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Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus
In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity ov...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9368937/ https://www.ncbi.nlm.nih.gov/pubmed/35955844 http://dx.doi.org/10.3390/ijms23158711 |
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author | Li, Xiaoyu Ito, Masahiko Aoyagi, Haruyo Murayama, Asako Aizaki, Hideki Fukasawa, Masayoshi Kato, Takanobu Wakita, Takaji Suzuki, Tetsuro |
author_facet | Li, Xiaoyu Ito, Masahiko Aoyagi, Haruyo Murayama, Asako Aizaki, Hideki Fukasawa, Masayoshi Kato, Takanobu Wakita, Takaji Suzuki, Tetsuro |
author_sort | Li, Xiaoyu |
collection | PubMed |
description | In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle. |
format | Online Article Text |
id | pubmed-9368937 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93689372022-08-12 Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus Li, Xiaoyu Ito, Masahiko Aoyagi, Haruyo Murayama, Asako Aizaki, Hideki Fukasawa, Masayoshi Kato, Takanobu Wakita, Takaji Suzuki, Tetsuro Int J Mol Sci Article In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle. MDPI 2022-08-05 /pmc/articles/PMC9368937/ /pubmed/35955844 http://dx.doi.org/10.3390/ijms23158711 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Li, Xiaoyu Ito, Masahiko Aoyagi, Haruyo Murayama, Asako Aizaki, Hideki Fukasawa, Masayoshi Kato, Takanobu Wakita, Takaji Suzuki, Tetsuro Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus |
title | Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus |
title_full | Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus |
title_fullStr | Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus |
title_full_unstemmed | Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus |
title_short | Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus |
title_sort | development and use of a kinetical and real-time monitoring system to analyze the replication of hepatitis c virus |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9368937/ https://www.ncbi.nlm.nih.gov/pubmed/35955844 http://dx.doi.org/10.3390/ijms23158711 |
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