PDF(Pubmed)
Abstract：
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.
摘要：
基孔肯雅病毒(CHIKV),蚊子传播的人类病原体，导致一种以严重关节痛为特征的致残疾病，这种疼痛可以持续数周，月，甚至几年的病人。非结构蛋白3（nsP3）在急性感染中发挥重要作用，但对nsP3在慢性病中的功能知之甚少。这里,我们使用亚衍射多色显微镜对持续复制复制子RNA的人类细胞中的CHIKVnsP3进行时空分析。各种大小的圆形细胞质颗粒（i）包含nsP3和应激颗粒组装因子1和2（G3BP1/2），（ii）紧挨着双链RNA病灶和nsP1阳性结构，和（iii）靠近核膜和核孔复合物蛋白Nup98。通过活细胞显微镜分析蛋白质的周转和流动性表明，颗粒可以持续数小时至数天，积累新合成的蛋白质，并以不同的速度穿过细胞质。颗粒还具有静态内部结构并且在细胞裂解物中是稳定的。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,nsP3可以形成在宿主细胞内长期持续存在的独特稳定的颗粒状结构。病毒和细胞蛋白质复合物的持续存在对于研究持续的CHIKV感染的致病后果以及减轻医学上重要的节肢动物传播的病毒（虫媒病毒）带来的慢性肌肉骨骼疾病负担的策略的开发具有意义。重要性基孔肯雅病毒（CHIKV）是一种由蚊子传播的重新出现的甲病毒，会引起短暂的疾病，但也会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有助于确定新的治疗靶点.显微镜的进展和非细胞毒性复制子的发展（A.Utt,P.K.Das,M.Varjak,V.Lulla,A.Lulla,A.优点,JVirol89：3145-3162，2015，https://doi.org/10.1128/JVI.03213-14）允许研究人员在受控的实验室环境中研究病毒蛋白。在这里，我们建立了稳定复制复制子RNA并表达标记的非结构蛋白3（nsP3）的人细胞。在宿主细胞内和在持续复制期间跟踪nsP3的能力可以有益于基础研究工作，以更好地理解病毒蛋白复合物持久性的长期后果，从而为控制CHIKV感染和治疗慢性疾病症状的新治疗靶标提供基础。
