Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication

ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins med...

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Autores principales:	Stacia L. Phillips, Erik J. Soderblom, Shelton S. Bradrick, Mariano A. Garcia-Blanco
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6692dfaaaefb4536a4256eec9f61b088
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spelling	oai:doaj.org-article:6692dfaaaefb4536a4256eec9f61b0882021-11-15T15:49:40ZIdentification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication10.1128/mBio.01865-152150-7511https://doaj.org/article/6692dfaaaefb4536a4256eec9f61b0882016-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01865-15https://doaj.org/toc/2150-7511ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. IMPORTANCE Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.Stacia L. PhillipsErik J. SoderblomShelton S. BradrickMariano A. Garcia-BlancoAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Stacia L. Phillips Erik J. Soderblom Shelton S. Bradrick Mariano A. Garcia-Blanco Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication
description	ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. IMPORTANCE Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.
format	article
author	Stacia L. Phillips Erik J. Soderblom Shelton S. Bradrick Mariano A. Garcia-Blanco
author_facet	Stacia L. Phillips Erik J. Soderblom Shelton S. Bradrick Mariano A. Garcia-Blanco
author_sort	Stacia L. Phillips
title	Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication
title_short	Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication
title_full	Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication
title_fullStr	Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication
title_full_unstemmed	Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication
title_sort	identification of proteins bound to dengue viral rna <italic toggle="yes">in vivo</italic> reveals new host proteins important for virus replication
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/6692dfaaaefb4536a4256eec9f61b088
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Identification of Proteins Bound to Dengue Viral RNA <italic toggle="yes">In Vivo</italic> Reveals New Host Proteins Important for Virus Replication

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