Visualizing Influenza A Virus vRNA Replication
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 encodin...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2022-06-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.812711/full |
| _version_ | 1811244448714063872 |
|---|---|
| author | Ya-Fang Chiu Ya-Fang Chiu Ya-Fang Chiu Yi-Wen Huang Chi-Yuan Chen Yu-Chia Chen Yu-Nong Gong Yu-Nong Gong Rei-Lin Kuo Chung-Guei Huang Shin-Ru Shih |
| author_facet | Ya-Fang Chiu Ya-Fang Chiu Ya-Fang Chiu Yi-Wen Huang Chi-Yuan Chen Yu-Chia Chen Yu-Nong Gong Yu-Nong Gong Rei-Lin Kuo Chung-Guei Huang Shin-Ru Shih |
| author_sort | Ya-Fang Chiu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T14:25:12Z |
| format | Article |
| id | doaj.art-60daeb5b7eb24ecca377a59ee77eb7d1 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-12T14:25:12Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-60daeb5b7eb24ecca377a59ee77eb7d12022-12-22T03:29:28ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-06-011310.3389/fmicb.2022.812711812711Visualizing Influenza A Virus vRNA ReplicationYa-Fang Chiu0Ya-Fang Chiu1Ya-Fang Chiu2Yi-Wen Huang3Chi-Yuan Chen4Yu-Chia Chen5Yu-Nong Gong6Yu-Nong Gong7Rei-Lin Kuo8Chung-Guei Huang9Shin-Ru Shih10Department of Microbiology and Immunology, Chang Gung University, Taoyuan, TaiwanResearch Center for Emerging Viral Infections, Chang Gung University, Taoyuan, TaiwanDepartment of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, TaiwanDepartment of Microbiology and Immunology, Chang Gung University, Taoyuan, TaiwanDepartment of Microbiology and Immunology, Chang Gung University, Taoyuan, TaiwanDepartment of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, TaiwanResearch Center for Emerging Viral Infections, Chang Gung University, Taoyuan, TaiwanDepartment of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, TaiwanResearch Center for Emerging Viral Infections, Chang Gung University, Taoyuan, TaiwanDepartment of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, TaiwanResearch Center for Emerging Viral Infections, Chang Gung University, Taoyuan, TaiwanInfluenza 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.https://www.frontiersin.org/articles/10.3389/fmicb.2022.812711/fullapoptosisinfluenza A viruslive-cell imagingviral-host interactionvRNA |
| spellingShingle | Ya-Fang Chiu Ya-Fang Chiu Ya-Fang Chiu Yi-Wen Huang Chi-Yuan Chen Yu-Chia Chen Yu-Nong Gong Yu-Nong Gong Rei-Lin Kuo Chung-Guei Huang Shin-Ru Shih Visualizing Influenza A Virus vRNA Replication Frontiers in Microbiology apoptosis influenza A virus live-cell imaging viral-host interaction vRNA |
| title | Visualizing Influenza A Virus vRNA Replication |
| title_full | Visualizing Influenza A Virus vRNA Replication |
| title_fullStr | Visualizing Influenza A Virus vRNA Replication |
| title_full_unstemmed | Visualizing Influenza A Virus vRNA Replication |
| title_short | Visualizing Influenza A Virus vRNA Replication |
| title_sort | visualizing influenza a virus vrna replication |
| topic | apoptosis influenza A virus live-cell imaging viral-host interaction vRNA |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.812711/full |
| work_keys_str_mv | AT yafangchiu visualizinginfluenzaavirusvrnareplication AT yafangchiu visualizinginfluenzaavirusvrnareplication AT yafangchiu visualizinginfluenzaavirusvrnareplication AT yiwenhuang visualizinginfluenzaavirusvrnareplication AT chiyuanchen visualizinginfluenzaavirusvrnareplication AT yuchiachen visualizinginfluenzaavirusvrnareplication AT yunonggong visualizinginfluenzaavirusvrnareplication AT yunonggong visualizinginfluenzaavirusvrnareplication AT reilinkuo visualizinginfluenzaavirusvrnareplication AT chunggueihuang visualizinginfluenzaavirusvrnareplication AT shinrushih visualizinginfluenzaavirusvrnareplication |