Sen Jayita, Jacobs Amy, Jiang Haiqing, Rong Lijun, Caffrey Michael
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Protein Sci. 2007 Jun;16(6):1236-41. doi: 10.1110/ps.072771407.
The importance of the HIV gp41 conserved disulfide loop to envelope function has been examined by mutational and functional analyses. Based on a luciferase-reporter entry assay, mutants gp41-CC/AA (C598A/C604A) and gp41-Delta (deletion of residues 596-606) result in a nonfunctional envelope protein. Western blot analysis shows both mutants to be properly expressed but not processed to form gp120 and gp41, which explains their nonfunctionality. The presence of mutant gp160 on the cell surface, as well as their ability to bind to sCD4, suggests that the mutations have disrupted processing at the furin recognition site encoded within the gp120 conserved domain 5, without resulting in an overall misfolding of the protein. With respect to the furin recognition site, the mutations are sequentially distant, which implies that the gp41 disulfide loop is interacting with gp120 C5 in gp160. In addition, we have modeled the gp120-gp41 interaction in unprocessed precursor gp160 using structural data available for gp120 and gp41 domains in isolation, supplemented by mutagenesis data. We suggest that the mutations have altered the interaction between gp120 C5 and the gp41 disulfide loop, resulting in decreased accessibility of the furin recognition site and implying that the interaction between the gp120 C5 and gp41 loop is a conformational requirement for gp160 processing. The sensitivity of this interaction could be exploited in future antivirals designed to disrupt HIV pathogenesis by disrupting gp160 processing.
通过突变和功能分析，研究了HIV gp41保守二硫键环对包膜功能的重要性。基于荧光素酶报告基因进入试验，突变体gp41-CC/AA（C598A/C604A）和gp41-Delta（缺失596-606位氨基酸）导致包膜蛋白无功能。蛋白质印迹分析表明，这两种突变体均能正常表达，但未加工形成gp120和gp41，这解释了它们的无功能状态。突变体gp160在细胞表面的存在及其与可溶性CD4（sCD4）结合的能力表明，这些突变破坏了gp120保守结构域5内编码的弗林蛋白酶识别位点的加工过程，但未导致蛋白质整体错误折叠。就弗林蛋白酶识别位点而言，这些突变在序列上距离较远，这意味着gp41二硫键环与gp160中的gp120 C5相互作用。此外，我们利用单独的gp120和gp41结构域的可用结构数据，并辅以诱变数据，对未加工的前体gp160中的gp120-gp41相互作用进行了建模。我们认为，这些突变改变了gp120 C5与gp41二硫键环之间的相互作用，导致弗林蛋白酶识别位点的可及性降低，这意味着gp120 C5与gp41环之间的相互作用是gp160加工的构象要求。这种相互作用的敏感性可用于未来设计的抗病毒药物，通过破坏gp160加工来破坏HIV发病机制。
