【作者】 何智文；

【导师】 于晓方；

【作者基本信息】 浙江大学 ， 肿瘤学， 2010， 博士

【摘要】 胞嘧啶脱氨酶APOBEC3G（或称A3G）和其他一些胞嘧啶脱氨酶例如APOBEC3F（或称A3F）都是有效的逆转录病毒抑制因子。人免疫缺陷病毒-1（HIV-1）需要存在于病毒粒子中的病毒感染子,即Vif（Virion infectivity factor）抑制人体内多种胞嘧啶脱氨酶的抗病毒作用才能获得感染能力。HIV-1 Vif蛋白抑制APOBEC3家族蛋白抗病毒功能的机制是相似的,即Vif蛋白通过结合Cullin5、ElonginB和ElonginC形成E3泛素连接酶复合体,从而导致靶蛋白APOBEC3家族蛋白被泛素化最终通过蛋白酶体系统将其降解。然而,介导HIV-1 Vif蛋白与胞嘧啶脱氨酶APOBEC3相互作用的蛋白结构域到目前为止尚不明确。本研究发现,HIV-1 Vif蛋白中含有一个VxIPLx_4-5LxΦx₂YwxL保守结构域。通过大量的实验研究,我们发现该结构域在介导Vif蛋白与APOBEC3的有效结合,由Vif介导的A3G蛋白降解,以及Vif对A3G抗病毒活性的抑制均起着十分重要的作用。该研究发现,由HIV-1 Vif蛋白的中52到72氨基酸残基序列构成的多肽片段(包含了VxIPLx_4-5LxΦx₂YWxL结构域)能有效的与A3G相互结合,然而对该多肽片段中两个疏水性氨基酸残基L64和I66进行突变后能破坏这种相互结合作用。我们还发现,当该结构域的某个氨基酸残基位点发生突变而导致Vif蛋白失去对A3G抗病毒功能的抑制作用时,该Vif蛋白仍然具有结合Cullin5-E3泛素复合体和A3G的能力。该结果提示,Vif与A3G的结合作用在其抑制A3G抗病毒作用中是必要但不充分的条件。与以往研究发现的HIV-1 Vif结构域中40到44氨基酸残基只抑制A3G的功能有所不同,该研究所发现的VxIPLx_4-5LxΦx₂YWxL结构域对Vif蛋白与A3G和A3F的结合以及功能抑制都十分重要。同时,我们还发现了另一个HIV-1 Vif蛋白结构域TG/PER/KxW,即74到79氨基酸残基,只对Vif与A3F的结合和功能抑制方面起重要作用,而对A3G的功能无明显影响。另外,Vif蛋白的N末端还存着一个保守的蛋白结构构域SLV/Ix₄Yx₉Y,即Vif蛋白23到40氨基酸残基区域。从1992年该被发现以来,人们对SLV/Ix₄Yx₉Y结构域的生物学功能仍不清楚。通过深入的研究,我们发现SLV/Ix₄Yx₉Y结构域在Vif蛋白抑制APOBEC3抗病毒作用中起着至关重要的作用。研究结果显示Vif蛋白的K26、Y30和Y40氨基酸残基对Vif介导的A3G的降解和功能抑制非常重要,而对APOBEC3家族另一主要成员A3F无明显影响。值得注意的是,在HIV和猿类免疫缺陷病毒（SIV）的Vif蛋白中,SLV/Ix₄Yx₉Y区域内的极性氨基酸残基K26总是高度保守,而且该氨基酸残基在Vif蛋白对A3G的功能抑制中非常重要,然而该氨基酸残基的突变不影响Vif对A3F的抑制能力。该研究还发现,Vif蛋白的S23、L24和V25三个氨基酸残基对介导Vif蛋白对A3F功能的抑制作用远远大于A3G,提示SLV/Ix₄Yx₉Y结构域中的SLV氨基酸残基是Vif介导对A3F功能抑制的主要位点。通过对Vif蛋白的氨基酸序列比对分析发现,Vif蛋白的结构域VxIPLx_4-5LxΦx₂YWxL、TG/PER/KxW和SLV/Ix₄Yx₉Y在HIV-1、HIV-2和SIV等病毒的Vif蛋白中均高度保守。我们的研究结果提示,灵长类慢病毒的Vif蛋白是通过这些保守的结构域识别宿主APOBEC3蛋白的。Vif蛋白这些保守的结构域为研制有效的抗HIV病毒的抑制剂提供了新的重要靶点。更多还原

【Abstract】 Apolipoprotein B mRNA-editingi catalytic polypeptide-like 3G（APOBEC3G,or A3G） and related cytidine deaminases such as apolipoprotein B mRNA-editing catalytic polypeptide-like 3F（APOBEC3F,or A3F） are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus（HIV）-1 requires suppression of multiple cytidine deaminases by Vif.HIV-1 Vif suppresses various APOBEC3 proteins through a common mechanism by recruiting Cullin5,ElonginB,and elonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation.Domains in Vif that mediate APOBEC3 recognition have not been fully characterized.In the present study part one,we identified a VxIPLx_4-5LxΦx₂YWxL motif in HIV-1 Vif,which is required for efficient interaction between Vif and A3G, Vif-mediated A3G degradation and virion exclusion,and functional suppression of the A3G antiviral activity.Amino acids 52 to 72 of HIV-1 Vif(including the VxIPLx_4-5LxΦx₂YWxL motif) alone could mediate interaction with A3G,and this interaction was abolished by mutatuins of two hydrophobic amino acids in this region. We have also observed that a Vif mutant was ineffective against A3G,yet it retained the ability to interact with Cullin5-E3 ubiquitin complex and A3G,suggesting that interaction with A3G is necessary but not sufficient to inhibit its antiviral function. Unlike the previously identified motif of HIV-1 Vif amino acids 40 to 44,which is only important for A3G suppression,the VxIPLx_4-5LxΦx₂YWxL motif is also required for efficient A3F interaction and suppression.On the other hand,another motif,TGERxW, of HIV-1 Vif amino acids 74 to 79 was found to be mainly important for A3F interaction and inhibition.The amino-terminal region of the HIV-1 Vif molecule contains a conserved SLV/Ix₄Yx₉Y motif that was first described in 1992,but the importance of this motif for Vif function has yet not been examined.In our study part two our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that this region is critical for APOBEC3 suppression.Amino acids K26,Y30 and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G（A3G）.In particular,the positively charged K26 of HIV-1 Vif is invariably conserved within the Vif SLV/Ix₄Yx₉Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivatin.However,mutation of these residues had little effect on the Vif-mediated suppression of A3F.The SLV portion of the Vif SLV/Ix₄Yx₉Y motif was found to be required for optimal suppression of A3F.Thus,the Vif SLV/Ix₄Yx₉Y motif represent an important functional domain in the Vif-mediated defense against APOBEC3.All the three Vif motifs VxIPLx_4-5LxΦx₂YWxL and TGERxW and SLV/Ix₄Yx₉Y are highly conserved among HIV-1,HIV-2 and various simian immunodeficiency virus （SIV） Vif proteins.Our data suggest that primate lentiviral Vif molecules recognize their autologous APOBEC3proteins through conserved structural features.These structural motifs in HIV-1 Vif represent attractive targets for the development of novel inhibitors to combat HIV infection.更多还原