Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3.
Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3.
复制标题
人类细胞中Chikungunya病毒复制子的持续复制与存在含有非结构蛋白3的稳定细胞质颗粒有关。
DOI：
10.1128/jvi.00477-18
复制
发表时间：
2018-08-15
影响因子：
5.4
通讯作者：
Harris M
中科院分区：
文献类型：
--
作者：
Remenyi R;Gao Y;Hughes RE;Curd A;Zothner C;Peckham M;Merits A;Harris M
基孔肯雅病毒（CHIKV）是一种由蚊子传播的重新出现的甲病毒，可引起短暂性疾病，但也可引起影响肌肉和关节的慢性疾病。没有批准的疫苗或抗病毒药物可用。因此，更好地了解病毒的生命周期和病毒蛋白的作用可以帮助确定新的治疗靶点。在显微镜和非细胞毒性复制子的发展（A。乌特峰达斯，M。Varjak，V. Lulla，A. Lulla，A. Merits，J Virol 89：3145-3162，2015，https：//doi.org/10.1128/JVI.03213-14）已经允许研究人员在受控的实验室环境中在延长的持续时间内研究病毒蛋白。在这里，我们建立了稳定复制子RNA和表达标记的非结构蛋白3（nsP 3）的人类细胞。在宿主细胞内和持续复制期间跟踪nsP 3的能力可以使基础研究工作受益，以更好地理解病毒蛋白复合物持续存在的长期后果，从而为控制CHIKV感染和治疗慢性疾病症状的新治疗靶点提供基础。基孔肯雅病毒（CHIKV）是一种由蚊子传播的人类病原体，可导致一种致残性疾病，其特征是严重的关节疼痛，患者可持续数周、数月甚至数年。非结构蛋白3（nsP 3）在急性感染期间发挥重要作用，但对nsP 3在慢性疾病期间的功能知之甚少。在这里，我们使用亚衍射显微镜对持续复制复制子RNA的人类细胞内的CHIKV nsP 3进行空间和时间分析。不同大小的圆形细胞质颗粒（i）含有nsP 3和应激颗粒组装因子1和2（G3 BP 1/2），（ii）靠近双链RNA灶和nsP 1阳性结构，（iii）靠近核膜和核孔复合物蛋白Nup 98。通过活细胞显微镜对蛋白质周转率和流动性的分析表明，颗粒可以持续数小时至数天，积累新合成的蛋白质，并以不同的速度在细胞质中移动。颗粒还具有静态内部结构，并且在细胞裂解物中稳定。清除了非细胞毒性复制子的难治性细胞重新获得了对亚砷酸盐诱导的应激反应的能力。总之，nsP 3可以形成独特的稳定颗粒结构，在宿主细胞内长期存在。这种病毒和细胞蛋白复合物的持续存在对于研究挥之不去的CHIKV感染的致病后果以及制定减轻由医学上重要的节肢动物传播病毒（虫媒病毒）带来的慢性肌肉骨骼疾病负担的策略具有意义。基孔肯雅病毒（CHIKV）是一种由蚊子传播的重新出现的甲病毒，可引起短暂性疾病，但也可引起影响肌肉和关节的慢性疾病。没有批准的疫苗或抗病毒药物可用。因此，更好地了解病毒的生命周期和病毒蛋白的作用可以帮助确定新的治疗靶点。在显微镜和非细胞毒性复制子的发展（A。乌特峰达斯，M。Varjak，V. Lulla，A. Lulla，A. Merits，J Virol 89：3145-3162，2015，https：//doi.org/10.1128/JVI.03213-14）已经允许研究人员在受控的实验室环境中在延长的持续时间内研究病毒蛋白。在这里，我们建立了稳定复制子RNA和表达标记的非结构蛋白3（nsP 3）的人类细胞。在宿主细胞内和持续复制期间跟踪nsP 3的能力可以使基础研究工作受益，以更好地理解病毒蛋白复合物持续存在的长期后果，从而为控制CHIKV感染和治疗慢性疾病症状的新治疗靶点提供基础。
Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145–3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms. Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months, or even years in patients. The nonstructural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used subdiffraction multicolor microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2), (ii) were next to double-stranded RNA foci and nsP1-positive structures, and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that the granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at various speeds. The granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the noncytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus). IMPORTANCE Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145–3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.