Identification and characterization of drug resistance mutations in the reverse transcriptase of HIV-1 CRF08_BC subtype

Abstract

The emergence of drug resistant virus variants containing one or more mutations has become one of the major reasons causing long-term treatment failure and leads to rapid progress into the acquired immunodeficiency syndrome (AIDS). Polymorphisms among different subtypes can affect the magnitude of drug resistance and the propensity to acquire certain resistance mutations. Most previous studies have focused on HIV-1 subtype B. Therefore, identification and characterization of the novel mutations in non-B subtype are necessary for developing novel efficient antiviral agents and optimizing therapies for the patients in the non-B subtype prevalent regions.

HIV-1 CRF08_BC is one of the major predominant subtypes in China, but studies of the drug resistance mutations in this subtype are limited so that the relative information is still poor. Previously, we selected some novel mutations in the viral reverse transcriptase (RT) presenting as accumulated patterns in a clinical isolate 2007CNGX-HK of CRF08_BC subtype during in vitro cultures with increasing concentration of NVP, one of the mostly used NNRTIs in the first-line therapy for HIV-1 infection. The purpose of this study is to further characterize these novel mutations either alone or in combination with the other reported mutations. We first constructed a wild type replication-competent infectious clone pBRGX from the isolated virus 2007CNGX-HK and confirmed CCR5 was the co-receptor for HIV-1 CRF08_BC subtype virus infection. Besides, we also found that a low-copy number vector might be better to construct a large molecular clone (>10k) than a high-copy number vector, and parental long terminal repeats (LTRs) were supposed to play a vital role in HIV-1 replication.

Based on pBRGX, novel mutations were induced either alone or together with other reported mutations by site-mutagenesis. Twenty-one virus variants were obtained from transfection and used for phenotypic assays, replication capacity and RT activity tests. Results showed that: 1) D404N in the connection subdomain of RT was firstly demonstrated to be a novel resistance-related mutation to non-nucleoside reverse transcriptase inhibitors (NNRTIs), which also resulted in great loss of replication capacity (RC). Modeling study suggested that the mutation D404N might abolish the hydrogen bonds between residue 404 and K30 in p51 or K431 in p66, leading to impaired RT subunit structure and resulting in drug resistance and reduced RC. 2) L228I, A139V and Y232H were new accessory drug resistance mutations to NNRTIs at cost of RC loss. 3) C162Y enhanced the drug resistance level of E138R to NNRTIs but not impair viral RC. 4) Reported mutations E138G/K/R, Y181C, Y188C and H221Y in subtype B were also critical for viral susceptibility to NNRTIs in subtype CRF08_BC.

In conclusion, our data indicate that novel mutations may play an active role in the regulation of NNRTI resistance in non-B subtype, reinforcing the complexity of RT inhibitor resistance. Several reported drug-resistance mutations in B subtype also exhibited similar effects in CRF08_BC subtype. These mutations might be considered for predicting clinical response to antiretroviral therapy and for assessing the efficacies of new drugs. Further clinical studies are necessary to demonstrate the clinical significance of these novel mutations.