The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity.
The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity.
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DOI：
10.1371/journal.ppat.1008850
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发表时间：
2020-09
期刊：
影响因子：
6.7
通讯作者：
Cosset FL
中科院分区：
文献类型：
--
作者：
Freitas N;Enguehard M;Denolly S;Levy C;Neveu G;Lerolle S;Devignot S;Weber F;Bergeron E;Legros V;Cosset FL
克里米亚-刚果出血热病毒（CCHFV）是一种蜱传正鼻病毒，已成为严重威胁公共卫生的重要病原。CCHFV具有由L、M和S片段组成的单链三重RNA基因组。M多聚蛋白前体的切割产生两个包膜糖蛋白（GP）以及三个分泌的非结构蛋白GP 38和GP 85或GP 160，代表仅GP 38或GP 38连接到粘蛋白样蛋白（MLD）和称为NSm的双跨膜蛋白。在这里，我们研究了每个M-节段的非结构蛋白在病毒组装，出口和感染性的相关性，使用一个完善的CCHFV病毒样颗粒系统（tc-VLP）。MLD蛋白的缺失对感染性没有影响，尽管它减少了60%的GP掺入颗粒中。GP 38的额外缺失消除了感染性tc-VLP的产生。感染性的丧失与受损的Gc成熟和高尔基体的排斥有关，表明Gn不足以将CCHFV GP靶向组装位点。与此一致，在反式表达MLD-GP 38的细胞中实现了有效的互补，其中preGc至Gc的转化水平增加，共靶向高尔基体，导致颗粒掺入并恢复感染性。相反，保留在ER中的MLD-GP 38变体允许preGc切割，但未能挽救错误定位或感染性。相反，NSm缺失不影响Gc的运输，但干扰Gc加工、颗粒形成和分泌。NSm表达影响不同病毒蛋白的N-糖基化，最有可能是由于通过分泌途径的运输速度增加。这突出了NSm在克服高尔基体保留和促进CCHFV出口中的潜在作用。因此，GP 38或NSm的缺失表明它们对CCHFV颗粒产生和感染性的重要作用。GP 85是前Gc切割、运输和Gc掺入颗粒的必需病毒因子，而NSm蛋白参与CCHFV组装和病毒粒子分泌。正鼻病毒，如致命的克里米亚-刚果出血热病毒（CCHFV），除了通过病毒M区段编码的多蛋白的内部切割加工的病毒粒子包膜糖蛋白（Gn和Gc）之外，还编码分泌的糖蛋白，如GP 38。CCHFV MLD-GP 38蛋白（GP 160/GP 85）还包括N-末端结构域，其包含通过弗林蛋白酶从GP 38释放的粘蛋白样蛋白。先前针对GP 38的非中和性单克隆抗体对致死性CCHFV攻击的保护作用突出了GP 38在CCHFV复制中的重要性。CCHFV还编码一个功能未知的双跨膜蛋白（NSm），位于多聚蛋白上的Gn和Gc之间。为了研究这些所谓的辅助蛋白编码的CCHFV的M-节段在病毒的形成和感染性的作用，我们产生了几个M-节段缺失突变体，并测试它们在CCHFV的转录进入能力的病毒样颗粒（tc-VLP）系统。在这里，我们表明，GP 38是至关重要的GC生物合成，与Gn和运输到高尔基体的相互作用，它的删除废除感染性颗粒的形成。我们还表明，NSm通过分泌途径增加蛋白质运输的速率，改变N-糖基化概况，有利于有效的病毒释放。这些数据推进了我们对GP 38和NSm作用以及CCHFV-宿主相互作用的理解。
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion. Orthonairoviruses, like the lethal Crimean-Congo hemorrhagic fever virus (CCHFV), encode secreted glycoproteins, such as GP38, in addition to virion envelope glycoproteins (Gn and Gc) that are processed by internal cleavage of the viral M segment encoded polyprotein. CCHFV MLD-GP38 proteins (GP160/GP85) also include an N-terminal domain encompassing a mucin-like protein that is released from GP38 by Furin. The protective effect of non-neutralizing monoclonal antibodies targeting GP38 against lethal CCHFV challenge previously highlighted the importance of GP38 in CCHFV replication. CCHFV also encodes a double-membrane-spanning protein (NSm) of unknown function, located between the Gn and Gc on the polyprotein. To investigate the roles of these so-called accessory proteins encoded by the CCHFV M-segment in virus formation and infectivity, we generated several M-segment deletion mutants and tested them in a CCHFV transcription-entry-competent virus-like particle (tc-VLP) system. Here, we demonstrate that GP38 is crucial for Gc biogenesis, interaction with Gn and trafficking to the Golgi, and that its deletion abrogates formation of infectious particles. We also show that NSm increases the rate of protein trafficking through the secretory pathway with altered N-glycosylation profiles that are advantageous for efficient virus release. These data advanced our understanding of GP38 and NSm roles and CCHFV-host interactions.
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影响因子：
16.6
作者：
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