Thermostable HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase
Résumé
Wild-type human immunodeficiency virus type 1 (HIV-1) group O reverse transcriptase (RT) shows increased thermostability in comparison with HIV-1 group M subtype B RT and murine leukemia virus (MLV) RT. However, its utility in the amplification of RNA targets is limited by the reduced accuracy of lentiviral RTs versus oncoretroviral RTs (i.e., MLV RT). The effects of mutations K65R, R78A and K65R/V75I on the fidelity of HIV-1 group O RTs were studied by using gel-based and M13mp2 lacZ forward mutation fidelity assays. Forward mutation assays demonstrated that mutant RTs K65R, R78A and K65R/V75I showed <9-fold increased accuracy in comparison with the wild-type enzyme, and were about two times more faithful than the MLV RT. Compared with MLV RT, all tested HIV-1 group O RT variants showed decreased frameshift fidelity. However, K65R RT showed a higher tendency to introduce one-nucleotide deletions in comparison with other HIV-1 group O RT variants. R78A had a destabilizing effect on the RT, either in the presence or absence of V75I. At temperatures above 52ºC, K65R, K65R/V75I and R78A retained similar levels of DNA polymerase activity to the wild-type HIV-1 group O RT but were more efficient than HIV-1 group M subtype B and MLV RTs. K65R and K65R/V75I RTs showed decreased misinsertion and mispair extension fidelity in comparison with the wild-type enzyme for most base pairs studied. These assays revealed that nucleotide selection is mainly governed by kpol in the case of K65R, while both kpol and Kd affect nucleotide discrimination in the case of K65R/V75I.
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