Wong Jerry, Zhang Jingchun, Si Xiaoning, Gao Guang, Mao Ivy, McManus Bruce M, Luo Honglin
The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia-St. Paul's Hospital, 1081 Burrard St., Vancouver, British Columbia, Canada.
J Virol. 2008 Sep;82(18):9143-53. doi: 10.1128/JVI.00641-08. Epub 2008 Jul 2.
Recent studies suggest a possible takeover of host antimicrobial autophagy machinery by positive-stranded RNA viruses to facilitate their own replication. In the present study, we investigated the role of autophagy in coxsackievirus replication. Coxsackievirus B3 (CVB3), a picornavirus associated with viral myocarditis, causes pronounced intracellular membrane reorganization after infection. We demonstrate that CVB3 infection induces an increased number of double-membrane vesicles, accompanied by an increase of the LC3-II/LC3-I ratio and an accumulation of punctate GFP-LC3-expressing cells, two hallmarks of cellular autophagosome formation. However, protein expression analysis of p62, a marker for autophagy-mediated protein degradation, showed no apparent changes after CVB3 infection. These results suggest that CVB3 infection triggers autophagosome formation without promoting protein degradation by the lysosome. We further examined the role of the autophagosome in CVB3 replication. We demonstrated that inhibition of autophagosome formation by 3-methyladenine or small interfering RNAs targeting the genes critical for autophagosome formation (ATG7, Beclin-1, and VPS34 genes) significantly reduced viral replication. Conversely, induction of autophagy by rapamycin or nutrient deprivation resulted in increased viral replication. Finally, we examined the role of autophagosome-lysosome fusion in viral replication. We showed that blockage of the fusion by gene silencing of the lysosomal protein LAMP2 significantly promoted viral replication. Taken together, our results suggest that the host's autophagy machinery is activated during CVB3 infection to enhance the efficiency of viral replication.
最近的研究表明，正链RNA病毒可能会接管宿主的抗菌自噬机制以促进自身复制。在本研究中，我们调查了自噬在柯萨奇病毒复制中的作用。柯萨奇病毒B3（CVB3）是一种与病毒性心肌炎相关的小RNA病毒，感染后会引起明显的细胞内膜重组。我们证明，CVB3感染会诱导双膜囊泡数量增加，同时LC3-II/LC3-I比值升高以及表达点状GFP-LC3的细胞积累，这是细胞自噬体形成的两个标志。然而，自噬介导的蛋白质降解标志物p62的蛋白质表达分析显示，CVB3感染后没有明显变化。这些结果表明，CVB3感染触发自噬体形成，但不促进溶酶体介导的蛋白质降解。我们进一步研究了自噬体在CVB3复制中的作用。我们证明，用3-甲基腺嘌呤或靶向自噬体形成关键基因（ATG7、Beclin-1和VPS34基因）的小干扰RNA抑制自噬体形成会显著降低病毒复制。相反，雷帕霉素或营养剥夺诱导自噬会导致病毒复制增加。最后，我们研究了自噬体-溶酶体融合在病毒复制中的作用。我们表明，通过溶酶体蛋白LAMP2的基因沉默阻断融合会显著促进病毒复制。综上所述，我们的结果表明，宿主的自噬机制在CVB3感染期间被激活，以提高病毒复制效率。
