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

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.

Positive-stranded RNA viruses are important and emerging pathogens that greatly depend on the host during infection. The host uses conserved innate and cell-intrinsic restriction factors as a first line of defense to combat viruses. Among the most intriguing host restriction factors are the family of DEAD-box RNA helicases, which can function as viral RNA sensors or directly as effectors by inhibiting RNA virus replication. RNA  translation [42,43]. In case of plant viruses, turnip mosaic virus and brome mosaic virus have been described to co-opt cellular DEAD-box helicases for proviral function in translation or replication [39,42,44]. Altogether, cellular helicases are important co-opted host factors for several viruses, playing critical roles in virus-host interactions. However, cellular RNA helicases also act as antiviral restriction factors, including functioning as viral RNA sensors (e.g., Dicer or RIG-I) or directly inhibiting RNA virus replication as effectors [45][46][47]. For example, DDX17 restricts Rift Valley fever virus [48], while DDX21 helicase inhibits influenza A virus and DDX3 blocks Dengue virus infections [49][50][51][52]. Thus, the emerging picture is that host helicases are important for the host to restrict RNA virus replication, but the mechanism of their activities or substrates are not well characterized.
In this work, we find that the plant DDX17-like RH30 DEAD-box helicase plays a strong restriction factor function against tombusviruses and related plant viruses. RH30 DEADbox helicase is expressed in all plant organs, but its cellular function is not known yet [53]. We find that RH30 is re-localized from the nucleus to the sites of tombusvirus replication via interacting with the TBSV p33 and p92 pol replication proteins. Several in vitro assays provide evidence that RH30 inhibits tombusvirus replication through blocking several steps in the replication process, including VRC assembly, viral RdRp activation and the specific interaction between p33 replication protein and the viral (+)RNA. RH30 knockout lines of Arabidopsis supported increased accumulation level for the related turnip crinkle virus, confirming the restriction factor function of RH30 against a group of plant viruses. This is the first identification and characterization of a plant helicase with an effector type restriction factor function against plant viruses. Since plant genomes codes for over 100 RNA helicases, it is likely that additional helicases have CIRF function against plant viruses.

The host RH30 RNA helicase is a potent restriction factor of tombusvirus replication in yeast and plants
To test if the host RH30 RNA helicase could affect tombusvirus replication, we expressed the Arabidopsis RH30 using agroinfiltration in Nicotiana benthamiana plants. Interestingly, expression of AtRH30 blocked TBSV replication by~90% in the inoculated leaves ( Fig 1A). The closely-related cucumber necrosis virus (CNV), which also targets the peroxisomal membranes for VRC formation, was also inhibited by~4-fold through the expression of AtRH30 ( Fig 1B). Replication of another tombusvirus, carnation Italian ringspot virus (CIRV), which builds the replication compartment using the outer membranes of mitochondria, was inhibited by~9-fold by the transient expression of AtRH30 in N. benthamiana (Fig 1C).
To test if RH30 was also effective against TBSV when expressed in yeast cells, we launched the TBSV repRNA replication assay in wt yeast by co-expressing the viral components with RH30. After 24 h of incubation, TBSV repRNA analysis revealed strong inhibition of viral replication by RH30 expression (Fig 1F), suggesting that RH30 is a highly active inhibitor against TBSV replication even in a surrogate host.
To learn if the putative helicase function of RH30 is required for its cell intrinsic restriction factor (CIRF) function, we expressed a motif IV helicase core mutant of RH30(F 416 L) in N. bentamiana via agroinfiltration. Mutation of the highly conserved F residue within the helicase core domain (see S1 Fig) has been shown to greatly decrease both ATP binding/hydrolysis and strand displacement activities in Ded1 and other DEAD-box helicases [54]. Northern blot analysis revealed the lack of inhibition of TBSV replication, and only partial inhibition of CIRV replication by RH30(F 416 L) (Fig 1D and 1E, lanes 9-12). Thus, we suggest that the full helicase/ATPase function of RH30 is required for its CIRF function against tombusviruses.
VIGS-based silencing of the endogenous RH30 in N. benthamiana led to~5-fold,~3-fold and~11-fold increased accumulation of TBSV, CNV and CIRV, respectively, in the inoculated leaves (Fig 2). The leaves of virus-infected and VIGS-treated plants showed severe necrotic symptoms earlier and died earlier than the control plants (i.e., TRV-cGFP treatment) in case of all three tombusvirus infections (Fig 2). On the other hand, the VIGS-treated plants became only slightly smaller than the TRV-cGFP treated control plants (Fig 2). Based on these and the Ethidium-bromide stained gel shows 18S ribosomal RNA as a loading control. (D-E) Expression of the helicase core mutant of RH30 (RH30m, F 416 L) inhibited TBSV or CIRV replication, respectively, to a lesser extent, demonstrating the requirement of the helicase/ATPase function of RH30 for its full virus restriction function. See further details in panel A. Each experiment was repeated at least three times. (F) Expression of RH30 inhibits TBSV replication in yeast. Top panel: Northern blot analysis of TBSV repRNA using a 3' end specific probe shows reduced accumulation of repRNA in WT yeast strain expressing RH30. Viral proteins His 6 -p33 and His 6 -p92 pol were expressed from plasmids from the CUP1 promoter, while DI-72(+) repRNA was expressed from the GAL1 promoter. His 6 -RH30 was expressed from a plasmid. Middle panel: Northern blot with 18S ribosomal RNA specific probe was used as a loading control. Bottom images: Western blot analysis of the level of His 6 -p33, His 6 -p92 pol and His 6 -RH30 with anti-His antibody. RH30 over-expression data, RH30 DEAD-box helicase seems to act as a major restriction factor against tombusviruses in plants and yeast.

RH30 DEAD-box helicase is re-localized into the tombusvirus replication compartment in plants
To identify the cellular compartment where RH30 DEAD-box helicase performs its CIRF function, first we used co-localization studies in N benthamiana protoplasts co-expressing  GFP-RH30, p33-BFP (to mark the site of viral replication) and RFP-tagged H2B, which is a nuclear marker protein. We detected the re-localization of GFP-RH30 into the large p33 containing replication compartment from the nucleus during CNV replication (Fig 3A, top panel  versus second panel). Both the p33-BFP and RFP-SKL (a peroxisomal matrix marker) showed the re-localization of GFP-RH30 into the large TBSV replication compartment, which consists of aggregated peroxisomes. Part of the ER is also recruited to the p33 and RH30 containing replication compartment (Fig 3A bottom panel), as shown previously [55,56].
Similar re-localization pattern of RH30 was observed in epidermal cells of whole plants infected with CNV ( Fig 3B, top panel versus second panel). The expression of only p33-BFP was satisfactory to recruit the RH30 into the replication compartment ( Fig 3B). RH30 was also re-targeted in CIRV-infected N. benthamiana cells into the p36 and p95 pol containing replication compartment (Fig 3B, bottom panel), which consists of aggregated mitochondria [57,58]. Based on these experiments, we propose that the mostly nuclear localized RH30 helicase is capable of entering the tombusvirus replication compartment via interaction with the replication proteins. However, the formation of large tombusvirus-induced replication compartments seemed to be normal in the presence of RH30, indicating the lack of interference with the biogenesis of the replication compartment by RH30.

Nuclear retention of RH30 DEAD-box helicase blocks its antiviral function in plants
To test if the cytosolic localization of RH30 is required for its CIRF function, we fused RH30 with a nuclear retention signal (NRS) [59] to enrich RH30 in the nucleus at the expense of the cytosolic pool of RH30. Interestingly, unlike WT RH30, expression of NRS-RH30 did not result in inhibition of TBSV replication in N. benthamiana (Fig 4A). Confocal microscopy experiments confirmed that NRS-RH30-GFP is localized exclusively in the nucleus (Fig 4B). Infection of the N. benthamiana protoplasts with CNV did not result in the re-targeting of NRS-RH30-GFP from the nucleus to the replication compartment visualized via p33-BFP. The nuclear retention of NRS-RH30-GFP was also confirmed in N. benthamiana epidermal cells infected with CNV or mock inoculated ( Fig 4C). Altogether, these experiments demonstrated that re-localization of RH30 helicase from the nucleus to the replication compartment is critical for its CIRF function in plants.

RH30 helicase interacts with the viral replication proteins in yeast and plants
To learn about the tombusviral target of RH30 DEAD-box helicase, we co-expressed the His 6tagged RH30 with Flag-tagged p33 and Flag-p92 pol replication proteins and the TBSV repRNA in yeast, followed by Flag-affinity purification of p33/p92 pol from the detergent-solubilized membrane fraction of yeast, which is known to harbor the tombusvirus replicase [20,60]. Western blot analysis of the affinity-purified replicase revealed the effective co-purification of His 6 -RH30 (Fig 5A, lane 3), suggesting that RH30 targets the VRCs for its CIRF function. Interestingly, His 6 -RH30 was co-purified from yeast co-expressing either Flag-p33 or Flag-p92 pol replication proteins ( Fig 5A, lanes 1-2), suggesting that RH30 likely directly interacts with the tombusvirus replication proteins in a membranous compartment.
To show direct interaction between RH30 DEAD-box helicase and the TBSV p33 replication protein, we performed pull-down assay with MBP-tagged RH30 and GST-tagged p33 proteins from E. coli. We found that MBP-RH30 captured GST-p33 protein on the maltosecolumn ( Fig 5B, lane 2), indicating direct interaction between the host RH30 and the viral p33 protein. In the pull-down assay, we used truncated TBSV p33 replication protein missing its  N-terminal region including the membrane-binding region to aid its solubility in E. coli [61]. Interestingly, the helicase core mutant RH30(F 416 L) also bound to p33 replication protein as efficiently as the wt RH30 (Fig 5B, lane 3 versus 2). Altogether, these data suggest that the direct interaction between RH30 host protein and the replication protein of TBSV occurs within the viral protein C-terminal domain facing the cytosolic compartment.
To provide additional evidence that RH30 helicase interacts with the tombusvirus replication protein, we have conducted bimolecular fluorescence complementation (BiFC) experiments in N. benthamiana leaves. The BiFC experiments revealed interaction between RH30 and the TBSV p33 replication protein within the viral replication compartment, marked by the peroxisomal matrix marker RFP-SKL ( Fig 5C). Altogether, these experiments revealed direct interaction between the cellular RH30 DEAD-box helicase and the TBSV p33 replication protein, which results in re-targeting of RH30 into the viral replication compartment.

RH30 DEAD-box helicase interferes with the assembly of tombusvirus VRCs and activation of p92 RdRp
To gain insight into the mechanism of CIRF function of RH30 helicase, we affinity-purified the recombinant RH30 and tested its activity in vitro in a TBSV replicase reconstitution assay, which is based on yeast cell-free extract [26,62]. Addition of RH30 to the replicase reconstitution assay led to inhibition of TBSV repRNA replication by~10-fold ( Fig 6A, lanes 9-10). The in vitro production of double-stranded repRNA replication intermediate was also inhibited bỹ 10-fold by RH30, indicating that RH30 likely inhibits an early step, such as the VRC assembly during TBSV replication.
We then used a step-wise TBSV replicase reconstitution assay [26,29], in which RH30 was added at different stages of VRC assembly (schematically shown in Fig 6B). RH30 showed significant inhibitory activity when added at the beginning of the TBSV replicase reconstitution assay ( Fig 6B, lanes 3-4 versus 1-2). On the contrary, RH30 was ineffective, when added to TBSV replicase reconstitution assay after the VRC assembly step and prior to RNA synthesis ( Fig 6B, lanes 7-8). These in vitro data support the model that the inhibitory role of RH30 is performed during or prior to the VRC assembly step, but RH30 is ineffective at the latter stages of TBSV replication.
We also utilized an in vitro RdRp activation assay based on the purified recombinant TBSV p92 RdRp, which is inactive and requires Hsp70 chaperone and the viral (+)RNA template to become an active polymerase [21]. Addition of the recombinant RH30 helicase strongly inhibited the polymerase activity of the p92 RdRp (Fig 6C), suggesting that RH30 blocks the critical RdRp activation step during tombusvirus replication.
Several RNA helicases are involved in regulation of cellular translation [63]. Therefore, we tested if RH30 affected the translation of tombusvirus genomic RNA, which is uncapped and lacks poly(A) tail [64]. CIRV genomic RNA was used in this in vitro assay based on wheat (transgenic plants expressing nucleus marker RFP-H2B or ER marker RFP-ER) were agro-infiltrated to express p33-BFP, GFP-RH30, and CNV 20KSTOP gRNA. Leaves without the expression of p33-BFP and CNV 20KSTOP gRNA were used as controls. The agro-infiltrated leaves were collected to isolate protoplasts for confocal imaging 2.5 days post agro-infiltration. Scale bars represent 10 μm. (B) Confocal microscopy images show co-localization of TBSV p33-BFP or CIRV p36-BFP replication proteins and the GFP-RH30 in planta. The large replication compartment was visualized via expression of TBSV p33-BFP or CIRV p36-BFP. Expression of the above proteins from the 35S promoter was done after co-agroinfiltration into N. benthamiana leaves. The leaves of N. benthamiana plants were agroinfiltrated to express TBSV p33-BFP or the CIRV p36-BFP, GFP-RH30, and CNV 20KSTOP or CIRV gRNAs. Leaves without the expression of p33-BFP or p36-BFP and the viral RNAs were used as controls. The agro-infiltrated leaves were collected for confocal imaging 2.5 days post agro-infiltration. Scale bars represent 10 μm. Each experiment was repeated. https://doi.org/10.1371/journal.ppat.1007771.g003 Cellular DEAD-box helicase acts as a restriction factor against TBSV  Co-purification of His 6tagged RH30 with Flag-p33 and Flag-p92 pol replication proteins from subcellular membranes. Top panels: Western blot analysis of co-purified His 6 -RH30 (lanes 1, 2, and 3) with Flag-affinity purified replicase, Flag-p33 and Flag-p92 pol replication proteins, respectively as shown. His 6 -p33, His 6 -p92 pol and His 6 -RH30 were detected with anti-His antibody, while Flag-p33 and Flag-p92 pol replication proteins were detected with anti-FLAG antibody. The negative control was from yeast expressing His 6 -RH30, His 6 -p33 and His 6 -p92 pol purified in a FLAG-affinity column (lane 4). Bottom panel: blot of total His 6 -p33 and His 6 -p92 pol and His 6 -RH30 in the total yeast extracts detected with anti-His antibody. (B) Pull-down assay including TBSV GST-p33 replication protein and the MBP-tagged RH30. Note that we used the soluble C-terminal region of TBSV p33 replication protein, which lacked the N-terminal sequence, including the trans-membrane TM domain. Top panel: Western blot analysis of the captured GST-p33C with the MBPaffinity purified MBP-RH30 or the helicase core mutant of RH30 (RH30mut, F 416 L) was performed with anti-His antibody. The negative control was MBP (lane 1). Middle panel: Coomassie-blue stained SDS-PAGE of the captured MBP-RH30 and MBP. Bottom panel: Western blot analysis of GST-p33C in total E. coli lysates. Each experiment was repeated three times. (C) Interactions between TBSV p33 replication protein and the RH30 helicase was detected by BiFC. The TBSV p33-cYFP replication protein and the nYFP-RH30 and the RFP-SKL peroxisomal marker protein were expressed via agro-infiltration. The merged image shows the efficient co-localization of the peroxisomal RFP-SKL with the BiFC signals, indicating that the interaction between the tombusvirus replication protein and the recruited RH30 helicase occurs in the large viral replication compartments, which consist of aggregated peroxisomes. Scale bars represent 5 μm.
https://doi.org/10.1371/journal.ppat.1007771.g005 Step #1 promotes the assembly of the functional tombusvirus replicase, whereas step #2 supports viral RNA synthesis in the presence of all four ribonucleotides. Note that MBP-RH30 or MBP (1.9 and 5.7 μM), as a control, were added to the reactions either at step #1 or step #2, as shown. The 32 P-labeled TBSV repRNA products of the reconstituted replicases were detected by denaturing PAGE. (C) The in vitro RdRp activation assay is based on (+)repRNA and p92-Δ167N RdRp protein in the presence of the soluble fraction of yeast CFE. Purified MBP-RH30 and MBP were added in increasing amounts. Denaturing PAGE analysis of the 32 P-labeled RNA products obtained in an in vitro assay with recombinant p92-Δ167N RdRp. (D) In vitro translation assay with wheat germ extract programmed with CIRV gRNA. Purified MBP-RH30 and MBP were added in increasing amounts (1.9 μM and 3.8 μM). The 35 S-methionine-labeled p36 replication protein translation product is detected by SDS-PAGE. Tdh2 mRNA was used as a control. Each experiment was repeated three times. https://doi.org/10.1371/journal.ppat.1007771.g006 Cellular DEAD-box helicase acts as a restriction factor against TBSV germ extract [65]. Addition of recombinant RH30 to the in vitro translation assay inhibited slightly the production of p36 replication protein from the gRNA when RH30 was used in high amount (Fig 6D). The highest amount of RH30 also had minor inhibition on translation of the control Tdh2 mRNA (Fig 6D). Thus, RH30 is unlikely to specifically affect the translation of the tombusvirus RNAs during infection.

RH30 helicase binds to critical cis-acting elements in the viral RNA
Since the canonical function of RNA helicases to bind RNA substrates and unwind base-paired structures [36], we tested if RH30 DEAD-box helicase could perform these functions with the TBSV RNA in vitro. First, we used gel-mobility shift assay with purified recombinant RH30, which showed that RH30 bound to both the (+) and (-)repRNA (Fig 7A and 7B). Since each of the four regions in the TBSV repRNA contains well-defined cis-acting elements, we performed template competition assay with the four regions separately in the presence of recombinant RH30 helicase. This assay defined that the best competitors for binding to RH30 was RII(+) and RII(-), whereas RI(+), RIV(+) and RI(-), RIV(-) also become competitive when added in high amounts (Fig 7C). Because RII(+) contains a critical cis-acting stem-loop element, termed RII(+)SL, which is involved in p33-mediated recruitment of the TBSV (+)RNA template [24], and the activation of the p92 RdRp [21], we tested if the purified RH30 could bind to this stem-loop element in vitro. Interestingly, RH30 bound to RII(+)SL in the absence of added ATP (Fig 7D). However, the presence of extra ATP enhanced the binding of RH30 to RII(+) SL, suggesting that RH30 binds to RNAs in an ATP-dependent fashion, similar to other DEAD-box helicases [36,54,66]. The control p33 (an N-terminally-truncated, soluble version) bound to RII(+)SL more efficiently and in an ATP-independent manner (Fig 7D), as also shown previously [24]. This highlights the possibility that RH30 and p33 replication protein compete with each other in binding to this critical cis-acting element.
To test the RNA helicase function of RH30, we performed strand separation assays, where parts of the TBSV repRNA was double-stranded as shown schematically in Fig 7E and 7F. The RNA helicase activity of RH30 in the presence of ATP was found to efficiently separate the partial dsRNA templates, involving RI and RII sequences (Fig 7E and 7F). RH30 was much less efficient to separate the partial dsRNA templates in the absence of ATP or when we added its helicase core mutant RH30(F 416 L) (Fig 7E, lanes 6-9; 7F, lanes 5-8). It is possible that the residual strand-separation activity of RH30(F 416 L) might come from its RNA binding and RNA chaperone activity with the TBSV RNA substrates. Additional biochemical assays will be needed to test if the partial activity of RH30 in the absence of added ATP is due to the possibly copurified residual ATP bound to RH30.
To test if RH30(F 416 L) helicase core mutant still has antiviral activity, we performed a TBSV replicase reconstitution assay with yeast cell-free extract [26,62]. Addition of RH30(F 416 L) to the replicase reconstitution assay led to minor inhibition of TBSV repRNA replication ( Fig  7G, lanes 1-2). Thus, mutation within the helicase core region of RH30 affected its antiviral activity on TBSV replication in vitro.

RH30 helicase inhibits the binding of the viral replication proteins to the template recruitment element in the viral (+)RNA
To further characterize the restriction function of RH30 during tombusvirus replication, we tested if RH30 helicase could inhibit the selective binding of p33 replication protein to the viral RNA template in vitro. To this end, we biotin-labeled RII(+) sequence of the TBSV RNA, which represents RII(+)-SL RNA recognition element required for template recruitment into replication by p33 replication protein [24]. Moreover, RII(+)-SL RNA is also essential part of an assembly platform for the replicase complex [67]. The biotin-labeled RII(+) RNA was then pre-incubated with purified RH30 (Fig 8A). Then, purified p33C (the soluble C-terminal region, including the RNA-binding and p33:p33/p92 interaction region of p33 replication protein) was added, which can bind specifically to RII(+)-SL if the hairpin structure with the C•C mismatch in the internal loop was formed [24]. After a short incubation, the biotin-labeled RII(+) RNA was captured on streptavidin-coated magnetic beads. After thorough washing of the streptavidin beads, the proteins bound to the RNA were eluted. Western blot analysis with anti-p33 antibody revealed that RH30 in the presence of ATP inhibited the binding of p33C to RII(+)-SL by 50% (Fig 8A, lane 2 versus lane 3) when compared with the control containing the MBP protein that does not bind to RII(+)-SL [24]. RH30 was less inhibitory of the p33C-RII(+)-SL interaction in the absence of ATP (Fig 8A, lane 4). We also performed the experiments when RH30 and p33C were incubated with biotin-labeled RII(+) RNA simultaneously. Western-blot analysis showed that RH30 was still inhibitory of p33C binding to RII(+)-SL ( Fig  8B), but less effectively than above when RH30 was pre-incubated with the RII(+) RNA. These in vitro results suggest that one of the mechanisms by which RH30 helicase inhibits tombusvirus replication is to inhibit the binding of p33 to the critical RII(+)-SL RNA recognition element required for template recruitment into replication. This inhibition is likely due to local unwinding RII(+)-SL, because the presence of ATP enhanced the inhibitory effect of RH30.
In another set of experiments, we first incubated biotin-labeled RII(+) RNA with p33C, followed by capturing the RNA-p33 complex with streptavidin-coated magnetic beads and then, the addition of RH30 helicase to the beads (Fig 8C). Here we tested the released p33C from the beads in the eluted fraction by Western blotting. Interestingly, increasing the amounts of RH30 added in the presence of ATP led to the release of p33C from the RII(+) RNA (Fig 8C,  lane 3-4), whereas RH30 was less efficient in replacing p33C in the absence of ATP (lanes 1-2). Based on these in vitro data, we suggest that RH30 helicase could replace the RNAbound p33C by likely remodeling the RNA-p33 complex in an ATP-dependent manner.

RH30 helicase is co-localized with the viral dsRNA replication intermediate within the tombusvirus replication compartment in plants
We also tested the localization of RH30 helicase in comparison with the viral repRNA in N. benthamiana. The TBSV repRNA carried six copies of the RFP-tagged coat protein recognition sequence from bacteriophage MS2 in either plus or minus polarity [68]. CNV served as a helper virus in these experiments. Interestingly, RH30 was co-localized with both (-)repRNA and (+)repRNA, which were present in the replication compartment decorated by the TBSV p33-BFP (Fig 9A and 9B). The RFP signal within the replication compartment was usually weaker when RH30 helicase was expressed, likely due to the inhibitory effect of RH30 on Schematic representation of the four regions carrying cis-acting sequences in the DI-72 (+)repRNA. In vitro RNA binding assay with purified MBP-RH30 (1.9 and 5.7 μM) and the 32 P-labeled RII(+)-SL was performed in the presence or absence of 1 mM ATP. MBP-p33C (1.9 and 5.7 μM) representing the C-terminal soluble portion of TBSV p33 replication protein was used as a positive control, whereas MBP was the negative control. See further details in panel A. (E-F) Top: Schematic representation of the partial RNA/RNA duplexes used in the strand separation assay. The unlabeled template consists of DI-72 (+)repRNA and a short 32 P-labeled complementary (-)RNA (representing either RI or RII in DI-72), which anneals to the 621 nt DI-72 (+)repRNA. Increasing amounts of purified recombinant MBP-RH30, a helicase core mutant of MBP-RH30m or MBP, as a control, were added to the reactions in the presence or absence of ATP. Bottom: Representative native gel of 32 P-labeled RNA products after the in vitro strand separation assay. Quantification of the partial dsRNA probe was done with a Phosphorimager. This experiment was repeated two times. (G) Increasing amounts (1.9 and 3.8 μM) of purified MBP-fusion protein or MBP (as a control) were added to the in vitro CFE assay #1. The 32 P-labeled RNA products were detected by nondenaturing PAGE. The bottom image shows the contrasted image of the dsRNA bands of the top image. https://doi.org/10.1371/journal.ppat.1007771.g007 Cellular DEAD-box helicase acts as a restriction factor against TBSV  [69], was co-localized with RH30 helicase within the replication compartment (Fig 10). These data demonstrate that RH30 helicase relocates to the replication sites where tombusvirus RNA synthesis takes place.

RH30 DEAD-box helicase inhibits the accumulation of related and unrelated plant and insect viruses in yeast or plants
To learn if RH30 has restriction function against additional plant viruses, we tested the effect of RH30 expression on TCV carmovirus and red clover necrosis mosaic virus (RCNMV) dianthovirus, both of which belong to the Tombusviridae family. Expression of AtRH30 in N. benthamiana plants led to complete block of TCV gRNA accumulation and~4-fold reduction in RCNMV RNA1 accumulation (Fig 11A and 11B). On the contrary, two separate transgenic RH30 knock-out lines of Arabidopsis thaliana supported increased levels of TCV gRNA accumulation by up to 2-fold ( Fig 11C).
The Arabidopsis-TCV system was also used to estimate if TCV infection could induce RH30 gene transcription. RT-PCR analysis revealed induction of RH30 mRNA transcription in TCV-infected versus mock-inoculated plants (Fig 11D). All these data are in agreement that RH30 is a strong restriction factor against tombusviruses and related viruses in plants.
To learn if RH30 also has restriction function against an unrelated plant virus, we overexpressed AtRH30 in N. benthamiana and measured the accumulation of the unrelated tobacco mosaic tobamovirus (TMV). We observed a~3-fold reduction in TMV RNA accumulation in N. benthamiana leaves expressing the WT RH30, but not in those leaves expressing the helicase core mutant of RH30(F 416 L) (Fig 11E). Expression of WT RH30, but not that of the RH30(F 416 L) helicase core mutant, also inhibited the accumulation of the insect-infecting Nodamura virus (NoV) by~3-fold in yeast (S2A Fig). Interestingly, the accumulation of Flock House virus (FHV), an alphanodavirus, which is related to NoV, was only slightly inhibited by the expression of WT RH30 in yeast (S2B Fig). Based on these observations, we suggest that the plant RH30 DEAD-box helicase has a broad-range CIRF activity against several RNA viruses.

Discussion
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 labeled RII(+) RNA-protein complex was captured on streptavidin-coated magnetic beads and washed the beads with a buffer. We eluted the proteins from the beads and measured the amounts of MBP-p33C in the eluates by Western blotting using anti-p33 antibody. Reduced amounts of MBP-p33C in the eluates mean that RH30 prevented the binding of p33C to the viral RNA, likely due to remodelling the RNA structure that could not be recognized by p33 any longer. Nonbiotinylated RNA (lane 1) was used as a control. (B) The scheme shows that the biotin-labeled RII(+) RNA, MBP-RH30 and MBP-p33C were added simultaneously to the in vitro assay. See additional details in panel A. (C) Top: The scheme shows that the biotin-labeled RII(+) RNA probe and MBP-p33C was allowed to form an RNP complex for 30 min, followed by capturing the biotin-labeled RII(+) RNA-protein complex on streptavidin-coated agarose beads. Then, we added MBP-RH30 protein with or without ATP, followed by incubation for 15 min and washing the beads with a small amount of buffer. Then, we measured the amount of MBP-p33C in the eluates by Western blotting using anti-p33 antibody. Increased amounts of MBP-p33C in the eluates mean that RH30 displaced p33C from the viral RNA, likely due to remodelling the RNA structure that could not be recognized by p33 any longer. Nonbiotinylated RNA (lane 6) was used as a control. Each experiment was repeated four times. https://doi.org/10.1371/journal.ppat.1007771.g008 Cellular DEAD-box helicase acts as a restriction factor against TBSV Cellular DEAD-box helicase acts as a restriction factor against TBSV replicase complex, a pull down assay, and BiFC in N. benthamiana. We propose that the interaction of RH30 helicase with the viral replication proteins might be important for the targeting of RH30 into the viral replication compartment (Fig 12). Accordingly, RH30 is recruited into the viral replication compartment from the cytosol and the nucleus based on live imaging in plant cells (Fig 3). The targeting of RH30 into the replication compartment is critical for its antiviral function, because fusion of a nuclear retention signal with RH30, which leads to its enrichment in the nucleus at the expense of the cytosolic pool of RH30, in turn, cancelled out the antiviral effect of RH30. Yeast CFE-based replicase reconstitution assays showed that RH30 acts in the early steps of replication, since both (-) and (+)RNA synthesis was inhibited by RH30 (Fig 6). Moreover, the in vitro RdRp activation assay demonstrated that RH30 inhibited the TBSV RdRp activation step during the replication process as well (Fig 6C). In contrast, the CFE-based TBSV replication was not inhibited by RH30 after replicase assembly was completed (see step 2, Fig 6B). These data suggest that RH30 DEAD-box helicase must act at the earliest steps in the replication process to inhibit TBSV replication.  Based on our current and previous data, we propose that the DDX17-like RH30 helicase interferes with several major steps during TBSV replication. First, RH30 interferes with the recruitment of the viral (+)RNA through unwinding RII(+)-SL cis-acting RNA element, which specifically binds to p33 replication protein only when the stem-loop structure is formed. Also, RH30 can potentially remodel the p33-(+)RNA complex, thus displacing p33 from the RH30 also binds to the viral RNA, including the 5' UTR (i.e., RI) and RII internal sequence present within the p92 pol coding region (Fig 7). Using in vitro interaction and replication assays between RNA-p33 replication protein, we show that RH30 inhibits several steps in tombusvirus replication. These include the RH30-based inhibition of (i) the specific recognition of the critical RII(+)-SL cis-acting element in the viral (+)RNA by p33 replication protein, which is absolutely required for template recruitment into VRCs, (ii) the activation of the viral p92 RdRp, and (iii) the assembly of the VRCs [21,26,73]. Moreover, RH30 helicase could disassemble viral RNA-p33 complexes by likely remodeling the RNA structure in an ATP-dependent manner (Fig 8). However, RH30-mediated disassembly of viral RNA-p33 complexes is unlikely to occur after VRC assembly is completed, because RH30 helicase was not an effective restriction factor when added at a late step of TBSV replication (step 2, Fig 6B). We propose that the membrane-bound TBSV VRCs are protecting the viral RNA-p33 complexes by restricting accessibility of the VRC complex to RH30 DEAD-box helicase. Accordingly, we have shown before that the fully-assembled TBSV VRCs are resistant to cellular ribonucleases [74]. Therefore, RH30 helicase might only be able to disassemble viral RNA-p33 complexes before the vesicle-like spherule formation, which is the characteristic structure of the TBSV VRCs in yeast and plants [75]. Altogether, the in vitro assays provide plentiful data on the direct inhibitory effect of RH30 helicase on TBSV replication, indicating that RH30 functions as an effectortype, not signaling-type, DEAD-box helicase, which detect viral RNA and send signals to downstream components of the innate immunity network [72]. Future experiments will address if RH30 might have additional mechanisms to restrict tombusvirus replication.
A recently emerging concept in innate immunity is the significant roles of DEADbox helicases expressed by host cells that greatly reduce virus replication and facilitate combating viruses and making the induced and passive innate immune responses more potent. Many of the identified yeast DEAD-box helicases with restriction functions against TBSV are conserved in plants and mammals. Altogether, the genome-wide screens performed with animal viruses have shown that helicases are the largest group of host proteins affecting RNA virus replication. For example, in case of HIV, the involvement of several cellular helicases has been demonstrated, including DDX17 and DDX3 [71,76,77]. Yet, the functions of the cellular helicases during virus replication are currently understudied.
The emerging pricture in plant-virus interactions, similar to animal-virus interactions, is the diverse roles of various host RNA helicases. Different plant viruses have been shown to coopt plant RNA helicases for pro-viral functions. These include RH8 and RH9 for potyvirus replication and RH20, RH2 and RH5 for TBSV replication [27][28][29]39,44,78]. However, this paper shows evidence that a plant DEAD-box helicase, RH30, can also be utilized by host plants for antiviral functions. Thus, in addition to the previously identified Dicer-like RNA helicases [16,[79][80][81], additional plant RNA helicases might function as CIRFs by recognizing plant virus RNAs. The DDX17-like RH30 DEAD-box helicase characterized here opens up the possibility that among the more than 100 helicases of plants, there are additional ones with antiviral functions, serving as effector-type or sensor-like RNA helicases. The discovery of the antiviral role of RH30 helicase illustrates the likely ancient roles of RNA helicases in plant innate immunity. In summary, we have demonstrated that the plant DDX17-like RH30 DEAD-box helicase acts as a major restriction factor against tombusvirus replication when complex. Second: Inhibition of p33-(+)RNA complex formation by RH30 also leads to blocking the activation of the p92 RdRp, which requires the (+)RNA with the stem-loop structure in RII(+)-SL formed. Third, displacing p33 from the p33-(+)RNA complex by RH30 inhibits VRC assembly as well. This is because the stem-loop structure in RII(+)-SL is essential part of the VRC assembly platform. The cytosolic pool of RH30 is essential for the antiviral activity. https://doi.org/10.1371/journal.ppat.1007771.g012 Cellular DEAD-box helicase acts as a restriction factor against TBSV expressed in plants and yeast surrogate host. We show that RH30 DEAD-box helicase is targeted to the large TBSV replication compartment. In addition, we find that RH30 blocks the assembly of viral replicase complex, the activation of the RNA-dependent RNA polymerase function of p92 pol and binding of p33 replication protein to critical cis-acting element in the TBSV RNA (Fig 12). Altogether, the plant DDX17-like RH30 DEAD-box helicase is a potent, effector-type, restriction factor of tombus-and related viruses. Assay #3: For the detection of p33 released from protein-biotinylated RNA complex, 1.9 μM of recombinant MBP-p33C was incubated with 0.1 μg of biontinylated RII of DI-72(+) RNA at 25˚C for 15 min, followed by the addition of 20 μl of Promega Streptavidin Magne-Sphere Paramagnetic Particles for another 30 min incubation at room temperature. After collection of the beads and washing with biotin-RNA binding buffer for five times, the particles were incubated with either 0.95 or 3.8 μM of MBP-RH30 or MBP (used as control) in the presence of biotin-RNA binding buffer containing 1 mM ATP at 25˚C for 15 min. The supernatant of the mixture was collected after collecting the particles in a magnetic stand and was analyzed by Western blot with anti-p33 antibody.

Gel mobility shift assay (EMSA) and dsRNA strand-separation assay
The conditions for the EMSA experiments were described previously [24]. Briefly, the EMSA assay was performed with 0.1 pmol of 32 P-labeled RNA probes along with different concentrations (0.4, 1.9, and 5.7 μM) of purified recombinant MBP-fusion proteins or MBP in the presence of RNA binding buffer (10 mM HEPES [pH7.4], 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% Glycerol, 2.5 mM MgCl 2 ), 2 U of RNase inhibitor, as well as 0.1 μg of tRNA in a total of 10 μl reaction volume. Two different amounts (2 and 4 pmol) of unlabeled RNAs together with 5.7 μM of either MBP-RH30 or MBP were used for template competition.
To study if purified proteins could unwind partial dsRNA duplex, the dsRNA strand-separation assay was performed as described [28]. Firstly, the unlabeled single-stranded DI-72 (-) or DI-72 (+) RNAs were synthesized via T7 polymerase-based in vitro transcription. The 32 Plabeled single-stranded RI(-) or RII(+) RNAs were synthesized by T7-based in vitro transcription using 32 P-labeled UTP. To prepare partial dsRNA duplexes, consisting of either RI(-)/DI-72 (+) or RII(+)/DI-72 (-) (see Fig 7E and 7F), 2 pmol of 32 P -labeled RI(-) or RII(+) were annealed to 6 pmol of unlabeled DI-72(+) or DI-72 (-) in STE buffer (10 mM TRIS [pH 8.0], 1 mM EDTA, and 100 mM NaCl) by slowly cooling down the samples (in a total volume of 20 μl) from 94˚C to 25˚C in 30 min. To test if the purified recombinant proteins could separate the partial dsRNA duplex, 1.9 and 5.7 μM purified MBP fusion proteins or MBP as a negative control were added separately to the partial dsRNA duplex in the RNA binding buffer (10 mM HEPES [pH7.4], 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% Glycerol, 2.5 mM MgCl 2 ) along with 1mM ATP, followed by incubation at 25˚C for 25 min. The reaction mixtures were then treated with Proteinase K (2 μg/per reaction) at 37˚C for 20 min, followed by loading onto 5% nondenaturing polyacrylamide gel with 200V for 1 h. Additional methods can be found in S1 Text and the primers used are listed in S1 Table. Supporting information S1 Text. Materials and methods. (DOCX) S1 Table. Sequences of primers used in this study.