HIV跨膜蛋白gp41和gp36原核表达及抗原性研究

目的 构建HIV跨膜蛋白gp41和gp36原核表达载体,诱导表达HIV抗原,为HIV快速诊断和蛋白疫苗研究提供材料基础.方法 用PCR方法对HIV-1包膜蛋白gp41和HIV-2包膜蛋白gp36基因序列进行全长、截短扩增,分别或以融合表达方式克隆入原核表达载体pQE30、pET32a(+)及pGST-MOLUC,重组质粒经异丙基硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)诱导表达,并经Ni-NAT或谷胱甘肽-Sepharose-4B层析柱亲和纯化HIV跨膜糖蛋白抗原,对获得的纯化抗原进行Western blot检测,利用HIV抗原制备ELISA检测试剂盒,并分析HIV临床样本. 结果 成功构建了HIV包膜蛋白的原核表达载体,利用亲和层析纯化得到了纯度较高的HIV包膜蛋白;Western blot分析结果证实表达纯化的HIV跨膜蛋白gp41(aa.538～718)、gp36(aa.533～764)以及gp41-gp36均能和HIV阳性血清反应;ELISA试剂盒检测临床样本显示,特异性达95%以上,灵敏度达100%.结论 获得了有生物学活性的HIV截短包膜蛋白,并可用于HIV快速检测试剂盒的研发.     

Association of variations of NAb 2F5 and 4E10 epitopes and disease progression in Chinese antiretroviral treatment-naïve patients infected with HIV-1 clade B’

Background Studies on human immunodeficiency virus type 1 （HIV-1） vaccines have recently focused on targeting the conserved neutralizing epitopes 2F5 and 4E10, and hence it is important to understand the extent of mutations in these two viral epitopes. Here, we investigated the amino acid mutations in epitopes of 2F5 （ELDKWA, HIV-1 HXB2 env 662-667 aa） and 4E10 （NWFDIT, HIV-1 HXB2 env 671-676 aa） in the membrane proximal-external region of gp41 from clade B＇ HIV-1-infected individuals living in Henan province, China. We also examined the frequency of a mutation and its relation to disease progression.Methods A cohort of 54 treatment-na（i）ve HIV-1-infected individuals was recruited in this study, and 16 individuals were selected for a short-term longitudinal study on sequence evolution. The HIV-1 env gp41 gene was amplified, cloned, and sequenced, and predicted amino acid sequences were aligned for analysis.Results The mutations E662A and K665E on the 2F5 epitope and N671S and T676S on the 4E10 epitope were seen.Simultaneous RNA sequencing showed some discrepancies with proviral DNA sequences. In our longitudinal study,mutation levels of these two neutralizing epitopes were low but diverse and persistent. The frequencies of mutations within the 4E10 peptide NWFDIT in slow progressors were noticeably lower than those in AIDS patients （P ＜0.05）.Conclusions Antigenic variation of the neutralizing epitopes 2F5 and 4E10 is limited in subtype B＇ infection, and that 4E10 peptide mutation is correlated with disease progression. Monitoring epitope mutations will offer useful data for development of the candidate 2F5-like and 4E10-like antibodies to prevent and treat AIDS.     

Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant “core” structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD “core” do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD “core” delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.     

The effect of the membrane-proximal tyrosine-based sorting signal of HIV-1 gp41 on viral infectivity depends on sequences within gp120

The cytoplasmic domain of the HIV-1 Env glycoprotein (gp41) contains sequences that affect the trafficking of Env within the host cell. We previously showed that the membrane-proximal tyrosine-based adaptor protein (AP)-binding signal of gp41 (Y712XXL) is required for optimal viral infectivity and entry into target cells. Because these effects were not attributable to an effect on the incorporation of Env into virions, we hypothesized that they involved targeting of viral assembly to specific endosomal membranes that conferred greater fusogenicity. To further elaborate this hypothesis, we mutated the C-terminal leucine-based AP-binding signal of gp41 (LL855/856). In contrast to Env Y712, the leucine signal was dispensable for viral infectivity in both single cycle assays and during spreading infections within cultures of peripheral blood mononuclear cells (PBMCs). To test the hypothesis that these AP-binding motifs target Env to endosomes during viral morphogenesis, we compared the subcellular localization of wild-type Env to mutants of the Y712 and LL855/856 signals. The results failed to support the hypothesis that these signals target viral assembly to specific endosomal membranes. Strikingly, in the context of a C2-V3 region that confers macrophage-tropism, mutation of Y712 no longer markedly affected viral infectivity in either single cycle assays or during spreading infection within PBMCs, and it did not impair viral entry. These data indicate that the importance of the tyrosine-based sorting signal in gp41 for optimal viral infectivity depends on sequences in gp120. This observation is consistent with the hypothesis that the Y712 residue is part of the ectodomain of gp41 in virion-associated Env. We speculate that as part of the ectodomain, Y712 could affect specifically the conformation of the more positively charged CXCR4-tropic V3 loop in a manner that augments viral fusogenicity and infectivity.