Identification of Proteins Bound to Dengue Viral RNA In Vivo 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 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.

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

“…Host proteins bind to RNA secondary structures to modulate lifecycle processes for many positive-sense RNA viruses, such as hepatitis C virus (HCV) (22), hepatitis A virus (23), poliovirus type 1 (24), dengue viruses (25), bovine coronavirus (26), and murine hepatitis virus (27)(28)(29). However, few host protein-viral RNA interactions have been characterized for negative-sense, single-stranded RNA viruses.…”

Unconventional RNA‐binding proteins step into the virus–host battlefront

“…System-wide approaches to discover the vRNA-host RBP landscape have contributed to our understanding of the role of host RBPs in infected cells (Knoener et al, 2017;LaPointe, Gebhart, Meller, Hardy, & Sokoloski, 2018;Lenarcic, Landry, Greco, Cristea, & Thompson, 2013;Phillips, Soderblom, Bradrick, & Garcia-Blanco, 2016;Viktorovskaya, Greco, Cristea, & Thompson, 2016). Although the currently available methods have technical limitations, we are confident that global analyses of protein-RNA interactions can determine the arsenal of RBPs deployed in the host-virus battlefield.…”

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

“…Flaviviruses may also sequester factors that ordinarily heighten translation of RNAs involved in an antiviral response; as one example, 3′‐UTR interactions with G3BP1, G3BP2, and CAPRIN1 may serve to dampen the interferon response . vRNA‐binding proteins that either increase or decrease vRNA levels in cells have been identified via protein–RNA crosslinking, followed by vRNA‐specific pulldown, identification of relevant proteins via mass spectrometry, and knockdown of proteins of interest . In one case, knockdown of host‐factor YBX1 resulted in an increase in vRNA inside cells despite a decrease in released viral particles, suggesting deficiencies in viral packaging.…”

“…In one case, knockdown of host‐factor YBX1 resulted in an increase in vRNA inside cells despite a decrease in released viral particles, suggesting deficiencies in viral packaging. We would also point out the presence of numerous RNA helicases capable of remodeling vRNA in the handful of studies in which mass spectrometry has been utilized to examine flaviviral RNA‐interacting proteins …”