Xiao-Min Zhang1, Hao Wu1, Qiwei Zhang2, Terrence Chi-Kong Lau3, Hin Chu1, Zhi-Wei Chen1, Dong-Yan Jin4, Bo-Jian Zheng5. 1. Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China. 2. Biosafety Level-3 Laboratory, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China. 3. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, SAR, China. 4. Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China. 5. Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China bzheng@hkucc.hku.hk.
Abstract
OBJECTIVES: Growing evidence suggests that mutations in the connection domain of the HIV-1 reverse transcriptase (RT) can contribute to viral resistance to RT inhibitors. This work was designed to determine the effects of a novel mutation, D404N, in the connection subdomain of RT of HIV-1 CRF08_BC subtype on drug resistance, viral replication capacity (RC) and RT activity. METHODS: Mutation D404N, alone or together with the other reported mutations, was introduced into an HIV-1 CRF08_BC subtype infectious clone by site-directed mutagenesis. Viral susceptibility to nine RT inhibitors, viral RC and the DNA polymerase activity of viral RT of the constructed virus mutants were investigated. A modelling study using the server SWISS-MODEL was conducted to explore the possible structure-related drug resistance mechanism of the mutation D404N. RESULTS: Single mutations D404N and H221Y conferred low-level resistance to nevirapine, efavirenz, rilpivirine and zidovudine. Double mutations Y181C/D404N and Y181C/H221Y significantly reduced susceptibility to NNRTIs. The most pronounced resistance to NNRTIs was observed with the triple mutation Y181C/D404N/H221Y. Virus containing D404N as the only mutation displayed ∼50% RC compared with the WT virus. The modelling study suggested that the D404N mutation might abolish the hydrogen bonds between residues 404 and K30 in p51 or K431 in p66, leading to impaired RT subunit structure and enhanced drug resistance. CONCLUSIONS: These results indicate that D404N is a novel NNRTI-associated mutation in the HIV-1 subtype CRF08_BC and provides information valuable for the monitoring of clinical RTI resistance.
OBJECTIVES: Growing evidence suggests that mutations in the connection domain of the HIV-1 reverse transcriptase (RT) can contribute to viral resistance to RT inhibitors. This work was designed to determine the effects of a novel mutation, D404N, in the connection subdomain of RT of HIV-1 CRF08_BC subtype on drug resistance, viral replication capacity (RC) and RT activity. METHODS: Mutation D404N, alone or together with the other reported mutations, was introduced into an HIV-1 CRF08_BC subtype infectious clone by site-directed mutagenesis. Viral susceptibility to nine RT inhibitors, viral RC and the DNA polymerase activity of viral RT of the constructed virus mutants were investigated. A modelling study using the server SWISS-MODEL was conducted to explore the possible structure-related drug resistance mechanism of the mutation D404N. RESULTS: Single mutations D404N and H221Y conferred low-level resistance to nevirapine, efavirenz, rilpivirine and zidovudine. Double mutations Y181C/D404N and Y181C/H221Y significantly reduced susceptibility to NNRTIs. The most pronounced resistance to NNRTIs was observed with the triple mutation Y181C/D404N/H221Y. Virus containing D404N as the only mutation displayed ∼50% RC compared with the WT virus. The modelling study suggested that the D404N mutation might abolish the hydrogen bonds between residues 404 and K30 in p51 or K431 in p66, leading to impaired RT subunit structure and enhanced drug resistance. CONCLUSIONS: These results indicate that D404N is a novel NNRTI-associated mutation in the HIV-1 subtype CRF08_BC and provides information valuable for the monitoring of clinical RTI resistance.
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