Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture

Affiliations

• Université de Paris-Cité, Biology of Emerging Viruses Team, INSERM U944/CNRS UMR 7212, Institut de Recherche Saint-Louis, Hôpital Saint Louis, Paris, France.
• European Molecular Biology Laboratory, Grenoble, France.
• CSSB Centre for Structural Systems Biology, Deutsches Elektronen Synchrotron DESY, Hamburg, Germany.
• Aix-Marseille Université, CNRS, AFMB UMR 7257, Marseille, France.
• Université́ de Tours, INSERM U1259 MAVIVH et Plateforme IBiSA de Microscopie Electronique, Tours, France.
• Aix-Marseille Université, CNRS, AFMB UMR 7257, Turing Centre for Living Systems, 13288, Marseille, France.
• Viral Macromolecular Complexes Team, ERL-INSERM U1324, Marseille, France.
• Institute of Virology, Medical Faculty, University of Bonn, Bonn, Germany.
• Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, IBS, Grenoble, France.
• Université de Paris-Cité, Biology of Emerging Viruses Team, INSERM U944/CNRS UMR 7212, Institut de Recherche Saint-Louis, Hôpital Saint Louis, Paris, France. ali.amara@inserm.fr.
• Aix-Marseille Université, CNRS, AFMB UMR 7257, Marseille, France. juan.reguera@inserm.fr.
• Viral Macromolecular Complexes Team, ERL-INSERM U1324, Marseille, France. juan.reguera@inserm.fr.

PMID:  39285216  DOI: 10.1038/s41467-024-51952-z

Abstract

Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function. Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 Å resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection.

基孔肯雅病毒（CHIKV）等甲病毒是由蚊子传播的病毒，在当前全球变暖的背景下对人类健康构成严重威胁。甲病毒的有效感染依赖于非结构蛋白3（nsP3）的活性，nsp3的功能尚不清楚，其在感染中的作用在很大程度上仍不为人所知。NsP3 是质膜结合的病毒 RNA 复制复合物（vRC）的一个组成部分，对 RNA 扩增至关重要，它还存在于功能未知的大型细胞质聚集体中。在这里，我们报告了分辨率为 2.35 Å 的 CHIKV nsP3 的冷冻电子显微镜（cryo-EM）结构。我们发现 nsP3 通过其甲病毒独特结构域 (AUD) 的螺旋排列组装成管状结构。nsP3 螺旋支架与成熟病毒 RC 的断层扫描重建中发现的冠状结构一致。此外，nsP3螺旋组装成细胞质颗粒，形成管状结构网络，其中包含病毒基因组RNA和囊膜以及生产性感染所需的宿主因子。结构诱导突变确定了阻止或干扰 nsP3 组装的残基，从而导致病毒复制或转录受损。总之，我们的研究结果揭示了一种意想不到的依赖 nsP3 的分子组织，它对甲病毒感染的不同阶段至关重要。