HIV-1逆转录酶H221Y突变对Y181C突变株复制能力影响研究

Impact of HIV-1 reverse transcriptase H221Y mutation on replication capacity of Y181C mutant viral strain

目的明确中国经抗病毒治疗HIV-1感染者中Y181c突变与H221Y突变发生情况及H221Y突变对Y181C突变株复制能力的影响。方法选取河南省经高效抗逆转录病毒治疗出现Y181C和H221Y双突变的B’亚型HIV-1感染者3例。回顾性检测其2004--2006年间抗病毒治疗后每6个月一次的随访血样，从血浆中扩增病毒pol区基因，克隆人T载体并测序，分析克隆中Y181C及H221Y突变的存在情况。构建Y181C单突变质粒及Y181C和H221Y双突变质粒，分别与包膜蛋白质粒pVSV-G共转染HEK293T细胞，包装两种突变型假病毒。分别单轮感染TZM—b1细胞，分析两种突变型假病毒单轮复制能力差异。结果共获得3例患者来自6个随访点的57条TA克隆序列，其中Y181C单突变克隆46个，Y181C和H221Y双突变克隆35个，H221Y突变均出现于已经存在Y181C突变的克隆中。在2例患者的两组假病毒中，携带Y181C和H221Y双突变假病毒的复制能力分别是Y181C单突变假病毒的1．42倍和1．62倍。结论在经抗病毒治疗的B’亚型HIV-1感染者中，H221Y突变很可能为Y181C突变的补偿突变，提高Y181C突变株复制能力。这一发现对中国B’亚型HIV-1感染者耐药基因型检测结果的解读及指导临床进行抗病毒治疗具有重要意义。

Objective To explore the development of Y181C and H221Y mutations among HIV-1 in- fected patients upon antiretroviral therapy in China and to investigate the impact of H221Y mutation on replica- tion capacity of Y181C mutant. Methods Three HIV-1 subtype B＇ infected patients with Y181C and H221Y mutations upon highly active antiretroviral therapy from Henan province were enrolled in this study. Follow-up blood samples collected every six months from the patients after therapy between 2004 to 2006 were detected for viral load evaluation. Fragments of pol gene were amplified by nested PCR. The purified PCR products were li- gated into pMD18-T vector to construct recombinant plasmids, which were further sequenced and compared with the HIV reference strain from Stanford HIVDB for analyzing the development of Y181C and H221Y mutations. Plasmids containing Y181C mutation alone and plasmids containing Y181C and H221Y double mutations were constructed and cotransfected respectively with envelope-expression plasmid pVSV-G into HEK 293T cells to ob- tain HIV-1 pseudotyped viruses. Replication capacity of each pseudotyped virus was analyzed after a single-cycle infection in TZM-bl cells. Results A total of 57 TA cloned sequences from six follow-up blood samples of three patients were obtained. Y181C mutation alone was found in 46 clones and Y181C and H221Y mutations were found in 35 clones. Moreover, H221Y mutation was only found in clones in which Y181C mutation had already developed. Between two pairs of pseudotyped virus from two patients, replication capacity of those viruses con- taining Y181C and H221Y mutations were 1.42 and 1.62 fold higher than the viruses containing YI81C muta- tion alone, respectively. Conclusion H221Y mutation might be a compensatory mutation of Y181C mutation occurred to improve the replication capacity of viral strains with Y181C mutant in HIV-1 subtype B＇ infected pa- tients upon antiretroviral therapy. The finding suggests that it＇s significantly important to further perform the gen- otypic test for HIV-1 subtype B＇ drug resistance in order to guide antiretroviral treatment in clinical in China.