Lutz Lindy M, Pace Chandler R, Arnold Michelle M
Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USAInstituto de Biotecnologia/UNAM.
Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USAInstituto de Biotecnologia/UNAM
J Virol. 2016 Jun 10;90(13):6036-48. doi: 10.1128/JVI.00704-16. Print 2016 Jul 1.
The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.
The ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.
轮状病毒非结构蛋白NSP1通过诱导激活干扰素（IFN）产生所需的关键蛋白降解，充当宿主抗病毒反应的拮抗剂。NSP1诱导的蛋白降解依赖于蛋白酶体，并且在N端附近存在一个RING结构域，这导致了NSP1是一种E3泛素连接酶的假说。为了检验这一假说，进行了下拉实验，随后进行质谱分析以鉴定与NSP1相关的宿主泛素化机制的成分。鉴定出了作为必需多亚基泛素化复合物的cullin RING连接酶（CRL）的多个成分与NSP1相关。在转染细胞和感染细胞中均验证了质谱分析，结果表明来自不同轮状病毒株的NSP1蛋白与CRL复合物的关键成分相关，最显著的是cullin支架蛋白Cul3和Cul1。使用纯化蛋白进行的体外结合实验证实NSP1与Cul3特异性相互作用，并且Cul3的N端区域负责与NSP1结合。为了测试NSP1是否利用CRL3诱导靶蛋白IRF3或β-TrCP的降解，使用小干扰RNA（siRNA）方法敲低了Cul3的水平。出乎意料的是，Cul3的缺失并未使感染细胞中的IRF3或β-TrCP免于降解。结果表明，NSP1可能不是通过积极利用CRL复合物来诱导IFN产生所需的靶蛋白降解，而是利用含cullin的复合物来阻止另一种细胞活性。
泛素-蛋白酶体途径在众多细胞功能中发挥重要的调节作用，许多病毒已经进化出利用或操纵该途径以增强复制和传播的机制。轮状病毒是幼儿严重胃肠炎的主要病因，每年在全球导致约42万人死亡，它利用泛素-蛋白酶体系统通过诱导干扰素（IFN）产生所需的关键成分降解来颠覆宿主的先天免疫反应。在这里，我们表明来自不同轮状病毒株的NSP1蛋白与CRL泛素连接酶复合物的支架蛋白Cul1和Cul3相关。尽管如此，Cul1和Cul3的敲低表明NSP1诱导某些靶蛋白的降解与其与CRL复合物的关联无关，这强调需要进一步研究NSP1如何颠覆宿主IFN反应的机制细节。
