AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP

Abstract

Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher KM-values for polymerization but no change in KD-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.

Molecular biology of foamy viruses

“…At the biochemical level, it was found that SFVmac res excises misincorporated AZT from the growing chain in the presence of ATP similar to what has been observed for HIV-1 [78]. Further biochemical studies should reveal whether the mode of action of this resistant enzyme could indeed mimic the resistant phenotype of HIV-1.…”

“…Curiously, the PR-RT molecules of PFV and the related simian foamy virus from macaque (SFVmac) have a monomer state in solution, and only under very high salt conditions, activity can be measured [76][77][78][79]. Activity of both enzymes, however, essentially requires dimerization.…”

Regulation of Foamy Virus Protease Activity by Viral RNA: a Novel and Unique Mechanism among Retroviruses

“…SFVmac and PFV PR-RTs and the GB1 (immunoglobulin binding domain B1 of streptococcal protein G)-green fluorescent protein (GFP) PR substrate were purified as described previously (10,11,13).…”

“…1). PR activity in vitro was observed using a model substrate and recombinant monomeric PR-RTs of PFV and of SFVmac purified from Escherichia coli (10,11). The model substrate GB1-GFP contains the natural RT-IN PR cleavage site in the Pol precursor between the GB1 (immunoglobulin binding domain B1 of streptococcal protein G) and GFP domains (13).…”

Foamy Virus Gag p71-p68 Cleavage Is Required for Template Switch of the Reverse Transcriptase

“…First we wanted to determine whether the free p3 influences RT activity. Therefore, in vitro RT activity assays using protease-reverse transcriptase (PR-RT) either from prototype or simian FV were performed (13) in the absence of p3 (RT activity, 23.7 Ϯ 1.1 U/g) or in the presence of equimolar concentrations (12 nM) of PR-RT and p3 (PFV p3 peptide, 5/6-fluorescein-AGDSRAVNTV TQSATSSTDESSSAVTAASGGDQRD) (RT activity, 26.6 Ϯ 1.7 U/g) or in a 6-fold excess of p3 peptide (RT activity, 25.3 Ϯ 1.4 U/g). These results indicate that free p3 has no impact on RT activity, leading us to the hypothesis that Gag p71 might inhibit the RT reaction.…”