Modulations in the host cell proteome by the hantavirus nucleocapsid protein.
Modulations in the host cell proteome by the hantavirus nucleocapsid protein.
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DOI：
10.1371/journal.ppat.1011925
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发表时间：
2024-01
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
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汉坦病毒已经进化出一种独特的翻译策略来促进感染细胞中病毒mRNA的翻译。汉坦病毒核衣壳蛋白（NP）通过核糖体蛋白S19与病毒mRNA 5 ' UTR和40S核糖体亚基结合。NP相关核糖体选择性地装载在病毒转录本上以促进其翻译。在这里，我们证明NP表达上调宿主细胞因子的一个子集的稳态水平，主要参与内质网的蛋白质加工。对转染细胞和病毒感染细胞中表达上调的宿主因子之一Valosin-containing protein （VCP/p97）的详细研究表明，在VCP mRNA 5 ' UTR的辅助下，NP促进下游VCP ORF的翻译。VCP mRNA包含一个5 ' UTR 987核苷酸窝藏6不寻常的启动密码子上游的正确启动密码子的VCP位于第988位位置从5 '帽。体外翻译GFP记者记录窝藏VCP信使rna 5 ' UTR产生GFP和短多肽~ 14 KDa的翻译起始密码子开始位于5 ' UTR在542位置从5 '帽。翻译起始542 8月在UTR序列证实细胞使用表达mCherry和GFP的双报告结构。14KDa多肽的合成极大地抑制了ORF从5 ‘帽的下游正确起始密码子988位的翻译。我们报道纯化的NP与VCP mRNA 5 ’ UTR具有高亲和力，并且NP结合位点位于542和aug附近。NP结合关闭14KDa多肽的翻译，从而促进正确AUG密码子的翻译起始。敲除VCP使细胞质中传染性较差的汉坦病毒颗粒水平降低，其在人脐静脉内皮细胞中的表达受到显著抑制。我们证明了VCP在汉坦病毒糖蛋白Gn被整合到组装病毒粒子之前就与之结合，并在感染过程中促进病毒向邻近细胞传播。我们的研究结果表明，核糖体参与5 ' UTR中542 AUG密码子可能调节细胞内源性VCP的稳态水平。汉坦病毒阻断了这一调控机制，增强了病毒感染细胞中VCP的稳态水平。这种增强有利于病毒复制，支持病毒向邻近细胞传播，并促进感染性病毒颗粒从宿主细胞释放。Valosin-containing protein （VCP）是蛋白质质量控制系统的重要组成部分，它控制着多种细胞过程，包括高尔基-内质网膜融合和高尔基组装。VCP在汉坦病毒感染的细胞中翻译上调。在体外翻译系统中，VCP mRNA的5 ' UTR中一个不寻常的起始密码子的翻译起始产生约14KDa的短多肽。含有VCP mRNA 5 ' UTR的GFP报告基因的翻译表明，14KDa多肽的合成对GFP报告基因的翻译产生负向影响。我们提出14KDa多肽的合成可能是宿主通过控制VCP正确起始密码子核糖体接合来调节细胞中VCP稳态水平的机制。我们的研究结果表明，汉坦病毒编码的NP与VCP mRNA的5 ' UTR具有高亲和力。NP可关闭报告转录物中14KDa多肽的翻译，从而促进细胞内和体外翻译系统中的报告转录。汉坦病毒很可能在感染期间使用相同的策略来翻译上调VCP。利用基因敲低和化学抑制方法，我们证明了VCP在汉坦病毒复制的多个阶段起着至关重要的作用，并且是汉坦病毒颗粒从感染细胞出口所必需的，这是病毒传播到邻近细胞的关键事件。
Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5’ UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5’ UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5’ UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5’ cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5’ UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5’ UTR at 542nd position from the 5’ cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5’ cap. We report that purified NP binds to the VCP mRNA 5’ UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5’ UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell. Valosin-containing protein (VCP), a crucial component in the regulation of protein quality control system, governs diverse cellular processes, including Golgi-ER membrane fusion and Golgi assembly. VCP is translationally upregulated in hantavirus infected cells. Translation initiation from an unusual start codon in the 5’ UTR of the VCP mRNA generates a short polypeptide of ~14KDa in an in vitro translation system. Translation of GFP reporter mRNA, harboring the VCP mRNA 5’ UTR, revealed that synthesis of 14KDa polypeptide negatively impacts the translation of the GFP reporter. We propose that synthesis of 14KDa polypeptide might be a host mechanism to regulate the steady state levels of VCP in cells by controlling the ribosome engagement at the correct start codon for VCP. Our results demonstrate that hantavirus encoded NP binds to the 5’ UTR of VCP mRNA with high affinity. NP shuts down the translation of 14KDa polypeptide in the reporter transcript, which boosts the reporter translation both in cells and in in vitro translation system. It is likely that hantavirus use the same strategy to translationally upregulate the VCP during infection. Using gene knockdown and chemical inhibition approaches, we demonstrate that VCP plays a vital role at multiple stages of hantavirus replication and is required for the egress of hantavirus particles from infected cells, a critical event for viral dissemination to neighboring cells.
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影响因子：
5.6
作者：
Costantini S;Capone F;Polo A;Bagnara P;Budillon A
通讯作者：
Budillon A
DOI：
10.1073/pnas.192298899
发表时间：
2002-10-15
影响因子：
11.1
作者：
Geimonen, E;Neff, S;Mackow, ER
通讯作者：
Mackow, ER
DOI：
10.1038/nsb972
发表时间：
2003-10-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
DeLaBarre, B;Brunger, AT
通讯作者：
Brunger, AT
影响因子：
16.2
作者：
DOZIO, N;MICOSSI, P;SODOYEZ, JC
通讯作者：
SODOYEZ, JC
影响因子：
4
作者：
Meyer, Hemmo;Weihl, Conrad C.
通讯作者：
Weihl, Conrad C.