The Increased RNase H Activity Affects HIV-1 Replication And The Efficacy Of Polymerase Inhibitors & The Anti-HIV-1 Mechanism Study Of A Salvia Miltiorrhiza Sourced Natural Product NPB524

As a critical anti-HIV-1 drug target,reverse transcriptase(RT)plays three enzyme activities,including RNA-dependent DNA polymerase(RDDP)activity,DNA-dependent DNA polymerase(DDDP)activity,and RNase H activity.The reverse transcription proceeds with the spatial and temporal coordination of the three activities.However,the twelve marketed reverse transcriptase inhibitors,including 7 Nucleoside Reverse Transcriptase Inhibitors(NRTIs)and 5 non-Nucleoside Reverse Transcriptase Inhibitors(NNRTIs),are all polymerase inhibitors.Currently,there is no drug targeting RNase H,which provides us an opportunity to focus on RNase H to study its potential as a target for AIDS.The HIV-1 reverse transcription is catalyzed by RT,which proceeds by DNA polymerase and RNase H coordination and balance.The DNA polymerase inhibitors,NRTIs and NNRTIs,are recommended by the WHO as the first line regiments for AIDS,which indicates that the inhibition of RT polymerase activity is indispensable for AIDS therapy.Here,two hypotheses were raised and proved in this thesis:?As polymerase inhibitor is essential,if RNase H inhibitor is used simultaneously,the balance of the two enzymatic acitivities may be rebuilt,which may result in a poor therapeutic effect.?With the inhibition of polymerase activity,an increased RNase H activity may aggravate the imbalance between these two enzymatic activities,which may bring a better impact on anti-HIV-1 replication.In this research,we used a phenotypic mixing strategy to increase the relative RNase H activity in HIV-1 virion to study the effect of the increased RNase H activity on HIV-1 replication and on the efficacy of NRTIs and NNRTIs.The results showed that the increased RNase H activity inhibited HIV-1 replication and increased the sensitivity of HIV-1RT-K103N and HIV-1IRT-U181C to NVP and the sensitivity of HIV-1RT-D67N/K70R/T215F to AZT.In this study,it is also found that when 50%of the wild-type RT was introduced into HIV-1RT-M184V virions,the sensitivity of virions to 3TC was as the same as wild-type HIV-1,ie,the drug resistance was eliminated,this is consistent with the clinical observation by K.Van Laethem et al.,which suggests that this phenotype mixing virus model can be used to study the relationship between the proportion of mutant viruses and the emergence of drug resistance.Our result also addressed whether the RT in the cytoplasm can enter the viral capsid core to regulate the reverse transcription process.The results showed that the wild-type RT in the cytoplasm did not increase the sensitivity of HIV-1RT-Mi84V to 3TC,suggesting that the RT in the cytoplasm cannot enter the viral capsid core to regulate the reverse transcription process.Our results suggest that the coordination between polymerase and RNase H activities is important for HIV-1 reverse transcription.Firstly,the increased RNase H activity inhibits HIV-1 replication by breaking the balance between the two enzyme activities.Secondly,the increased RNase H activity can improve the efficacy of polymerase inhibitors on common HIV-1 mutants.Thirdly,providing exogenous RNase H in the cytoplasm cannot increase the RNase H activity,suggesting that searching for RNase H activator could be the direct and effective way to further break the balance when using NRTIs or NNRTIs.Traditional Chinese medicine sourced natural products are an important resource of antiviral drugs.Our previous study found that NPB524,the lipid-soluble component extracted from the cell culture of Salvia miltiorrhiza,exhibited a good anti-HIV-1 activity with IC50 value of 30 nM.The present study investigated its anti-HIV-1 mechanism and found that NPB524 is an HIV-1 transcription inhibitor.In summary,this thesis consists of two research projects.In the first project,it focused on the effect of the imbalance between RT polymerase activity and RNase H activity on HIV-1 replication and the efficacy of polymerase inhibitors.It was shown that the relatively increased RNase H activity could inhibit HIV-1 replication and improve the sensitivity of the common HIV-1 mutants to polymerase inhibitors.This research promises a horizon for anti-HIV-1 drug development targeting RNase H.In the second project,a natural product NPB524 was discovered as an HIV-1 transcription inhibitor,which provides new structural basis and mechanism of action for the development of anti-HIV drugs.