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可视化甲型流感病毒vRNA复制

Visualizing Influenza A Virus vRNA Replication.

作者信息

Chiu Ya-Fang, Huang Yi-Wen, Chen Chi-Yuan, Chen Yu-Chia, Gong Yu-Nong, Kuo Rei-Lin, Huang Chung-Guei, Shih Shin-Ru

机构信息

Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan.

Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, Taiwan.

出版信息

Front Microbiol. 2022 Jun 6;13:812711. doi: 10.3389/fmicb.2022.812711. eCollection 2022.

DOI:10.3389/fmicb.2022.812711
PMID:35733972
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9207383/
Abstract

Influenza A virus (IAV) has caused recurrent epidemics and severe pandemics. In this study, we adapted an MS2-MCP live-cell imaging system to visualize IAV replication. A reporter plasmid, pHH-PB2-vMSL, was constructed by replacing a part of the PB2-coding sequence in pHH-PB2 with a sequence encoding 24 copies of a stem-loop structure from bacteriophage MS2 (MSL). Binding of MS2 coat protein (MCP) fused to green fluorescent protein (GFP) to MSL enabled the detection of vRNA as fluorescent punctate signals in live-cell imaging. The introduction of pHH-PB2-vMSL into A549 cells transduced to express an MCP-GFP fusion protein lacking the nuclear localization signal (MCP-GFPdN), subsequently allowed tracking of the distribution and replication of PB2-vMSL vRNA after IAV PR8 infection. Spatial and temporal measurements revealed exponential increases in vRNA punctate signal intensity, which was only observed after membrane blebbing in apoptotic cells. Similar signal intensity increases in apoptotic cells were also observed after MDCK cells, transduced to express MCP-GFPdN, were infected with IAV carrying PB2-vMSL vRNA. Notably, PB2-vMSL vRNA replication was observed to occur only in apoptotic cells, at a consistent time after apoptosis initiation. There was a lack of observable PB2-vMSL vRNA replication in non-apoptotic cells, and vRNA replication was suppressed in the presence of apoptosis inhibitors. These findings point to an important role for apoptosis in IAV vRNA replication. The utility of the MS2-imaging system for visualizing time-sensitive processes such as viral replication in live host cells is also demonstrated in this study.

摘要

甲型流感病毒（IAV）已引发多次流行和严重大流行。在本研究中，我们采用了一种MS2-MCP活细胞成像系统来可视化IAV复制。通过用编码来自噬菌体MS2（MSL）的24个茎环结构拷贝的序列替换pHH-PB2中PB2编码序列的一部分，构建了一个报告质粒pHH-PB2-vMSL。与绿色荧光蛋白（GFP）融合的MS2外壳蛋白（MCP）与MSL的结合使得在活细胞成像中能够将vRNA检测为荧光点状信号。将pHH-PB2-vMSL引入转导以表达缺乏核定位信号的MCP-GFP融合蛋白（MCP-GFPdN）的A549细胞中，随后可以追踪IAV PR8感染后PB2-vMSL vRNA的分布和复制。空间和时间测量显示vRNA点状信号强度呈指数增加，这仅在凋亡细胞的膜泡化后观察到。在用携带PB2-vMSL vRNA的IAV感染转导以表达MCP-GFPdN的MDCK细胞后，也观察到凋亡细胞中类似的信号强度增加。值得注意的是，观察到PB2-vMSL vRNA复制仅在凋亡细胞中发生，且在凋亡开始后的一致时间出现。在非凋亡细胞中未观察到明显的PB2-vMSL vRNA复制，并且在存在凋亡抑制剂的情况下vRNA复制受到抑制。这些发现表明凋亡在IAV vRNA复制中起重要作用。本研究还证明了MS2成像系统在可视化活宿主细胞中诸如病毒复制等对时间敏感的过程方面的实用性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/cd020b69d875/fmicb-13-812711-g009.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/69be90e337e2/fmicb-13-812711-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/57833eb48658/fmicb-13-812711-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/8b7b93d8efec/fmicb-13-812711-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/cd020b69d875/fmicb-13-812711-g009.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/d4b6bbc79a0c/fmicb-13-812711-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/9f84fba2035f/fmicb-13-812711-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/433bde85eef3/fmicb-13-812711-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/69be90e337e2/fmicb-13-812711-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/57833eb48658/fmicb-13-812711-g007.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3394/9207383/cd020b69d875/fmicb-13-812711-g009.jpg

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