Drug Resistance Of HIV-1CRF_BC Strains And Identification Of Novel Drug-resistant Mutations

Objective:HIV-1CRF_BC has become one of mainly HIV-1subtypes strains circulating in China and spreading to other regions all over the world. The emergence of HIV drug resistance has been the most major obstacle in HIV-1treatment and prevention since free antiviral treatment in China put into effect in2003.Resistance to antivirals is a complex and dynamic phenomenon and the results of effect of host and drugs on HIV-1. This process involved more mutations than are currently known, including some unknown drug-resistant mutation. At present Most HIV-1drug-resistant data and the mechanisms come from the subtype B which is very popular in Europe and America. Furthermore, some in vitro and in vivo observations suggest that the various subtypes may respond differently to certain antiretroviral drugs. The frequency and pattern of mutations conferring resistance to these drugs differ among HIV-1subtypes and can influence therapeutic outcome. Therefore, focusing on the study of HIV-1CRF_BC drug resistance, especially subtypes’ specificity associated with drug resistance is very important.On the basis of three parts including the accordance analysis of genotypic and phenotypic results of drug resistance, discovery of new drug-resistant associated with drug resistance and identification of novel drug-resistant mutations, our study expounds drug resistance of HIV-1CRF_BC strains and effect of novel drug-resistant mutations. These will do much help to design suitable antiviral treatment regime and draw up national strategies of prophylaxis and treatment and then promote HIV-1clinical prevention and cure scientificly and effectively. 1. The concordance analysis of genotypic and phenotypic resistance among HIV-1CRF07BC in ChinaFirst, acquire HIV-1isolates from CRF07_BC treatment-failure patients and then analyze the concordance between genotypic and phenotypic resistant results of these HIV-1isolates. The results show that the concordances of HIV-1CRF07BC isolates on FTC, EFV, NVP are the best and90.9%,90.9%,100%separately. Those on AZT and TDF are the worst and36.4%and45.5%. Most of the differences are under the situation that genotypic resistance results show’Susceptibility’, but phenotypic resistance results appear’Resistance’. Those on3TC, D4T and DDI is72.7%,72.7%and81.8%respectively. Next, construct clone viruses whose sequence of RT region is similar with HIV-1isolates’and more compare the concordance between genotypic and phenotypic resistant. The results reveal that the concordances of clone virus are all above70%. That on FTC is the highest and the number is100%. Those on3TC, D4T, DDI, EFV and NVP take second place and the numbers are all80%. Those on AZT and TDF are the lowest and the number is70%.2. The discovery of new mutations associated with drug-resistance through comparing RT region’s sequences of HIV-1CRF_BC treatment-failure with that of treatment-naive patients.Compare the gene sequences of pol region of HIV-1CRFBC viruses isolated from631treatment-naive and363treatment-experienced patients and performed the selection pressure analysis to reveal the drug resistance mutations. We found that the frequencies of15polymorphism sites in RT of CRF_BC strains isolated from the treatment-experienced patients were significantly different from those isolated from the treatment-naive patients, especially W88C, K101Q, I132L, R135L, T139K/R, H221Y and L228R, Their frequencies were significantly increased in ART-treated group (P<0.01). They were presented in RT of CRF_BC strains isolated from the treatment-experienced patients, while they, except R135L, were completely absent in the RT of CRFBC strains isolated from ART-nai’ve patients. It reveals that these mutation sites may be associated with drug treatment and belong to drug-resistant mutations. And then conduct the conditional selection pressure analysis to identify novel sites of mutations potentially associated with major drug-resistance mutations. The results show that the mutation T139K may be induced by other mutations, including K103N, Y181C, M184V and G190A, and the selection pressure ratio from G190A to T139K was the greatest, reaching to42. Notably, T139K mutation had a significant influence to G190A, indicating a correlated mutation between the two mutations. The mutual influence between T139K and G190A hints that these two mutations may form as a mutation pattern to function synergistically. Interestingly, H221Y was associated with Y181C and/or K103N mutations in RT of CRF_BC strains isolated from the treatment-experienced patients. For example, K103N, Y181C and H221Y are three mutations formed by pairwise interactions. Y181C and H221Y, in particular, have strong mutual influence (conditional selection ratio of Y181Câ†'H221Y and H221Yâ†'Y181C was45and11, respectively), suggesting that H221Y and Y181C may form combinatorial mutation patterns to synergistically resist the drug treatment.3. The relationship between new mutations mentioned above and their susceptibility on reverse transcriptase inhibitors.First amplify pol region of infectious clone which contains HIV-1CRF_BC pol region by PCR and ligate to pGET-T vector(3kb). Then the mutations are introduced into the new T-vector containing HIV PR and RT regions by use of DNA polymerase and proper site mutation primers. After sequencing, digest with proper enzyme and ligate to BstE â…¡-and AgeI-digested pNL4-3. The transfected clone viruses containing site-mutation are used to test the susceptibility on RTIs. The result confirms that five mutation including K101Q, I132L, T139K/R, H221Y exhibited a significantly decreased the susceptibility of all four NNRTIs (TCM-125, DLV, NVP and EFV) tested(P<0.05). The virus with K101Q mutant shows3.50-,1.71-,12.57-,3.06-fold higher resistance to all NNRTIs tested. I132L mutant shows2.55-,19.35-,28.05-,6.13-fold T139K mutant shows4.67-,3.66-,7.35-,3.30-fold. T139R shows1.82-,4.69-,25.12-,1.89-fold. H221Y mutant shows nearly2-fold to all NNRTIs tested. The K101Q, I132L and T139K/R mutants exhibited significant (2～187-fold) increases in resistance to all the four NNRTIs tested (P<0.05), and H221Y mutant had a moderate increase (approximately2-fold) of resistance to these four NNRTIs (P<0.05), while the W88C, R135L, and L228R mutations had no significant effect on the viral resistance to RTIs. K103N+T139K mutant induce higher resistance to all four NNRTIs, with FC ranging from2.00to14.15. K103N+H221Y mutations exhibited an increased (1.69-to2.96-fold) resistance to the four NNRTIs tested (P<0.05), while K103N+K101Q/L228R mutants did not displayed a higher NNRTI-resistance than K103N alone mutant. K101Q and H221Y combined with Y181C can decrease the drug susceptibility significantly(P<0.05). Y181C+K101Q mutant showed a2.48-,4.37-, and4.69-fold higher resistance to TCM-125, NVP and EFV, respectively, than Y181C alone mutant. Y181C+H221Y mutations resulted in significantly higher resistance to all four NNRTIs than Y181C alone mutation, ranging in3.00-4.24FC. G190A+T139K also showed a higher increased (1.48-to7.21-fold) resistance to all four NNRTIs than G190A alone mutation.In summary, our study suggest that K101Q, I132L, T139K/R and H221Y are novel sites of mutations strongly associated with NNRTI-resistance in RT of CRFBC strains that are predominantly circulating in China. The co-presence of H221Y, T139K or K101Q with the well-known RTI-resistance mutations K103N, G190A or Y181C may strengthen the drug-resistance effect. When testing the drug resistance of HIV-1CRF_BC, genotypic resistance assay can take the place of phenotypic resistance assay to detect FTC, EFV, NVP,3TC, D4T, DDI. But it will be better to run phenotypic resistance assay to detect AZT and TDF.