WALDEMAR POPIK, PHD, LAB STARTUP PACKAGE: HIV

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞膜形成了专门的脂域的马赛克。其中，鞘磷脂和胆固醇富集区，称为脂筏，由于它们在膜运输、细胞形态发生、信号转导，特别是在包括细菌及其毒素和病毒在内的各种病原体的进入中所起的作用而受到广泛关注。尽管脂筏在HIV感染中的作用尚不清楚，但似乎脂筏代表了病毒与集中在这些区域的CD4受体结合的特权部位。因此，我的研究目标集中在A)了解HIV-1受体在HIV进入脂筏中的定位的意义，以及B)阐明针对主要HIV-1受体CD_4进入脂筏的分子机制(S)。 对CD4受体的广泛诱变发现了一个RAFT定位标记，它由一小段带正电的氨基酸残基组成，RHRRR，存在于CD4的膜-近端细胞质结构域。用丙氨酸残基替换RHRRR序列将CD4重定向到非筏子。令人惊讶的是，突变的CD4支持HIV-1的生产性进入相当于野生型CD4的水平，这表明病毒进入可能不需要RAFT定位的CD4。然而，我们不能排除HIV与位于筏外的CD4受体结合可以在与辅受体结合之前或之后或在融合/进入过程中刺激RAFT组装的可能性。进一步的研究旨在回答这些问题。 我们和其他人已经证明了APOBEC3G的抗逆转录病毒活性需要它通过与Gag核衣壳蛋白NC相互作用组装成HIV病毒粒子。然而，在病毒辅助蛋白Vif的存在下，这一过程受到严重限制，Vif与APOBEC3G结合并被蛋白酶体降解。因此，寻找逃避Vif介导的降解的方法已成为开发基于APOBEC3G的有效抗逆转录病毒治疗的挑战。最近，我们发现APOBEC3G可以有效地包装成外体，即细胞衍生的纳米囊泡，在癌症治疗临床试验中显示出良好的潜力。我们目前的研究集中在阐明APOBEC3G靶向外切体的分子机制(S)，以及评估外切体包裹的APOBEC3G作为一种新的抗HIV治疗方法的潜力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell membranes form a mosaic of specialized lipid domains. Among them, sphingolipid and cholesterol enriched domains, called lipid rafts, have received much attention due to the role they play in membrane trafficking, cell morphogenesis, signal transduction and particularly in entry of various pathogens including bacteria and their toxins as well as viruses. Although the role of lipid rafts in HIV infection is still unclear, it seems likely that rafts represent privileged sites for virus binding to CD4 receptors concentrated in these domains. Thus, my research goals have focused on A) understanding the significance of localization of HIV-1 receptors in lipid rafts in HIV entry and B) elucidating the molecular mechanism(s) targeting the primary HIV-1 receptor, CD4, into lipid rafts. Extensive mutagenesis of the CD4 receptor identified a raft-localizing marker, consisting of a short sequence of positively charged amino acid residues, RHRRR, and present in the membrane-proximal cytoplasmic domain of CD4. Substitution of the RHRRR sequence with alanine residues redirected CD4 to non-rafts. Surprisingly, the mutant CD4 supported productive HIV-1 entry to levels equivalent to that of wild type CD4, suggesting that raft localization of CD4 might not be required for virus entry. However, we cannot exclude the possibility that HIV binding to CD4 receptors localized outside rafts could stimulate raft assembly before or after engaging coreceptors or during fusion/entry process. Further studies are aimed to answer these questions. We and others have demonstrated that antiretroviral activity of APOBEC3G requires its packaging into assembling HIV virions through interaction with the Gag nucleocapsid protein NC. However, this process is severely limited in the presence of the viral accessory protein Vif, which binds to and target APOBEC3G for degradation by proteasomes. Thus, finding means of escaping of Vif-mediated degradation has become a challenge to developing an effective APOBEC3G-based antiretroviral therapy. Recently, we have found that APOBEC3G can be efficiently packaged into exosomes, cell-derived nanovesicles that showed promissing potential in cancer therapy clinical trials. Our current research is focused on the elucidation of the molecular mechanism(s) for targeting APOBEC3G into exosomes and evaluating the potential of exosome-encapsidated APOBEC3G as a novel approach in anti-HIV therapy.