Chung Hyo-Young, Morita Eiji, von Schwedler Uta, Müller Barbara, Kräusslich Hans-Georg, Sundquist Wesley I
University of Utah, Department of Biochemistry, 15 N. Medical Drive, East, Salt Lake City, UT 84112-5650, USA.
J Virol. 2008 May;82(10):4884-97. doi: 10.1128/JVI.02667-07. Epub 2008 Mar 5.
The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6(Gag) bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.
细胞内的ESCRT途径在伴随内体蛋白分选、胞质分裂和包膜RNA病毒出芽的膜重塑事件中发挥作用。在最后一种情况中，人类免疫缺陷病毒1型（HIV-1）p6（Gag）内的短序列基序（称为晚期结构域）结合并招募两种ESCRT途径蛋白TSG101和ALIX，以促进病毒出芽。我们现在报告，HECT泛素E3连接酶NEDD4L/NEDD4-2的过表达刺激了缺乏TSG101和ALIX结合晚期结构域的HIV-1构建体的释放，使感染滴度增加了20倍以上。此外，内源性NEDD4L的缺失抑制了这些缺陷病毒的释放，并导致胞质分裂缺陷。病毒出芽的刺激依赖于NEDD4L的泛素连接酶活性，并且只需要最小的HIV-1 Gag组装区域，这表明Gag在p6区域之外具有泛素依赖性的顺式作用晚期结构域活性。NEDD4L的刺激也需要TSG101，并导致包括TSG101在内的几种ESCRT-I亚基的泛素化。最后，我们发现TSG101/ESCRT-I是Mason-Pfizer猴病毒有效释放所必需的，该病毒主要通过使用PPXY晚期结构域招募NEDD4样蛋白来出芽。这些观察结果表明，NEDD4L以及可能的其他NEDD4样蛋白可以泛素化并激活ESCRT-I以在病毒出芽中发挥作用。
