Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2009
Publication Date: 8/26/2009
Citation: Lawrence, P.J., Rieder, A.E. 2009 Identification of RNA Helicase A (RHA) as a New Host Factor in the Replication Cycle of FMDV. Journal of Virology. 83 (21): 11356-11366. Interpretive Summary: In an effort to better understand the interaction of foot-and-mouth disease virus (FMDV) with its natural hosts, we sought to identify cellular factors that are subverted by the virus for its own purposes. Indeed, we discovered a novel host factor, RNA helicase A (RHA), that appears to play a critical role in FMDV virulence and potentially in pathogenesis. RHA has been described as a critical cellular factor in the life cycle of other groups of viruses including: retroviruses, flaviviruses, and adenoviruses. We found that FMDV triggers a profound redistribution of RHA to parts of the host cell where virus replication is taking place, specifically translocation from the nucleus to the cell cytoplasm. In fact, the translocated RHA co-localizes with and physically interacts with virus proteins that participate in the replication of the virus RNA genome as well as another cellular protein known to contribute to RNA replication. When the amount of available RHA is reduced, productive FMDV infection is significantly curtailed. These effects were observed for several serotypes of FMDV including: A24-Cruzeiro, 01-Campos, C3-Resende, and SAT2. Additionally, we obtained similar results using an unrelated picornavirus: bovine enterovirus 1 (BEV-1). We believe that FMDV is acquiring the RNA helicase activity of RHA to assist in the separation of double-stranded RNA intermediates that form during the course of the replication of the RNA. Identification of the cellular factors needed for FMDV replication will help in developing better countermeasures.
Technical Abstract: Foot-and-mouth disease virus (FMDV) as with other RNA viruses, recruits various host cell factors to assist in the translation and replication of the virus genome. In this study we investigated the role of RNA helicase A (RHA) in the life cycle of FMDV. Immunofluorescent microscopy combined with biochemical assays showed a change in the subcellular distribution of RHA from the nucleus to the cytoplasm in FMDV-infected cells as infection progressed. Unlike nuclear RHA, the RHA detected in the cytoplasm does not appear to be methylated on arginine-glycine-glycine (RGG) motifs in the C-terminus. In contrast to alterations in the subcellular distribution of nuclear factors observed during infection with similar picornaviruses, cytoplasmic accumulation of RHA did not require an active virus leader protease. Concomittant with the progress of FMDV infection, the cytoplasmic RHA co-localized with viral 2C, 3A, and to a lesser extent 3D polymerase, where replication complexes are assembled. RHA co-precipitated with viral 2C and 3A, indicating that RHA is situated on the membranous structures formed by these viral components of the replication complex. RNA-binding assays further demonstrated that RHA has specific RNA-binding activity, recognizing the S-fragment in the 5’NTR of FMDV. Treatment of cells with short interfering RNA (siRNA) selectively targeting the RHA gene knocked-down expression of this protein and concomitantly reduced FMDV replication, which was not observed using non-specific siRNA controls. Potentially, RHA facilitates the unwinding of double-stranded RNA intermediates during the replication of the virus genome.