, PMEG has antitumor activity (4) against intraperitoneal P388 leukemia and subcutaneously implanted B16 melanoma in mice. PMEG, also supresses human condylomas from papillomavirus (HPV-11) infected human foreskin in transplanted mice (5). We have investigated the cellular metabolism of PMEG and the viral and cellular target enzymes involved in the mode of action to provide rationale for the low antiviral selectivity and to assist with future design of more selective analogs of PMEG.

Nonnucleoside reverse transcriptase inhibitors that potently and specifically block human immunodeficiency virus type 1 replication.

Certain bis(heteroaryl)piperazines (BHAPs) are potent inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) at concentrations lower by 2-4 orders of magnitude than that which inhibits normal cellular DNA polymerase activity. Combination of a BHAP with nucleoside analog HIV-1 RT inhibitors suggested that together these compounds inhibited RT synergistically. In three human lymphocytic cell systems using several laboratory and clinical HIV-1 isolates, the BHAPs blocked HIV-1 replication with potencies nearly identical to those of 3'-azido-2',3'-dideoxythymidine or 2',3'-dideoxyadenosine; in primary cultures of human peripheral blood mononuclear cells, concentrations of these antiviral agents were lower by at least 3-4 orders of magnitude than cytotoxic levels. The BHAPs do not inhibit replication of HIV-2, the simian or feline immunodeficiency virus, or Rauscher murine leukemia virus in culture. Evaluation of a BHAP in HIV-1-infected SCID-hu mice (severe combined immunodeficient mice implanted with human fetal lymph node) showed that the compound could block HIV-1 replication in vivo. The BHAPs are readily obtained synthetically and have been extensively characterized in preclinical evaluations. These compounds hold promise for the treatment of HIV-1 infection.

Protease Inhibitors as Potential Therapeutic Agents for AIDS

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

Failure of Castanospermine to Reduce Spleen or Serum Virus Titres in Rauscher Murine Leukaemia Virus-Infected Mice

The effects of orally administered castanospermine (CS) and azidothymidine (AZT) on spleen and serum virus titres and on spleen weights of mice inoculated with Rauscher murine leukaemia virus (R MuLV) were evaluated. Virus titres of samples from CS-treated mice did not differ appreciably from saline-treated mice, despite a significant inhibition of splenomegaly. In contrast, AZT treatment resulted in significant inhibition of splenomegaly and a reduction of virus titres.

Evaluation of antiretroviral drugs against Rauscher MuLV by ELISA

A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Heme inhibits human immunodeficiency virus 1 replication in cell cultures and enhances the antiviral effect of zidovudine.

The effects of heme alone and heme administered together with 3'-azido-3'-deoxythymidine (AZT) on human immunodeficiency virus replication in human peripheral blood lymphocytes and in the H9 cell line were studied. Heme enhanced the antiviral action of AZT against both drug-resistant and drug-sensitive viral strains; the heme effect was more pronounced against the latter. Moreover, heme alone displayed a significant ability to inhibit viral replication in concentrations markedly smaller than those required to inhibit the reverse transcriptase of Rauscher murine leukemia virus. The results of this study extend the range of pharmacological actions that metalloporphyrins exert in biological systems and suggest that further study of the interactions of the natural compound heme and human immunodeficiency virus chemotherapeutic agents such as AZT may be useful.

Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells

We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge.

Unsaturated and carbocyclic nucleoside analogs: synthesis, antitumor and antiviral activity

A series of unsaturated analogues of nucleosides were prepared and their cytotoxic, antitumor, and antiviral activities were investigated. Alkylation of cytosine with (E)-1,4-dichloro-2-butene gave chloro derivative 2f, which was hydrolyzed to alcohol 2h. Cytosine, adenine, 2-amino-6-chloropurine, thymine, and (Z)-1,4-chloro-2-butene gave compounds 4c-f, which, after hydrolysis, afforded alcohols 4a, 4b, 4g, and 4h. Alkenes 4d and 4e were cyclized to heterocycles 12 and 13. Alkylation of 2,6-diaminopurine with 1,4-dichloro-2-butyne led to chloro derivative 6a, which was hydrolyzed to alcohol 6b. Allenic isomerization of 6b gave compound 5c. Chloro derivatives 2e-g, 4c-f, 5d, and 6c-e as well as pyrimidine oxacyclopentenes 9c and 9d are slow-acting inhibitors of murine leukemia L1210 of IC50 10-100 microM. The most active were analogues 4c, 4d, 4e, and 6e (IC50 10-20 microM). The corresponding hydroxy derivatives were less active of inactive. Inhibition of macromolecular synthesis with compounds 4c, 4d, 6e, 9c, and 9d follows the order: DNA greater than RNA greater than or equal to protein. Cytotoxic effects of 4c, 6e, and 9d are not reversed with any of the four basic ribonucleosides or 2'-deoxyribonucleosides. Inhibitory activity of cytosine derivative 9c is reversed with uridine and 2'-deoxyuridine but not with the corresponding cytosine nucleosides. Zone assays in several tumor cell lines show that active compounds are cytotoxic agents with little selectivity for tumor cells. Analogue 6c showed 16.7% ILS in leukemia P388/o implanted ip in mice at 510 and 1020 mg/kg, respectively. Cytallene (5b) and 6'beta-hydroxyaristeromycin (10) exhibited significant activity against Friend and Rauscher murine leukemia viruses. The rest of the hydroxy derivatives, with the exception of 4a, were moderately effective or inactive as antiviral agents. None of the chloro derivatives or oxacyclopentenes exhibited an antiviral effect at noncytotoxic concentrations. Z-Olefin 4b and 2-aminoadenallene (5c) are substrates for adenosine deaminase.

Castanospermine vs. Its 6-O-Butanoyl Analog

Inhibitors of glycoprotein processing, such as castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), have been shown previously to inhibit human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured human cells. In prior experiments, we have tested the toxicity and antiviral efficacy of castanospermine in mice infected with the Rauscher murine leukemia virus (RLV). When compared with 3'-azido-3'-deoxythymidine (AZT, zidovudine), castanospermine was less effective and more toxic. Since the 6-O-butanoyl analog of castanospermine was previously found to have a more favorable activity profile than the parent compound against HIV-1 in cultured cells, we compared the antiviral efficacy of both compounds in parallel in vitro and in vivo in the RLV system. Plaque formation in the XC assay was inhibited with a 50% inhibitory concentration (IC50) of 2.4 microM for the 6-O-butanoyl analog of castanospermine, as compared to 9 microM for castanospermine. For both compounds, concentrations resulting in significant cytotoxicity were about ten times higher. Both compounds significantly decreased HIV-1 env-induced syncytium formation in a novel in vitro assay. In RLV-exposed mice, the 6-O-butanoyl analog showed no advantage over the parent compound: both curves for toxicity as well as antiviral efficacy were super-imposable. We conclude that the 6-O-butanoyl analog of castanospermine as well as castanospermine itself are active antiviral agents in mice and that prolonged oral administration is tolerable. However, in comparison to AZT, their antiviral activity profiles are less favorable.

N-Carboxymethylchitosan-N,O-sulfate as an anti-HIV-1 agent

N-carboxymethylchitosan-N-O-sulfate (NCMCS), a sulfated polysaccharide derivative of chitin, inhibited the propagation of the human immunodeficiency virus type 1 (HIV-1) in human CD4+ cells and that of Rauscher murine leukemia virus (RLV) in murine fibroblasts. A dose-dependent inhibition of both viruses was observed without significant cytotoxicity. NCMCS blocked the binding of HIV-1 to human CD4+ target cells and competitively inhibited HIV-1 reverse transcriptase. Thus, NCMCS may prevent HIV-1 infection by inhibiting viral adsorption to the CD4 receptor and reverse transcription of the viral genome.