Taylor Adam, Liu Xiang, Zaid Ali, Goh Lucas Y H, Hobson-Peters Jody, Hall Roy A, Merits Andres, Mahalingam Suresh
Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia
Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia.
mBio. 2017 Feb 21;8(1):e01970-16. doi: 10.1128/mBio.01970-16.
Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. CHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain affecting millions of people. After a series of major outbreaks over the last 10 years, CHIKV and its mosquito vectors have been able to expand their range extensively, now making CHIKV a human pathogen of global importance. With no licensed vaccine or antiviral therapy for the treatment of CHIKV disease, there is a growing need to understand the molecular determinants of viral pathogenesis. These studies identify a previously uncharacterized nucleolar localization sequence (NoLS) in CHIKV capsid protein, begin a functional analysis of site-directed mutants of the capsid protein NoLS, and examine the effect of the NoLS mutation on CHIKV pathogenesis and its potential to influence CHIKV vaccine design. A better understanding of the pathobiology of CHIKV disease will aid the development of effective therapeutic strategies.
蚊子传播的基孔肯雅病毒（CHIKV）是一种致关节炎的甲病毒，属于该病毒科，可导致人类频繁爆发关节炎疾病。衣壳蛋白是由CHIKV RNA基因组编码的一种结构蛋白，能够转运至宿主细胞核仁。在脑炎甲病毒中，核转位会诱导宿主细胞转录关闭；然而，衣壳蛋白核仁定位在致关节炎甲病毒中的作用仍不清楚。我们使用重组增强绿色荧光蛋白（EGFP）标记的表达构建体和CHIKV感染性克隆，描述了衣壳蛋白N端区域中一个核仁定位序列（NoLS），该序列在CHIKV中以前未被鉴定。通过定点诱变对NoLS进行突变会降低CHIKV衣壳蛋白的核输入效率。在病毒中，衣壳蛋白NoLS（CHIKV-NoLS）的突变会减弱在哺乳动物细胞和蚊子细胞中的复制，产生小噬斑表型。CHIKV-NoLS的减毒可能是由于病毒RNA合成下游的病毒复制周期受到破坏。在小鼠中，与野生型CHIKV（CHIKV-WT）感染的小鼠相比，CHIKV-NoLS感染未引起疾病症状；缺乏疾病症状与病毒血症显著降低和促炎因子表达减少相关。用CHIKV-NoLS免疫的小鼠在免疫后30天用CHIKV-WT攻击，未出现疾病症状且未检测到病毒血症。来自CHIKV-NoLS免疫小鼠的血清能够有效中和CHIKV感染。此外，用相关甲病毒罗斯河病毒攻击CHIKV-NoLS免疫的小鼠后，其攻击后的早期和峰值病毒血症降低，表明有交叉保护作用。CHIKV-NoLS的高度减毒可能会改善CHIKV抗病毒和合理疫苗设计。CHIKV是一种蚊媒病原体，能够引发影响数百万人的使人丧失能力的关节疼痛的爆发性流行。在过去10年发生一系列重大疫情后，CHIKV及其蚊媒已能够广泛扩大其传播范围，现在使CHIKV成为具有全球重要性的人类病原体。由于没有用于治疗CHIKV疾病的许可疫苗或抗病毒疗法，对病毒发病机制分子决定因素的了解需求日益增加。这些研究鉴定了CHIKV衣壳蛋白中一个以前未被鉴定的核仁定位序列（NoLS），开始对衣壳蛋白NoLS的定点突变体进行功能分析，并研究NoLS突变对CHIKV发病机制及其影响CHIKV疫苗设计潜力的作用。更好地了解CHIKV疾病的病理生物学将有助于开发有效的治疗策略。
