Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription
Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription
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DOI：
10.1371/journal.ppat.1007239
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发表时间：
2019-01-01
期刊：
影响因子：
6.7
通讯作者：
Harris, Mark
中科院分区：
文献类型：
--
作者：
Gao, Yanni;Goonawardane, Niluka;Harris, Mark
基孔肯雅病毒（CHIKV）是一种重新出现的甲病毒，引起发热，关节疼痛，皮疹，关节痛，偶尔死亡。迫切需要抗病毒疗法和/或有效的疫苗。CHIKV生物学知之甚少，特别是非结构蛋白3（nsP 3）的功能。在这里，我们提出了nsP 3的甲病毒独特结构域（AUD）的致突变分析的结果。根据辛德比斯病毒AUD的结构和多种甲病毒的氨基酸序列比对，在CHIKV亚基因组复制子中产生了AUD的一系列突变。该分析揭示了AUD在CHIKV RNA复制中的重要作用，突变体表现出物种和细胞类型特异性表型。为了测试AUD是否在病毒生命周期的其他阶段发挥作用，在感染性CHIKV的背景下分析突变体。该分析表明，AUD也是病毒组装所必需的。特别地，一个突变体（P247 A/V248 A）表现出感染性病毒产生的显著减少。这种表型被证明是由于亚基因组RNA转录的阻断导致结构蛋白合成减少和伴随的病毒产生减少。这种表型可以通过P247 A/V248 A突变体nsP 3与病毒基因组RNA在体内的结合减少以及突变体AUD对亚基因组启动子RNA在体外的亲和力降低来进一步解释。我们认为AUD是一个多效性蛋白质结构域，在CHIKV RNA合成过程中具有多种功能。作者摘要基孔肯雅病毒（CHIKV）是一种新兴的威胁世界健康的病毒。它是由伊蚊属蚊子传播的，并在地球仪引起了大规模的流行病。这种病毒会引起发烧、皮疹、关节炎，有时可能致命。CHIKV的生物学知之甚少，为了解决这一缺陷，我们旨在鉴定病毒蛋白之一nsP 3的功能。我们专注于这种蛋白质的中心部分，称为甲病毒独特结构域（AUD），因为它是CHIKV所特有的病毒属-甲病毒-而不存在于其他相关病毒中。通过改变AUD的氨基酸序列（突变）并分析这些变化的影响，我们表明它参与了病毒生命周期的多个阶段。这些观察结果将nsP 3和AUD特别鉴定为抗病毒治疗或合理疫苗设计的潜在靶标。
Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.Author summary Chikungunya virus (CHIKV) is an emerging threat to world health. It is transmitted by Aedes species mosquitos, and has caused massive epidemics across the globe. The virus causes fever, rash, arthritis and can sometimes be fatal. The biology of CHIKV is poorly understood, to address this deficiency we aimed to identify functions of one of the viral proteins, nsP3. We focused on the central part of this protein, termed the alphavirus unique domain (AUD) because it is unique to the genus of viruses to which CHIKV belongs-the Alphaviruses-and not present in other related viruses. By making changes (mutations) in the amino acid sequence of the AUD and analysing the effects of these changes we show that it is involved in multiple stages of the virus lifecycle. These observations identify nsP3 and the AUD in particular as a potential target for antiviral therapy or rational vaccine design.