A Retrotranslocation Assay That Predicts Defective VCP/p97-Mediated Trafficking of a Retroviral Signal Peptide.
A Retrotranslocation Assay That Predicts Defective VCP/p97-Mediated Trafficking of a Retroviral Signal Peptide.
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一种预测缺陷VCP/p97介导的逆转录病毒信号肽运输的逆转录易位试验。
DOI：
10.1128/mbio.02953-21
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发表时间：
2022-02-22
期刊：
影响因子：
6.4
通讯作者：
Dudley JP
中科院分区：
文献类型：
--
作者：
Das P;Xu WK;Gautam AKS;Lozano MM;Dudley JP
对病毒复制的研究提供了对宿主过程的关键见解，包括蛋白质贩运和周转。小鼠乳腺肿瘤病毒(MMTV)是一种编码98个氨基酸的信号肽(SP)的β病毒。MMTV SP是由Rem和包膜前体蛋白在内质网(ER)膜上通过信号肽酶裂解产生的。我们先前的研究表明，SP是一种人类免疫缺陷病毒1型(HIV-1)Rev样蛋白，依赖于AAA ATPase Valosin-Holding Protein(VCP)/p97来颠覆ER相关降解(ERAD)。SP在N端的45个氨基酸中含有一个核定位序列(NLS)/核仁定位序列(NOLS)。为了直接确定膜提取和转运所需的SP区域，我们建立了生物素受体多肽(BAP)标记的SP蛋白的定量逆转位分析方法。用BAP标记的MMTV SP的丙氨酸替代突变体进行逆位分析，发现57和58位氨基酸(M57-58)的突变干扰了内质网膜的提取，而邻近突变不干扰内质网膜的提取。在免疫共沉淀实验中，M57-58突变体与VCP/p97的相互作用也减少。通过转染和报告实验检测BAP标记蛋白的活性，与野生型SP相比，M57-58和邻近的突变体(M59-61)都存在功能缺陷。共聚焦显微镜显示SP核转运存在缺陷，M57-58和M59-61定位异常。此外，纯化的谷胱甘肽S转移酶标记的M57-58和M59-61与标记的野生型SP相比，寡聚能力降低。这些实验表明，SP氨基酸57和58对于VCP/p97相互作用和反向易位是关键的，而57到61残基是与NLS/NOLS无关的寡聚和核运输的关键。我们的结果强调了复杂的宿主与长信号肽的相互作用。
Studies of viral replication have provided critical insights into host processes, including protein trafficking and turnover. Mouse mammary tumor virus (MMTV) is a betaretrovirus that encodes a functional 98-amino-acid signal peptide (SP). MMTV SP is generated from both Rem and envelope precursor proteins by signal peptidase cleavage in the endoplasmic reticulum (ER) membrane. We previously showed that SP functions as a human immunodeficiency virus type 1 (HIV-1) Rev-like protein that is dependent on the AAA ATPase valosin-containing protein (VCP)/p97 to subvert ER-associated degradation (ERAD). SP contains a nuclear localization sequence (NLS)/nucleolar localization sequence (NoLS) within the N-terminal 45 amino acids. To directly determine the SP regions needed for membrane extraction and trafficking, we developed a quantitative retrotranslocation assay with biotin acceptor peptide (BAP)-tagged SP proteins. Use of alanine substitution mutants of BAP-tagged MMTV SP in retrotranslocation assays revealed that mutation of amino acids 57 and 58 (M57-58) interfered with ER membrane extraction, whereas adjacent mutations did not. The M57-58 mutant also showed reduced interaction with VCP/p97 in coimmunoprecipitation experiments. Using transfection and reporter assays to measure activity of BAP-tagged proteins, both M57-58 and an adjacent mutant (M59-61) were functionally defective compared to wild-type SP. Confocal microscopy revealed defects in SP nuclear trafficking and abnormal localization of both M57-58 and M59-61. Furthermore, purified glutathione S-transferase (GST)-tagged M57-58 and M59-61 demonstrated reduced ability to oligomerize compared to tagged wild-type SP. These experiments suggest that SP amino acids 57 and 58 are critical for VCP/p97 interaction and retrotranslocation, whereas residues 57 to 61 are critical for oligomerization and nuclear trafficking independent of the NLS/NoLS. Our results emphasize the complex host interactions with long signal peptides.