Pastore Cristina, Nedellec Rebecca, Ramos Alejandra, Hartley Oliver, Miamidian John L, Reeves Jacqueline D, Mosier Donald E
Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Virol. 2007 Aug;81(15):8165-79. doi: 10.1128/JVI.02792-06. Epub 2007 May 16.
We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.
我们研究了人类免疫缺陷病毒1型（HIV-1）包膜功能在共受体从CCR5转换为CXCR4过程中的演变。采用定点诱变技术，在四种不同的共受体转换突变体的包膜中引入了大部分可能的中间突变，每个突变体都有独特的CCR5和CXCR4利用模式，从高效利用两种共受体到仅利用CXCR4。对在表达CCR5或CXCR4的靶细胞上仍保留部分进入功能的突变包膜，进一步检测其对CCR5或CXCR4抑制剂、可溶性CD4以及中和抗体b12-IgG和4E10的敏感性。还对一部分突变包膜进行了直接CD4或CCR5结合试验以及包膜介导的融合反应研究。共受体转换中间体对CCR5抑制剂的敏感性增加（V2或C2有少数突变的包膜除外），这与CCR5结合能力的丧失相关。随着CXCR4利用能力的提高，突变包膜介导的感染对可溶性CD4抑制的抵抗力增强，与CD4的直接结合增加。这些变化伴随着对CXCR4抑制剂AMD3100的抵抗力增强。对中和抗体的敏感性变化更大，尽管感染表达CXCR4的靶细胞通常比感染表达CCR5的靶细胞对b12-IgG和4E10的中和作用更敏感。包膜功能的这些变化在所有四个系列的包膜突变中都是一致的，因此与CCR5和CXCR4的最终利用情况无关。CCR5结合能力降低和CD4结合能力增加似乎是共受体转换中间体的共同特征。
