Jáuregui Paula, Logue Eric C, Schultz Megan L, Fung Stephanie, Landau Nathaniel R
Department of Microbiology, New York University School of Medicine, New York, New York, USA.
Department of Microbiology, New York University School of Medicine, New York, New York, USA
J Virol. 2015 May;89(10):5701-13. doi: 10.1128/JVI.03575-14. Epub 2015 Mar 11.
Sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.
SAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.
无菌α基序结构域和含HD结构域蛋白1（SAMHD1）限制人类免疫缺陷病毒1型（HIV-1）在髓系细胞和静息T细胞中的复制。诸如HIV-2和一些猴免疫缺陷病毒（SIV）等慢病毒通过编码Vpx或Vpr来对抗这种限制，Vpx或Vpr是包装在病毒粒子中的辅助蛋白，当病毒进入细胞质时，它们会诱导SAMHD1的蛋白酶体降解。作为研究这些机制的工具，我们构建了表达一种称为NLS.GFP.SAM595的融合蛋白的HeLa细胞系，其中SAMHD1的Vpx结合结构域与绿色荧光蛋白（GFP）的羧基末端融合，并且一个核定位信号与GFP的氨基末端融合。将含Vpx的病毒粒子与细胞一起孵育后，NLS.GFP.SAM595融合蛋白在数小时内被降解，并且由于CRL4 E3泛素连接酶的持续靶向作用，其水平在5天内一直保持较低。融合蛋白的降解要求其包含一个核定位序列。与细胞质蛋白muNS融合使该蛋白对Vpx介导的降解具有抗性，证实SAMHD1在细胞核中被靶向。用蛋白酶抑制剂处理的病毒粒子无法释放Vpx，表明病毒粒子脱壳对于Vpx从病毒粒子中释放是必需的。衣壳蛋白中的突变改变了病毒脱壳动力学，并且Gag结合药物PF74对Vpx介导的降解没有影响。这些结果表明，Vpx从病毒粒子中释放不需要衣壳脱壳，从而使得Vpx在进入细胞质后能够迅速转运至细胞核。
SAMHD1限制慢病毒在髓系细胞和静息T细胞中的复制。HIV-2等病毒编码一种包装在病毒粒子中且专门用于诱导SAMHD1降解的辅助蛋白，这一事实突出了其重要性。Vpx需要在感染后迅速发挥作用以使逆转录得以进行。病毒粒子中有限数量的Vpx分子还需要在较长时间内清除细胞内的SAMHD1。通过使用一种表达绿色荧光蛋白（GFP）-SAMHD1融合蛋白的工程化HeLa细胞系，我们表明Vpx依赖的降解无需病毒衣壳脱壳即可发生。此外，融合蛋白仅在定位于细胞核时才会被降解，证实SAMHD1在细胞核中被靶向，从而解释了Vpx为何也定位于细胞核。
