Identification of proteins bound to dengue viral RNA In Vivo reveals new host proteins important for virus replication

Stacia L. Phillips, Erik J. Soderblom, Shelton Bradrick, Mariano Garcia-Blanco

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 crosslinking 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.

Original languageEnglish (US)
Article numbere01865-15
JournalmBio
Volume7
Issue number1
DOIs
StatePublished - Jan 5 2016

Fingerprint

Dengue
Viral RNA
Virus Replication
Dengue Virus
Proteins
Ribonucleoproteins
RNA-Binding Proteins
Arthropods
Mass Spectrometry
Infection
RNA
Virology
Viral Genome
Gene Silencing
Virus Diseases
Life Cycle Stages
Immunoprecipitation
Small Interfering RNA

ASJC Scopus subject areas

  • Microbiology
  • Virology

Cite this

Identification of proteins bound to dengue viral RNA In Vivo reveals new host proteins important for virus replication. / Phillips, Stacia L.; Soderblom, Erik J.; Bradrick, Shelton; Garcia-Blanco, Mariano.

In: mBio, Vol. 7, No. 1, e01865-15, 05.01.2016.

Research output: Contribution to journalArticle

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