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Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties

Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenge...

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Autores principales: Dai, Haiyue, Wu, Jianan, Yang, Hongshu, Guo, Yongli, Di, Haoqing, Gao, Mingchun, Wang, Junwei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9304932/
https://www.ncbi.nlm.nih.gov/pubmed/35873151
http://dx.doi.org/10.3389/fcimb.2022.942987
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author Dai, Haiyue
Wu, Jianan
Yang, Hongshu
Guo, Yongli
Di, Haoqing
Gao, Mingchun
Wang, Junwei
author_facet Dai, Haiyue
Wu, Jianan
Yang, Hongshu
Guo, Yongli
Di, Haoqing
Gao, Mingchun
Wang, Junwei
author_sort Dai, Haiyue
collection PubMed
description Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenger RNA (mRNA) to interrupt host defense and facilitate the rapid proliferation of BHV-1. However, studies on the BHV-1 UL41 gene are limited, and BHV-1 defective virus construction using the CRISPR/Cas9 system is somewhat challenging. In this study, we rapidly constructed a BHV-1 UL41-deficient strain using the CRISPR/Cas9 system in BL primary bovine-derived cells. BHV-1 UL41-defective mutants were screened by Western blot analysis using specific polyclonal antibodies as the primary antibodies. During the isolation and purification of the defective strain, a mixed virus pool edited by an efficient single-guide RNA (sgRNA) showed a plaque number reduction. Viral growth property assessment showed that BHV-1 UL41 was dispensable for replication, but the UL41-defective strain exhibited early and slowed viral replication. Furthermore, the BHV-1 UL41-deficient strain exhibited enhanced sensitivity to temperature and acidic environments. The BHV-1 UL41-deficient strain regulated viral and host mRNA levels to affect viral replication.
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spelling pubmed-93049322022-07-23 Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties Dai, Haiyue Wu, Jianan Yang, Hongshu Guo, Yongli Di, Haoqing Gao, Mingchun Wang, Junwei Front Cell Infect Microbiol Cellular and Infection Microbiology Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenger RNA (mRNA) to interrupt host defense and facilitate the rapid proliferation of BHV-1. However, studies on the BHV-1 UL41 gene are limited, and BHV-1 defective virus construction using the CRISPR/Cas9 system is somewhat challenging. In this study, we rapidly constructed a BHV-1 UL41-deficient strain using the CRISPR/Cas9 system in BL primary bovine-derived cells. BHV-1 UL41-defective mutants were screened by Western blot analysis using specific polyclonal antibodies as the primary antibodies. During the isolation and purification of the defective strain, a mixed virus pool edited by an efficient single-guide RNA (sgRNA) showed a plaque number reduction. Viral growth property assessment showed that BHV-1 UL41 was dispensable for replication, but the UL41-defective strain exhibited early and slowed viral replication. Furthermore, the BHV-1 UL41-deficient strain exhibited enhanced sensitivity to temperature and acidic environments. The BHV-1 UL41-deficient strain regulated viral and host mRNA levels to affect viral replication. Frontiers Media S.A. 2022-07-08 /pmc/articles/PMC9304932/ /pubmed/35873151 http://dx.doi.org/10.3389/fcimb.2022.942987 Text en Copyright © 2022 Dai, Wu, Yang, Guo, Di, Gao and Wang https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Dai, Haiyue
Wu, Jianan
Yang, Hongshu
Guo, Yongli
Di, Haoqing
Gao, Mingchun
Wang, Junwei
Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties
title Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties
title_full Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties
title_fullStr Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties
title_full_unstemmed Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties
title_short Construction of BHV-1 UL41 Defective Virus Using the CRISPR/Cas9 System and Analysis of Viral Replication Properties
title_sort construction of bhv-1 ul41 defective virus using the crispr/cas9 system and analysis of viral replication properties
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9304932/
https://www.ncbi.nlm.nih.gov/pubmed/35873151
http://dx.doi.org/10.3389/fcimb.2022.942987
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