Phenotypic effects and predictions of HIV-1 subtype C reverse transcriptase C-terminal domain mutations on reverse transcriptase inhibitors.

Abstract

Antiretroviral drug therapy has been shown to reduce the death of HIV-1 infected individuals. However, the emergence of HIV-1 drug resistance has hindered the success of HIV-1 treatment. Genotyping tests mainly concentrate on the polymerase domain of HIV-1 RT leaving the rest understudied. Recently, data from HIV-1 C suggested that drug resistance could be caused by mutations in the connection and RNase H domains either alone or in combination with mutations in the polymerase region. Here, the phenotypic effects of the specific RNase H domain mutations in HIV-1 subtype C on RTIs were investigated. The predictions of HIV-1 subtype C reverse transcriptase C-terminal domain mutations on reverse transcriptase inhibitors were also investigated. Material and methods Viral RNA was extracted from 500μl of plasma using the Viral RNA extraction kit (Qiagen,Germany) according to the manafacturer’s instructions, and stored at -80oC until utilisation. The RNA was amplified using the Superscript III One-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, Life Technologies Corporation, Carlsbad, CA, California). The HIV-1 RT amplicon was cloned into a TOPO vector using the TOPO TA cloning kit (Invitrogen, Life Technologies). Viral mutants were constructed using site-directed mutagenesis, introducing D67N in the polymerase domain and E529D, L517I, T470S, and T470P mutations in the RNase H domain. Viral replication capacity and drug susceptibilities were determined using the single cycle luciferase assay in TZM-bl cells. To identify the HIV-1 connection domain (CN) mutations associated with drug resistance, HIV-1 subtype C sequences were downloaded from the Los Alamos and Stanford HIV- drug resistance xvi databases from drug naïve and treated-experienced patients. The presence of connection domain (CN) mutations were identified using REGA HIV-1 subtyping tools (Universiteit Leuven, Belgium). Bayesian Network (BN) analysis (B-course) was used to determine the association of connection domain (CN) mutations (condon 320-440) with other TAMS. The effect of RNase H domain mutations on the structure of reverse transcriptase was determined using Swiss Model and viewed in Chimera. Results The replication capacities of T470S, T470P, L517I, E529D RNase H domain mutations were lower than the HIV-1 subtype C wild type in the absence of drugs. The D67N mutation alone had a lower replication capacity compared with the wild-type. Combination of L517I and D67N showed a further decrease in replication capacity compared to the wild type in the absence of drugs. E529D mutation replication capacity was assessed in TZM-bl cell line in both HIV-1 subtype B and C. Although not statistically significant, both competent subtype B and C E529D mutant had a decreased growth and infectivity rate compared to their respective wildtypes. The RNase H domain mutation T470S showed a moderate level of resistance to NVP (10.2X), ETR (8.75X), d4T (5X) and no resistance to AZT and EFV. Interestingly, T470P showed a moderate level of resistance to NVP (6X) and no resistance to ETR and d4T, as well as EFV. It did however show a 5-fold change to AZT when compared to the wild-type virus. As expected, the thymidine analog mutation, D67N, showed a high level of resistance to AZT (103.3X), moderate level of resistance to d4T (6.2X) and low level of resistance to ETR (3.2X) and no resistance to NVP and EFV. The RNase H domain L517I mutation showed moderate level of resistance to AZT (5.2X), d4T (6.0X) and NVP (10.79X), and low level of resistance to ETR (3.50X). L517I mutation caused hypersusceptibility to EFV. The combination of RNase H and TAM (L517I+D67N) showed high xvii level of resistance to AZT (157.3X), moderate level of resistance to NVP (11.3X), low level of resistance to d4T (3.9X) and no resistance to ETR and EFV. Subtype C E529D mutation conferred up to 2-fold stavudine (d4T), 3-fold zidovudine (AZT) and nevirapine (NVP) resistance, respectively. These findings demonstrate that RNase H mutation E529D can confer mild resistance to nucleotide (AZT and d4T) and non-nucleotide (NVP) reverse transcriptase inhibitors. New connection domain mutations identified were: D324G/N/P, T338S, I341F/L/V, M357R, E370D, M377T/L, A376S, I434M/L, A437V/I. E370D and A437V/I were directly associated with treatment in the BN, while N348I was only indirectly associated with treatment. Discussion and Conclusions Overall, the RNase H domain mutations impaired replication capacity in the absence of drugs, suggesting that they are acquired at a fitness cost (as withmost drug resistance mutations). While T470S decreased drug susceptibility to ETR, it was shown to be hypersusceptible to EFV. Interestingly, T470S is very common in subtype C RTI treated patients and could indicate that a switch to the newer NNRTI might not be as beneficial as expected. The phenotypic data also suggests that the resistance pathways for T470S and T470P could be different; however further studies are required to investigate their mechanism of resistance. The L517I mutation alone only minimally decreased drug susceptibility to both NRTI and NNRTIs. However it futher decreased drug susceptibility to the NRTIs when in combination with D67N, compared to D67N alone. D67N is known to affect the NRTIs, and the combined effect of D67N and L517I on NVP, a NNRTI, was surprising. The decreased replication capacity in the L517I indicated that it has additive effect in fitness loss of the viral. Further site-directed mutagenesis studies are needed to understand the effect of these RNase H mutations alone and in xviii combination with other polymerase domain mutations or with connection domain mutations on nucleoside reverse transcriptase inhibitors. Structural analysis showed that the T470S/P mutations cause an inward movement of the RNase H active site amino acids residues, which may have an affect on RNase H activity. There was no interaction between L517I and E529D with the RNase H active site amino acid residues observed. The observed interaction was between the amino acids that form part of the RNase H primer grip and these RNase H mutations. New HIV-1 subtype C connection domain mutations were identified and phenotypic studies are required to investigate their role in HIV-1 drug resistance. In conclusion, this is the first study to show that T470S/P, L517I and E529D in HIV-1 subtype C affect NNRTI drug susceptibility. This provides further support for the monitoring of C-terminal domain mutations in relation to NNRTI, as well as NRTI drug resistance. In addition, these mutations need to be taken into account when designing newer NNRTI with the ability to retain activity against these mutants.