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Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen

As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identified and characterized. T...

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Autores principales: McCormick, Dominique, Lin, Yao-Tang, Grey, Finn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020295/
https://www.ncbi.nlm.nih.gov/pubmed/29946045
http://dx.doi.org/10.1128/mBio.00716-18
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author McCormick, Dominique
Lin, Yao-Tang
Grey, Finn
author_facet McCormick, Dominique
Lin, Yao-Tang
Grey, Finn
author_sort McCormick, Dominique
collection PubMed
description As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identified and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which independently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identified in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Furthermore, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efficient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identification of host-virus interactions, identifies multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection.
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spelling pubmed-60202952018-06-29 Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen McCormick, Dominique Lin, Yao-Tang Grey, Finn mBio Research Article As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identified and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which independently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identified in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Furthermore, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efficient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identification of host-virus interactions, identifies multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection. American Society for Microbiology 2018-06-26 /pmc/articles/PMC6020295/ /pubmed/29946045 http://dx.doi.org/10.1128/mBio.00716-18 Text en Copyright © 2018 McCormick et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
McCormick, Dominique
Lin, Yao-Tang
Grey, Finn
Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
title Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
title_full Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
title_fullStr Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
title_full_unstemmed Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
title_short Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
title_sort identification of host factors involved in human cytomegalovirus replication, assembly, and egress using a two-step small interfering rna screen
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020295/
https://www.ncbi.nlm.nih.gov/pubmed/29946045
http://dx.doi.org/10.1128/mBio.00716-18
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