HIV-1 Distinctive Drug Resistance-associated Mutations And Viral Replicative Fitness

Up to date, HIV infection still extensive spread. Fortunately, the increasing availability of antiretroviral therapy (ART) worldwide has significantly improved quality of life and prolonged life-span for HIV-infected patients, also achieved the goal of reducing AIDS transmission. However, the therapeutic effect of ART is weakened by the emergence of drug-resistant mutant viruses. There may be an extremely complicated and dynamic progress participated in the occurrence of drug resistance, including several environmental factors and viral genes, e.g combined action of multi-drug-resistant sites. The viral gene sites played a role in drug resistance include drug resistance associated (DR-associated) mutations and overlapping polymorphisms, which created an environment for the virus to change the distribution of quasi-species in patients and reach the purpose of optimal fitness through changing the replicative ability of mutated viruses.Most DR-associated mutations may result in significantly decreased replicative fitness. There is a hypothesis but lack of experimental evidence, that is, polymorphisms sites with covariation may happen in the viral evolution, and compensate the reduced fitness induced by drug resistance mutation sites. In this study, we focused on the sequence analysis of subtypes CRF01_AE, CRF07_BC and B’isolated in China, and proved the covariation of polymorphisms and drug resistance associated mutation sites using the bioinformatics methods. Subsequently, the influence of these distinctive drug resistance associated mutation sites in viral fitness was further verified in vitro, and the related mechanisms were also primarily explored. The results of this study have an important significance in understanding the mechanisms for drug resistance, and provide basic data for improvement of antiviral therapy program and effective prevention and control of HIV.1. Investigation of HIV-1 infection in MSM in Chaoyang district, Beijing from 2011 to 2013, and analysis of HIV-1 transmitted drug resistance-associated mutationsIn this study, we analyzed the HTV-1 pol gene sequences of ART treatment-naive MSM in Beijing diagnosed from 2011 to 2013, and made clear the distribution of HIV-1 subtypes and characterization of drug resistance genes. Further, through comparison of HIV-1 sequences with or without drug resistance sites, we observed the phenomenon of covariation of polymorphism and drug resistance associated mutation sites in ART treatment-naive MSM with CRFO1 AE infection.Results showed that HIV-1 still prevalence in MSM in Chaoyang district, Beijing with a high level. The largest proportion of the HIV-1 strains among them belonged to the CRF01_AE subtype, followed by CRF07_BC and subtype B. The proportion of samples with TDR-associated mutations over the sample period was determined to be 25.56%. Moreover, eight significant co-variation pairs were found between TDR-associated mutations (V179D/E) and seven overlapping polymorphisms (R238K, A272P, T11K, I173K, K174Q, S207Q, S211K) in subtype CRF01_AE.2. Co-variation analysis between DR-associated mutations and overlapping polymorphism in subtypes CRF07_BC and B’We analyzed the co-variation between DR-associated mutations and overlapping polymorphism among subtype B’and CRF07_BC using R and the CorMut package. The pol gene sequences of HIV-1 subtype CRF07_BC strains isolated from Xinjiang and Sichuan were identified, including 600 ART treatment-naive cases and 344 treated patients. Through comparison, we found the co-variation between DR-associated mutaions (K103N, M184V, Q197K, G190A, Y181C, L228R, M230L) and overlapping polymorphisms (A36E, R135I, R277K, L283I, D291E) in RT region, and the co-variation between DR-associated mutaions (L101) and overlapping polymorphisms (I132L) in PR region in treated patients. Likewise, we also found the co-variation between DR-associated mutaions (Y188C, K103N, E138G, V108I, L74F) and overlapping polymorphisms (M16K, K166R, E248V, L283I, E297A, T200M) in RT region, and the co-variation between DR-associated mutaions (L101) and overlapping polymorphisms (164V, A71V) in PR region in treatment-naive cases.The pol gene sequences of HIV-1 subtype B’strains isolated from Henan and Anhui were identified, including 178 ART treatment-naive cases and 327 treated patients. Through comparison, we found the co-variation between DR-associated mutaions (Y181C, L210W, M41L, E44A, E203D, H208Y, T215Y, L228R, K65R, Q151M, V75T) and overlapping polymorphisms (S162C, Q207E, R211K, L214F, I293V) in RT region in treated patients. Likewise, we also found the co-variation between DR-associated mutaions (V106I, M41L, L210W, T215Y, H221Y) and overlapping polymorphisms (1135V, S68G, I178L, R277K) in RT regionin treatment-naiVe cases.3. The impact of DR-associated mutations on viral fitnessThe recombinant HIV-1 CRF07BC virus was constructed to contain the DR-associated mutations sites, combined with previously established recombinant HIV-1 B virus. Subsequently, the infection ability assay and a multiple-cycle, recombinant-virus, growth competition assay were applied to determine the impact of distinctive drug resistance-associated mutations on fitness. In addition, the related mechanism was also primarily explored through detection of concentration and activity of reverse transcriptase.The results showed that the fitness, relative content and activity of reverse transcriptase of recombinant virus with T215Yand I178L co-variation increased more than that with single T215Y mutation in ART treatment-naive HIV-1 B’infection group. It indicates that I178L mutation could compensate the reduction of fitness induced by T215Y mutation through adjusting the P51 relative content and activity of reverse transcriptase. In the treated group of HIV-1 B’infection, the fitness of recombinant virus with T215Y and R211K co-variation increased more than that with single T215Y mutation in vitro without drug treatment. Moreover, the fitness of recombinant virus with T215Y and R211K co-variation increased more than wild type in vitro with AZT treatment. It indicates that R211K mutation could increase the fitness and may speed the progress of drug resistance.