Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Department of Biophysics, University of Michigan, Ann Arbor, Michigan, USA
J Virol. 2015 Jan;89(1):454-67. doi: 10.1128/JVI.02178-14. Epub 2014 Oct 15.
HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however, the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of the uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in nonpolarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color superresolution localization microscopy. Consistent with the findings of the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with the cytoplasmic tail of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants.
Nascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote the association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using superresolution localization microscopy suggest the presence of a novel molecular mechanism promoting the association of PSGL-1, CD43, and CD44 with assembling HIV-1 which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein.
HIV-1 在质膜处进行粒子组装时会整合各种宿主膜蛋白；然而，介导这种整合过程的机制仍知之甚少。我们之前曾表明，HIV-1 结构蛋白 Gag 定位于极化 T 细胞的尾部结构尿足，并且在非极化 T 细胞中，组装的 Gag 共斑与尿足定向蛋白的一部分而非全部（即 PSGL-1、CD43 和 CD44）共定位。后一种观察结果表明存在一种促进这些细胞蛋白与病毒粒子结合的机制。为了解决这个可能性并确定分子决定因素，在本研究中，我们使用定量双色超分辨率定位显微镜检查了 T 细胞和 HeLa 细胞中 Gag 与跨膜蛋白之间的共聚类。与 T 细胞共斑研究的结果一致，我们发现 Gag 基质结构域内的碱性残基是 Gag-PSGL-1 共聚类所必需的。值得注意的是，PSGL-1 细胞质结构域中多碱性序列的存在显著增强了这种共聚类。我们还发现，CD43 和 CD44 细胞质尾部存在的多碱性基序也促进了它们与 Gag 的共聚类。ICAM-1 和 ICAM-3 是与 Gag 在 T 细胞中不共斑的尿足定向蛋白，而 CD46 是一种非尿足定向蛋白，与 Gag 无或几乎无共聚类。然而，用 PSGL-1 的细胞质尾巴代替它们的细胞质尾巴显著增强了它们与 Gag 的共聚类。总之，这些结果确定了一种新的宿主膜蛋白与组装中的 HIV-1 Gag 结合的机制，其中膜蛋白和 Gag 中存在的多碱性序列是主要决定因素。
在组装过程中，新生的 HIV-1 颗粒会整合许多宿主质膜蛋白。然而，很大程度上尚不清楚是什么机制促进了这些蛋白与质膜内病毒组装部位的关联。值得注意的是，我们之前的研究表明，HIV-1 结构蛋白 Gag 与 T 细胞质膜上的一组尿足定向跨膜蛋白 PSGL-1、CD43 和 CD44 共定位。使用超分辨率定位显微镜获得的研究结果表明，存在一种新的分子机制促进了 PSGL-1、CD43 和 CD44 与组装中的 HIV-1 的结合，该机制依赖于 HIV-1 Gag 中的多碱性序列和跨膜蛋白的细胞质结构域。这些信息增进了我们对宿主质膜蛋白病毒粒子掺入的理解，其中一些蛋白正向或负向调节病毒的传播，并提出了一种可能的新策略，即用所需的跨膜蛋白来富集 HIV-1 为基础的慢病毒载体。
