Effects Of Elevated Ribonuclease H Activity On Pharmacodynamics Of HIV-1 Reverse Transcriptase Inhibitors

Reverse transcription is a critical process for HIV-1 replication. Double-stranded DNA is synthesized with the viral genome RNA as the template. Reverse transcriptase (RT) catalyzes this process. RT possesses three enzymatic activities:RNA dependent DNA polymerase (RDDP) activity, DNA dependent DNA polymerase (DDDP) activity and Ribonuclease H (RNase H) activity. Since reverse transcription is a unique process, RT has been an important target for antiretroviral drug developemnt and drugs targeting RT are AIDS patients’essential treatment. There are nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Both NRTIs and NNRTIs act on p66 palm domain, which lead to DNA polymerase activity reduction.Drugs resistance, a major factor causes clinical treatment failure, appears after a period time of treatments due to no proof reading in HIV replication. However, there is still no drug functioning on HIV RT RNase H. As one of the three catalytical enzamatic activities, RNase H might become a drug target for blocking reverse transcription process, which may benefit all patients carrying NNRTIs-or NRTIs-resistant HIV-1.Reverse transcription is a complicated process which requires the balance between RT polymerase and RNase H acitivities. RT DNA polymerase activity inhibition is essential for AIDS therapy. According to the global guideline for antiretroviral therapy, combined two NRTIs as regimens are recommended for AIDS first-line therapy. Here, we bring forth a hypothesis:whether increasing RNase H activity to further breaking the balance between DNA polymerase and RNase H activities would give a better RT inhibition? In this study, we investigated NRTIs/NNRTIs activities on HIV-1 with higher RNase H activity.We managed two means to improve RNase H activity:looking for RNase H activators and RT with high RNase H activity. As the result, there is no RNase H activators either reported or obtained by our screen. Therefore, we steped on RNase H mutations isolated from AIDS patients. There were 6 mutants were selected, they share the follow characteristics:① Since they were isolated from patients, their DNA polymerase and RNase H activities are functional. ②There was no drug resistance appeared in patients, which indicated that there was little change of the binding affinity of NRTIs/NNRTIs to RTs p66 subunit, ie. the mutant RTs’DNA polymerase activity had no change or changed slightly. ③ Mutations in RT RNase H might affect their RTs RNase H activity, possibilly increasing.The six mutant RTs, RT5326, RT5329, RT5512, RT5331, RT6316, and RT6853 were subcloned and expressed in p6HRT-Pr with wild type RT as backbone. Two plasmids, pDMI and p6HRT-Pr, were transformed into E.coli M15 strain to overexpress wild type and mutant RTs. Then the RTs were purified by Ni-NTA affinity chromatography. RNase H and RDDP activities were tested by fluorescence assay and ELISA, respectively. As the result, only RT5329 RNase H activity increased 20% and RDDP activity declined of 6% comparing to RTwt, whereas, the other five mutant RTs RNase H activities declined or changed slightly. Hence, the RT5329, RTN460D/T468S/K530R/A554T/V559I, was used for further study. RT5329 gene was synthesized and subcloned into HIV-1pNL4-3. Recombinant VSVG/HIV-15329 and VSVG/HIV-1wt were used as modeled viruses to compare NRTIs/NNRTIs activities on HIV-1 with higher RNase H. The results showed that the inhibitory acitivities of NRTIs/NNRTIs on VSVG/HIV-15329is similar to that on VSVG/HIV-1wt, which indicates that little affect on NRTIs/NNRTIs pharmacodynamics as RNase H acitivity increasing 20%.