Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Li, Xiaoyu, Ito, Masahiko, Aoyagi, Haruyo, Murayama, Asako, Aizaki, Hideki, Fukasawa, Masayoshi, Kato, Takanobu, Wakita, Takaji, Suzuki, Tetsuro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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
_version_ 1784766299209990144
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
work_keys_str_mv AT lixiaoyu developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT itomasahiko developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT aoyagiharuyo developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT murayamaasako developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT aizakihideki developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT fukasawamasayoshi developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT katotakanobu developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT wakitatakaji developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus
AT suzukitetsuro developmentanduseofakineticalandrealtimemonitoringsystemtoanalyzethereplicationofhepatitiscvirus