Tabata Keisuke, Arimoto Masaru, Arakawa Masashi, Nara Atsuki, Saito Kazunobu, Omori Hiroko, Arai Arisa, Ishikawa Tomohiro, Konishi Eiji, Suzuki Ryosuke, Matsuura Yoshiharu, Morita Eiji
Laboratory of Viral Infection, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
Cell Rep. 2016 Aug 30;16(9):2339-47. doi: 10.1016/j.celrep.2016.07.068. Epub 2016 Aug 18.
Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.
黄病毒感染会诱导内质网（ER）膜重排，以形成一个用于病毒基因组复制和病毒颗粒组装的区室。利用定量质谱分析，我们鉴定出了几种被招募到内质网上病毒复制位点的内体分选转运所需复合体（ESCRT）蛋白。系统性的小干扰RNA（siRNA）筛选显示，单独敲低TSG101或共同敲低特定组合的ESCRT-III蛋白会显著降低登革病毒和日本脑炎病毒的释放，导致病毒滴度降低≥1000倍。相比之下，敲低包括VPS4在内的一些核心ESCRT蛋白对病毒释放没有影响。将ESCRT蛋白重新引入经siRNA处理的细胞中，揭示了ESCRT蛋白之间对黄病毒出芽至关重要的相互作用。电子显微镜研究表明，CHMP2和CHMP4蛋白直接在内质网的膜变形过程中发挥作用。因此，ESCRT的一个独特且特定的亚群在黄病毒感染期间有助于内质网膜生物发生。
