Murine leukemia virus as a model for the development of anti-retroviral drugs

This thesis describes work on exploring the feasibility of using a murine system to assist in anti-human immunodeficiency virus drug development at both the designing and evaluation phases. Specifically, the research focused on murine leukemia virus (MuLV) and inbred mice.; In chapter 2, the reverse transcriptase (RT) of Moloney MuLV (M-MuLV) was purified, characterized and compared to HIV-1 RT. The enzymatic activity of the RNase H domain of M-MuLV RT was separated and studied extensively using the Scintillant Proximity Assay, an in situ gel assay and a specific activity gel assay. The M-MuLV RNase H activity was different from its HIV-1 counterpart in that it could be isolated as a separate domain with non-specific ribonuclease activity, but its specificity of cleavage could not be reconstituted by adding back the DNA polymerase domain in vitro. Furthermore, results from the specific activity gel assay and affinity column chromatography suggested the formation of heterodimers between the wild-type M-MuLV RT and its DNA polymerase domain, which was similar to the HIV-1 enzyme.; In chapter 3, a quantitative infectivity assay for M-MuLV (the XC plaque assay) was used to evaluate various compounds for their antiviral activity on M-MuLV in vitro. Among the compounds tested, AZT and AG337 (an inhibitor of the cellular enzyme thymidylate synthase) were the most potent, while ddI, ddC and two potent HIV-1 protease inhibitors showed little or no antiviral activity. When tested in combinations, AZT and AG337 appeared to be mildly synergistic, with a calculated interaction parameter of 0.36 +/{dollar}-{dollar} 0.32 (positive values indicate synergism and negative values indicate antagonism).; In chapter 4, the combination of AZT and AG337 was further tested in vivo. Balb/c mice were infected by the Rauscher strain of MuLV via intravenous injection and then treated with combinations of various dosages of AZT and/or AG337. The therapeutic effect was evaluated by measuring both the inhibition of splenomegaly and the reduction of infectious cells in the spleen. Although potential mild synergy was observed when the lowest dosage of AZT (0.0005 mg/ml in drinking water) was used in combination with 50 mg/kg/day AG337, further experiments using a larger number of animals need to be performed before the therapeutic effect of this combination can be properly assessed.; In conclusion, this murine/MuLV model could be useful for designing compounds that block common retroviral functions such as reverse transcription, integration and protease. This model can also be used to evaluate the specificity as well as antiviral activity of potential anti-HIV compounds both in vitro and in vivo. On the other hand, if a compound appears to be inactive in this model, further tests in other animal models may be advisable before eliminating it as candidate for development.