Wang Zheng, Hong Kunxue, Zhang Jing, Zhang Lei, Li Dan, Ren Li, Liang Hua, Shao Yiming
State Key Laboratory for Infectious Disease Prevention and Control, Division of Research of Virology and Immunology, National Center for AIDS/STD Control and Prevention, China CDC, Beijing, China ; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
PLoS One. 2013 Nov 18;8(11):e79177. doi: 10.1371/journal.pone.0079177. eCollection 2013.
Among the various subtypes of the M group of human immunodeficiency virus type 1 (HIV-1), clade CRF07_BC is the most prevalent in China. To date, no strong replicable CRF07_BC infectious clone has been constructed. Here we report on the construction and characterization of highly replicable infectious molecular clones from the isolate XJDC6291 of this HIV-1 subtype. Four full-length clones pXJDC2-7, pXJDC3-7, pXJDC2-6 and pXJDC3-6 were successfully produced, but only pXJDC2-7 presented detectable infectivity and replication capability. To improve the replication capability of pXJDC2-7, a 4.8 kb region spanning from the pol Integrase to nef gene of the clone was replaced by PCR products of the corresponding fragments from the original isolate XJDC6291, which produced two clones pXJDC13 and pXJDC17 that exhibited strong replication capability. The viral stocks obtained by pXJDC-13 and pXJDC-17 transfection into 293T cells replicated efficiently in human PBMCs, human primary CD4(+) T cells and displayed CCR5 tropism. Sequence alignment between pXJDC13, pXJDC17 and pXJDC2-7 suggested that polymorphisms in the V1V2 region may influence infectivity, and reverse genetic experiment showed that V1V2 polymorphisms may influence the infectivity of the clones but did not affect the replication capability at a significant level. pXJDC13 and pXJDC17 displayed strong replication capability and are the first full-length infectious clones of HIV-1 CRF07_BC clade in the world. The availability of CRF07_BC infectious clones provides a useful tool for a wide range of studies, including antiretroviral drug and vaccine research as related to this HIV subtype.
在人类免疫缺陷病毒1型（HIV-1）M组的各种亚型中，CRF07_BC分支在中国最为流行。迄今为止，尚未构建出具有强大复制能力的可复制CRF07_BC感染性克隆。在此，我们报告了从该HIV-1亚型的XJDC6291分离株构建具有高度复制能力的感染性分子克隆及其特性。成功构建了四个全长克隆pXJDC2-7、pXJDC3-7、pXJDC2-6和pXJDC3-6，但只有pXJDC2-7表现出可检测到的感染性和复制能力。为提高pXJDC2-7的复制能力，用原始分离株XJDC6291相应片段的PCR产物替换了该克隆从pol整合酶到nef基因的4.8 kb区域，产生了两个具有强大复制能力的克隆pXJDC13和pXJDC17。通过将pXJDC - 13和pXJDC - 17转染到293T细胞中获得的病毒株在人外周血单核细胞（PBMC）、人原代CD4(+) T细胞中高效复制，并表现出CCR5嗜性。pXJDC13、pXJDC17与pXJDC2-7之间的序列比对表明，V1V2区域的多态性可能影响感染性，反向遗传实验表明V1V2多态性可能影响克隆的感染性，但对复制能力没有显著影响。pXJDC13和pXJDC17表现出强大的复制能力，是世界上首个HIV-1 CRF07_BC分支的全长感染性克隆。CRF07_BC感染性克隆的可用性为广泛的研究提供了有用工具，包括与该HIV亚型相关的抗逆转录病毒药物和疫苗研究。
