A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

ABSTRACT While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YF...

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Autores principales:	Harish N. Ramanathan, Shuo Zhang, Florian Douam, Katrina B. Mar, Jinhong Chang, Priscilla L. Yang, John W. Schoggins, Alexander Ploss, Brett D. Lindenbach
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	flavivirus nucleocapsid uncoating viral entry Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/aee8a1244e744296af80d6a1ac424a93
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spelling	oai:doaj.org-article:aee8a1244e744296af80d6a1ac424a932021-11-15T15:57:01ZA Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating10.1128/mBio.00467-202150-7511https://doaj.org/article/aee8a1244e744296af80d6a1ac424a932020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00467-20https://doaj.org/toc/2150-7511ABSTRACT While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.Harish N. RamanathanShuo ZhangFlorian DouamKatrina B. MarJinhong ChangPriscilla L. YangJohn W. SchogginsAlexander PlossBrett D. LindenbachAmerican Society for Microbiologyarticleflavivirusnucleocapsiduncoatingviral entryMicrobiologyQR1-502ENmBio, Vol 11, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	flavivirus nucleocapsid uncoating viral entry Microbiology QR1-502
spellingShingle	flavivirus nucleocapsid uncoating viral entry Microbiology QR1-502 Harish N. Ramanathan Shuo Zhang Florian Douam Katrina B. Mar Jinhong Chang Priscilla L. Yang John W. Schoggins Alexander Ploss Brett D. Lindenbach A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
description	ABSTRACT While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
format	article
author	Harish N. Ramanathan Shuo Zhang Florian Douam Katrina B. Mar Jinhong Chang Priscilla L. Yang John W. Schoggins Alexander Ploss Brett D. Lindenbach
author_facet	Harish N. Ramanathan Shuo Zhang Florian Douam Katrina B. Mar Jinhong Chang Priscilla L. Yang John W. Schoggins Alexander Ploss Brett D. Lindenbach
author_sort	Harish N. Ramanathan
title	A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
title_short	A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
title_full	A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
title_fullStr	A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
title_full_unstemmed	A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
title_sort	sensitive yellow fever virus entry reporter identifies valosin-containing protein (vcp/p97) as an essential host factor for flavivirus uncoating
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/aee8a1244e744296af80d6a1ac424a93
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A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

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