Research Article: Blocking tombusvirus replication through the antiviral functions of DDX17-like RH30 DEAD-box helicase

Date Published: May 28, 2019

Publisher: Public Library of Science

Author(s): Cheng-Yu Wu, Peter D. Nagy, Savithramma P. Dinesh-Kumar.

http://doi.org/10.1371/journal.ppat.1007771

Abstract

Positive-stranded RNA viruses replicate inside cells and depend on many co-opted cellular factors to complete their infection cycles. To combat viruses, the hosts use conserved restriction factors, such as DEAD-box RNA helicases, which can function as viral RNA sensors or as effectors by blocking RNA virus replication. In this paper, we have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. The helicase function of RH30 was required for restriction of tomato bushy stunt virus (TBSV) replication. Knock-down of RH30 levels in Nicotiana benthamiana led to increased TBSV accumulation and RH30 knockout lines of Arabidopsis supported higher level accumulation of turnip crinkle virus. We show that RH30 DEAD-box helicase interacts with p33 and p92pol replication proteins of TBSV, which facilitates targeting of RH30 from the nucleus to the large TBSV replication compartment consisting of aggregated peroxisomes. Enrichment of RH30 in the nucleus via fusion with a nuclear retention signal at the expense of the cytosolic pool of RH30 prevented the re-localization of RH30 into the replication compartment and canceled out the antiviral effect of RH30. In vitro replicase reconstitution assay was used to demonstrate that RH30 helicase blocks the assembly of viral replicase complex, the activation of the RNA-dependent RNA polymerase function of p92pol and binding of p33 replication protein to critical cis-acting element in the TBSV RNA. Altogether, these results firmly establish that the plant DDX17-like RH30 DEAD-box helicase is a potent, effector-type, restriction factor of tombusviruses and related viruses. The discovery of the antiviral role of RH30 DEAD-box helicase illustrates the likely ancient roles of RNA helicases in plant innate immunity.

Partial Text

Positive-stranded (+)RNA viruses replicate inside cells and depend on many co-opted cellular factors to complete their infection cycle. These viruses build elaborate membranous viral replication compartments, consisting of viral replication proteins, viral RNAs and recruited host factors, in the cytosol of the infected cells. The hijacked host factors participate in all steps of RNA virus replication, including the assembly of membrane-bound viral replicase complexes (VRCs), viral RNA-dependent RNA polymerase (RdRp) activation and viral RNA synthesis. The growing list of co-opted host factors facilitating VRC assembly includes translation initiation and elongation factors, protein chaperones, RNA-modifying enzymes, SNARE and ESCRT proteins, actin network, and lipids [1–9]. Many (+)RNA viruses extensively rewire metabolic pathways, remodel subcellular membranes and take advantage of intracellular trafficking.

DEAD-box RNA helicases are the most numerous among RNA helicases [33,37]. They are involved in all facets of RNA processes in cells. RNA viruses and retroviruses also usurp several DEAD-box helicases to facilitate their replication and other viral processes during infection [70,71]. However, the host also deploys DEAD-box helicases to inhibit RNA virus replication [70,72]. Accordingly, in this work we present several pieces of evidence that the DDX17-like RH30 DEAD-box helicase restricts tombusvirus replication, including the peroxisomal replicating TBSV and CNV and the mitochondrial-replicating CIRV in yeast and plants, and the more distantly related TCV and RCNMV and the unrelated TMV in plants. On the contrary, knock-down of RH30 enhances the replication of these three tombusviruses in N. benthamiana or the related TCV in RH30 knock-out lines of Arabidopsis. On the other hand, the helicase core mutant RH30 can only partially inhibit tombusvirus replication in plants or in vitro, suggesting that the helicase function of RH30 is needed for its full antiviral activity.

 

Source:

http://doi.org/10.1371/journal.ppat.1007771

 

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