https://orcid.org/0000-0003-4164-1237
Figures
Abstract
The intricate interplay between +RNA viruses and their hosts involves the exploitation of host resources to build virus-induced membranous replication organelles (VROs) in cytosol of infected cells. Previous genome- and proteome-wide approaches have identified numerous nuclear proteins, including restriction factors that affect replication of tomato bushy stunt virus (TBSV). However, it is currently unknown how cells mobilize nuclear antiviral proteins and how tombusviruses manipulate nuclear-cytoplasmic communication. The authors discovered that XPO1/CRM1 exportin plays a central role in TBSV replication in plants. Based on knockdown, chemical inhibition, transient expression and in vitro experiments, we show that XPO1 acts as a cellular restriction factor against TBSV. XPO1 is recruited by TBSV p33 replication protein into the cytosolic VROs via direct interaction. We find that blocking nucleocytoplasmic transport function of XPO1 inhibits delivery of several nuclear antiviral proteins, such as AGO2 and DRB4 RNAi factors and CenH3 and nucleolin restriction factors, into VROs resulting in dampened antiviral effects. The co-opted actin network is critical for XPO1 to deliver nuclear proteins to VROs for antiviral activities. We show that XPO1 and XPO1-delivered restriction factors accumulate in vir-condensates associated with membranous VROs. Altogether, the emerging theme on the role of vir-condensates is complex: we propose that vir-condensate serves as a central battleground between virus and the host for supremacy in controlling virus infection. It seems that the balance between co-opted pro-viral and antiviral factors within vir-condensates associated with membranous VROs could be a major determining factor of virus replication and host susceptibility. We conclude that XPO1 and nuclear antiviral cargos are key players in nuclear-cytoplasmic communication during cytosolic +RNA virus replication.
Authors summary
Tomato bushy stunt virus (TBSV), similar to other (+)RNA viruses, replicates in the cytosol and exploits organellar membrane surfaces to build viral replication organelles (VROs) that represent the sites of virus replication. The authors discovered that XPO1 exportin nuclear shuttle protein inhibited TBSV replication in plants. The conserved XPO1 is a central protein interaction nod, which propels nucleocytoplasmic transport of several viral restriction factors into the cytosolic VROs that restrict tombusviruses replication. The delivered virus restriction factors provide inhibitory functions within virus-induced condensates associated with membranous VROs. The authors propose that the VRO-associated condensate serves as a central battleground between virus and the host for supremacy in controlling virus infection. Altogether, XPO1 is a critical protein interaction hub with major implications in viral replication. The authors conclude that XPO1 and its nuclear antiviral cargos are key players in nuclear-cytoplasmic communication during cytosolic (+)RNA virus replication.
Citation: Sun B, Wu C-Y, Gonzalez PA, Nagy PD (2025) Mobilization of nuclear antiviral factors by exportin XPO1 via the actin network inhibits RNA virus replication. PLoS Pathog 21(8): e1012841. https://doi.org/10.1371/journal.ppat.1012841
Editor: Aiming Wang, Agriculture and Agri-Food Canada, CANADA
Received: December 18, 2024; Accepted: July 24, 2025; Published: August 19, 2025
Copyright: © 2025 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript and /or Supporting information files.
Funding: This work was supported in part by the National Science Foundation (IOS-1922895), USDA (NIFA, 2020-70410-32901) and a USDA hatch grant (KY012042) to PDN. The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interest exists.
Introduction
Positive-strand RNA viruses are abundant and cause serious diseases as amply demonstrated by the recent emergence of flaviviruses and the SARS-CoV-2 global pandemic. + RNA viruses also cause major losses in agriculture and threaten global food security. (+)RNA viruses exploit the abundant resources of the host cells to build viral replication compartments/organelles (VROs) in the cytosol. The assembled VROs support +RNA virus replication in a membranous protective microenvironment [1–5].
Virus – host interactions are currently among the most intensively studied research areas due to the promising new antiviral approaches emerging from these studies [3,5–10]. The host uses conserved innate and cell-intrinsic restriction factors as a first line of defense to fight viruses. Recent genome-wide screens with multiple viruses have identified dozens of restriction factors, which inhibit RNA virus accumulation [11–14]. Many restriction factors are nuclear RNA-binding proteins or shuttle proteins between the nucleus and the cytosol. How the nucleus might be able to mobilize nucleic acid binding or modifying proteins to fight against cytosolic RNA viruses is understudied.
Tomato bushy stunt virus (TBSV), a (+)RNA virus, is intensively studied to deepen our understanding of virus-host interactions, the mechanisms of virus replication and recombination [15–20]. An emerging theme with TBSV is that this virus dramatically remodels subcellular membranes and retargets various transport vesicles for VRO biogenesis [18,21,22]. Moreover, TBSV dramatically rewires cellular metabolism, and alters the lipid compositions of the targeted organelles [21–26]. TBSV co-opts the actin network and numerous host proteins, such as Hsp70 chaperone, translation factors eEF1A and eEF1Bγ, the ESCRT-associated proteins, Vps4 AAA ATPase and DEAD-box helicases needed for robust virus replication [27–34].
TBSV codes for two viral replication proteins, termed p33 and p92pol, which are essential for virus replication [35–37]. TBSV p33 protein with the help of co-opted host factors, rewires cellular pathways and optimizes the cellular milieu to support VRO biogenesis and viral RNA replication. Yet, the picture of virus-host interactions is further complicated by host responses, which include an arsenal of restriction factors to inhibit tombusvirus replication [16,38–42].
Previous genome- and proteome-wide studies based on yeast model host identified exportin Crm1/XPO1 and many other nuclear proteins, which might be involved in TBSV replication [16,38–42]. It remains unclear if only the cytosolic pool of nuclear proteins plays a role in tombusvirus replication or if there is a mechanism(s) involving active protein translocation from the nucleus into the viral replication compartments. Identification of the nuclear shuttle protein, called exportin-1 (XPO1 or Crm1 in humans and yeast) suggested a putative role of nuclear export function in the replication of the cytosolic TBSV [38]. The evolutionarily conserved XPO1 is essential for exporting hundreds of proteins from the nucleus to the cytoplasm [43,44]. This protein is so conserved in eukaryotes that the yeast XPO1/Crm1 can be complemented by human XPO1 [43,44]. Among the XPO1 cargos are many nuclear RNA-binding proteins and putative antiviral factors [22,45]. Nuclear export of proteins takes place through the nuclear pore complex with the help of nuclear export receptors, such as XPO1/Crm1 [43,44]. XPO1 binds the nuclear cargo, usually carrying a specific leucine-rich nuclear export signal (NES). The active export of cargos is stimulated by Ran-GTP that facilitates XPO1 to bundle cargos as transport complexes, followed by export to the cytosol. Then, GTP-hydrolysis stimulated by co-factors RanGAP and RanBP1 facilitates the disassembly of the transport complex, releasing the cargos into the cytosol, whereas XPO1 is recycled to the nucleus for new rounds of export [46,47].
In this paper, we studied the role of XPO1 in TBSV replication in plants and in vitro. Based on knockdown, chemical inhibition or over-expression and in vitro experiments, we showed that XPO1 acts as a cellular restriction factor against TBSV replication. XPO1 was found to be recruited by the TBSV p33 replication protein into the cytosolic VROs. BiFC and co-purification experiments demonstrated interaction between XPO1 and the viral p33 replication protein. We also demonstrated the nucleocytoplasmic transport function of XPO1 is needed to deliver nuclear antiviral proteins, such as DRB4, AGO2, nucleolin and CenH3, into VROs to inhibit TBSV replication. The co-opted actin network was critical in XPO1 antiviral activities. In addition, we showed that XPO1 and the XPO1-delivered restriction factors accumulated in vir-condensates associated with the membranous VROs. Altogether, we propose that XPO1 and its cargos are targeted into VROs, ultimately acting antiviral roles.
Results
XPO1 exportin restricts replication of three tombusviruses in plants
Crm1/XPO1 has been identified in yeast screens as a putative antiviral host factor [38]. Because Crm1/XPO1 is a major protein interaction hub protein, we further tested its role in TBSV replication. We knocked down XPO1 mRNA level based on a virus-induced gene silencing (VIGS) approach in Nicotiana benthamiana [48]. Because XPO1 knockdown resulted in smaller plants than the control (TRV-cGFP) plants after 12 days, we shortened the VIGS experiments to 7 days (Fig 1A). Replication of the peroxisome-associated TBSV (Fig 1B) and CNV (the closely-related cucumber necrosis virus) and the mitochondria-associated carnation Italian ringspot virus (CIRV) genomic (g)RNAs was increased by ~2-to-3-fold in XPO1 knockdown (KD) plants when compared to the nonsilenced control plants two-three days after inoculation (S1A and S1B Fig). The symptoms caused by all three tombusviruses became more severe in XPO1 KD than in control plants (Figs 1B, S1A and S1B). Thus, the XPO1 KD plants are highly supportive of tombusvirus replication, suggesting that XPO1 functions as a restriction factor for tombusvirus replication.
(A) Top panel: The phenotypes of XPO1-silenced N. benthamiana is shown at 7 days post agroinfiltration (7 dpai). VIGS was performed by agroinfiltration of tobacco rattle virus (TRV) vectors carrying partial sequence of XPO1 or 3ʼ terminal GFP sequence as the control. Middle panel: RT-PCR analysis of XPO1 mRNA level in the silenced and control plants. Bottom panel: RT-PCR analysis of TUBULIN mRNA level in the silenced and control plants. (B) Accumulation of TBSV genomic (g)RNA and subgenomic (sg)RNAs at 1.5 days after TBSV inoculation in XPO1-silenced (KD) plants was measured by northern blot analysis. The inoculation with TBSV was performed 7 days after VIGS. Second panel: The ribosomal RNA is shown as the loading control in agarose gel stained with ethidium-bromide. TBSV gRNA is visible in the gel. Right panel: Accelerated and more severe TBSV-induced symptom development was observed in XPO1-silenced N. benthamiana. The symptoms were documented at 5 days post TBSV inoculation. The experiment was repeated three times. (C) Transient expression of XPO1 inhibits TBSV accumulation in N. benthamiana. Top panel: Accumulation of TBSV RNAs at 1.5 dpi was measured by northern blot analysis. Bottom panel: The 18S ribosomal RNA is shown in an agarose gel stained with ethidium-bromide as the loading control. N. benthamiana leaves were agroinfiltrated for transient expression of XPO1 (pGD vector as control), in combination with with 0.5% ethanol (EtOH) as a control or 400 nM of Leptomycin B (LMB), a chemical inhibitor of XPO1. The LMB and EtOH treatments were repeated 24 h later. (D) Accumulation of TBSV replicon repRNA in srm1ts or wild type (wt) yeasts at permissive temperature (23 °C) or semi-permissive temperature (29 °C) was measured with northern blotting. His6-p33 and His6-p92 RdRp were expressed from CUP1 promoter and TBSV (+)repRNA from GAL1 promoter to launch replication of TBSV repRNA. Top panel: Northern blot analysis of TBSV repRNA accumulation 1.5 d time point was done using a 3ʼ end specific probe. Bottom panel: The 18S ribosomal RNA is shown as the loading control, which was detected by northern blot. (E) Addition of XPO1 inhibits in vitro replication of TBSV repRNA based on TBSV replicase reconstitution assay in yeast cell-free extract (CFE). The E. coli-expressed and affinity-purified recombinant TBSV p33 and p92pol replication proteins and TBSV (+)repRNA template were added to program CFE to support in vitro replication of TBSV repRNA. Increasing amounts (1.9 and 3.8 μM) of GST (as a control) or GST-XPO1 were added to the reactions. 5% polyacrylamide PAGE containing 8 M urea shows the produced 32P-labeled TBSV (+)repRNAs and dsRNA replication intermediate. (F) The transcriptional level of XPO1 is induced during TBSV replication. Transcriptional levels of XPO1 at six time points were estimated by RT-qPCR in total RNA samples obtained from mock-treated or TBSV-inoculated N. benthamiana. Each experiment was repeated three times. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001; *** represents P < 0.001; ** represents P < 0.01). Error bars represent standard deviation (SD).
To further test the restriction function of XPO1 during tombusvirus replication in plants, we transiently expressed AtXPO1 in N. benthamiana via agroinfiltration. The same leaves were separately inoculated with the three tombusviruses 18 h later. RNA analysis showed ~3-to-5-fold reduction in TBSV (Fig 1C, lanes 4–6 versus 1–3), CNV and CIRV gRNAs accumulation in the inoculated leaves (S2A and S2B Fig, lanes 5–8 versus 1–4).
Nucleus to cytosol export function of XPO1 can be blocked by inhibitors [49]. We treated the tombusvirus-inoculated leaves with Leptomycin B (LMB), which is a highly efficient and selective inhibitor of XPO1 and nuclear export. LMB binds to the NES-binding groove of XPO1 [43,49]. Interestingly, replication of TBSV (Fig 1C, lanes 7–9 versus 1–3) and CIRV (S2B Fig,S2C Fig, lanes 9–12 versus 1–4) was enhanced by ~1.5-to-2-fold after LMB treatment of plant leaves. LMB treatment also neutralized the restriction function of XPO1 expressed transiently in the treated and TBSV (Fig 1C), CNV or CIRV inoculated leaves (S2A and S2B Fig), without changing XPO1 levels (S2C Fig). The above data demonstrate that the restriction function of the plant XPO1 depends on its nucleocytoplasmic export function in tombusvirus replication. The enhanced replication of TBSV and CIRV after the LMB treatment indicates that XPO1 restriction function might be due to the nucleocytoplasmic export of one or more restriction factors from the nucleus to the cytosol, where they might inhibit tombusvirus replication.
The nucleocytoplasmic transport function of XPO1 also depends on regulatory factors in the nucleus. These factors include Ran GEF (guanine nucleotide exchange factor, called Srm1/Prp20 in yeast and RCC1 in humans) that promotes exchange of GDP to GTP in RanGTPase. Then, RanGTP facilitates the formation of XPO1-RanGTP-cargo complex prior to transportation out of the nucleus through the nuclear pore [46,47]. To test if this nuclear activity of RanGTP and XPO1 affects TBSV replication, we used a temperature-sensitive (ts) mutant of Srm1 in haploid yeast [50]. Growing the mutant srm1ts yeast at the semi-permissive temperature (29 °C) resulted in ~4-fold increased TBSV replication in comparison with wild type yeast (Fig 1D, lanes 13–16 vs 9–12). Thus, the RanGTP function and nucleocytosolic transport seems to be important for XPO1 to operate as a tombusvirus restriction factor in yeast.
To further test the restriction function of XPO1, we reconstituted TBSV replicase in vitro using yeast cell-free extract (CFE) programmed with purified p33, p92pol and a replicon (+)RNA [32,51]. In the CFE assay, we used purified recombinant XPO1, which was added in increasing amounts at the beginning of the assay. At the end of the assay, we performed nondenaturing PAGE analysis of the in vitro replicase products. The replication assay revealed up to ~5-fold reduction in both dsRNA replication intermediate [52] and in (+)ssRNA products in CFE with the highest amount of XPO1 in comparison with the RNA replication supported by WT CFE in the presence of GST control (Fig 1E, lanes 2–3 versus 1). Inhibition of both (-)RNA synthesis (i.e., dsRNA production) and (+)RNA synthesis by XPO1 suggests that XPO1 blocks the TBSV replicase assembly steps, which occur prior to (-)RNA and (+)RNA synthesis in vitro [51,53]. Because the CFE preparations are mostly free of the nuclear fraction, and XPO1 is purified from E. coli, it is likely that XPO1 has a direct restriction function against TBSV.
We found that transcription of XPO1 mRNA level was slowly increased during TBSV replication up to 7-fold by the fifth day of infection (Fig 1F). This might indicate that N. benthamiana increases nuclear protein export during TBSV replication as an antiviral response.
XPO1 interacts with the tombusvirus replication proteins and is recruited into the tombusvirus replication organelles in plants
TBSV replicates in the cytosolic side of clustered peroxisomes by assembling large viral replication organelles (VROs) [54–57]. To test if the restriction function of XPO1, which is a shuttle protein between the nucleus and cytosol (Fig 2C) [46], is performed in the cytosol, we co-expressed TBSV p33-BFP replication protein and the eGFP-tagged XPO1 in N. benthamiana leaves. Confocal laser microscopy analysis revealed the co-localization of p33-BFP and eGFP-XPO1 within the cytosolic VROs (marked by the peroxisomal luminar marker, RFP-SKL) in N. benthamiana cells replicating TBSV (Fig 2A). We also used transgenic N. benthamiana expressing the H2B-CFP (Histone2B) nuclear marker protein, which showed that eGFP-XPO1 partitioned between the nucleus and VROs in plant cells infected with TBSV (Fig 2B). Comparable studies with the CIRV p36 replication protein also showed the partial re-localization of eGFP-XPO1 to the CIRV VROs marked by CIRV p36-BFP or RFP-CoxIV mitochondrial marker protein (S3 Fig). Distribution of eGFP-XPO1 in a single cell showed partitioning of XPO1 between the mitochondria-associated VROs and the nucleus in CIRV-infected cells (S3C Fig). Confocal laser microscopy analysis revealed the partial re-localization of eGFP-XPO1 to the cytosolic VROs (marked by the peroxisomal luminar marker, RFP-SKL) in N. benthamiana cells infected with CNV (S3D Fig). Based on these results, we suggest that tombusvirus infections of N. benthamiana plants induce the partial re-localization of XPO1 nuclear shuttle protein into the cytosolic VROs.
(A) Confocal laser microscopy images show co-localization of TBSV p33-BFP replication protein and eGFP-XPO1 in N. benthamiana during TBSV replication. The VROs consisting of clustered peroxisomes are indicated by a RFP-SKL peroxisomal marker. Scale bars represent 10 μm. (B) eGFP-XPO1 partitions between the nucleus and cytosolic VROs in plant cells during TBSV replication. Transgenic N. benthamiana expressing H2B-CFP (Histone2B) was used to mark the nucleus. VROs were marked by white arrows, whereas nucleus was pointed at by yellow arrows. Scale bars represent 10 μm. (C) eGFP-XPO1 distributes in the nucleus and cytosol in healthy transgenic H2B-CFP N. benthamiana. Scale bars represent 50 μm. (D) XPO1 interacts with TBSV p33 replication protein. Interaction between TBSV p33-cYFP replication protein and nYFP-XPO1 was detected by BiFC during TBSV replication in H2B-CFP transgenic N. benthamiana. The green interaction signals surrounding the RFP-SKL marked peroxisomes are indicated by white arrows, demonstrating that interaction between p33-cYFP and nYFP-XPO1 occurs in VROs in planta. Nucleus is marked with yellow arrows. Scale bars represent 10 μm. (E) Copurification of TBSV Flag-p33 and Flag-p92pol replication proteins with His6-XPO1 from subcellular membranes of yeast. Top two panels: Co-purified His6-XPO1 (lane 2) and Flag-affinity-purified Flag-p33 were detected using western blot analysis. The negative control (lane 1) was based on yeast expressing His6-p33 and His6-p92pol together with His6-XPO1. Middle three panels: Protein expression levels in total samples of yeast were detected by western blotting using the shown antibodies. Bottom panel: Coomassie Brilliant Blue staining was used for the normalization of total proteins as loading control. Each experiment was repeated three times.
To test if the re-localization of XPO1 to VROs is dependent on the TBSV p33 replication protein, we used a BiFC approach in N. benthamiana plants. BiFC signals were observed within TBSV VROs, which formed from aggregated peroxisomes and located either at perinuclear or more frequently at distant cytosolic locations (Fig 2D). Similarly, we observed BiFC signals induced by the CIRV p36 replication protein together with XPO1 at the mitochondrial VROs (S4B Fig). The negative control showed no BiFC signal (S4A Fig). We conclude that the interaction between tombusvirus replication proteins and XPO1 mostly takes place in VROs that are occasionally close to perinuclear regions.
To confirm direct interaction between XPO1 and either TBSV p33 or CIRV p36 replication proteins, we performed co-purification experiments from yeast co-expressing Flag-tagged p33, Flag-p92pol or CIRV Flag-p36/Flag-p95pol replication proteins and His6-tagged XPO1. After detergent-solubilization of the membrane-fraction of yeast, the Flag-tagged replication proteins were immobilized on a Flag-column. Western blot analysis of the eluted proteins from the column identified the co-purified His6-XPO1 (Figs 2E, lane 2, and S4C). These co-purification experiments demonstrated the interaction involving TBSV p33 or CIRV p36 replication proteins and XPO1 occurs in the yeast membrane fraction.
Nucleocytoplasmic shuttling of XPO1 promotes XPO1 re-localization to VROs
We blocked the cargo exporting function of XPO1 by applying LMB inhibitor, which specifically binds to XPO1, preventing the binding of cargoes in the nucleus [46]. Confocal microscopy of N. benthamiana plants infected with either TBSV or CIRV and expressing eGFP-XPO1 revealed its mostly nuclear localization and poor re-localization of XPO1 from the nucleus into VROs in LMB-treated plant cells (Fig 3A and 3B). This XPO1 distribution pattern is different from its efficient localization in VROs in the control EtOH-treated plant cells infected with TBSV or CIRV. We suggest that the nucleocytoplasmic shuttling activity of XPO1 contributes to its recruitment into tombusvirus VROs.
(A) Confocal microscopic images show the absence of relocation of eGFP-XPO1 to VROs during TBSV replication when treated with 400 nM LMB inhibitor in H2B-CFP transgenic N. benthamiana. Bottom panel: Localization of eGFP-XPO1 when leaves were treated with 0.5% EtOH (control) in H2B-CFP transgenic N. benthamiana. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. Scale bars represent 10 μm. Right panel: The graphs show fluorescence intensity of eGFP-XPO1 in VROs during TBSV replication after LMB versus EtOH treatments of H2B-CFP transgenic N. benthamiana. The fluorescent intensity in VRO regions is quantified by Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001). Error bars represent standard deviation (SD). (B) Confocal microscopic images show the absence of relocation of eGFP-XPO1 to VROs during CIRV replication when leaves were treated with LMB inhibitor in H2B-CFP transgenic N. benthamiana. The VROs, consisting of clustered mitochondria, are visualized by CoxIV-RFP mitochondrial marker. See further details in panel A. Each experiment was repeated three times.
BiFC experiments between TBSV p33 and XPO1 in LMB-treated N. benthamiana leaves showed that the interaction between these proteins occurs in the VROs (S5A Fig). Similarly, LMB treatment did not prevent the interaction between CIRV p36 and XPO1 within the mitochondria-associated VROs (S5B Fig). These observations suggest that p33/p36-XPO1 interaction region is unlikely to include the canonical cargo-binding pocket in XPO1, which is targeted/blocked by LMB. Moreover, these data also support the model that XPO1 could be directly recruited by the tombusvirus p33/p36 replication proteins into VROs from the cytosol. Further works will define the proposed novel interaction sites/domains in TBSV p33 and XPO1.
XPO1 delivers RNAi restriction factors from the nucleus to VROs during tombusvirus infections
Increased tombusvirus replication in XPO1 KD plants (Fig 1) and by blocking of XPO1 re-location from the nucleus to the VROs by treatment with LMB (Fig 3) indicate that a plausible explanation of anti-tombusvirus activity of XPO1 is the delivery of a pool of restriction factors from the nucleus to VROs. To test this model, we decided to characterize the effect of XPO1 on the re-localization of known nuclear restriction factors to tombusvirus VROs.
First, we studied selected components of the RNAi machinery, which are known to present in both nucleus and the cytosol [58,59]. DRB4, which is a double-stranded RNA binding protein, is known to be re-targeted into TBSV VROs in Arabidopsis [60] and N. benthamiana (S6 Fig). We observed that DRB4 was mostly restricted to nucleus after LMB treatment (S6E Fig), suggesting that DRB4 localization is affected by nucleocytoplasmic transport function of XPO1. We also found that a fraction of DRB4 was re-targeted to VROs during TBSV (S6A–C Fig), CNV (S7A and S7B Fig), or CIRV infections (S8A–C Fig). Interestingly, knocking down XPO1 expression or inhibiting XPO1 activity by LMB treatment reduced the recruitment of DRB4 into CNV VROs by ~2.5-fold (Fig 4A and 4B) and in TBSV VROs by ~2–3-fold (S9 Fig). Similarly, XPO1 knockdown significantly affected the recruitment of DRB4 into CIRV VROs (S8E Fig). Transient expression of DRB4 inhibited CNV replication in N. benthamiana plants by 2-fold, confirming DRB4 antiviral function (Fig 4C, lanes 5–8 versus 1–4). However, treatment of N. benthamiana leaves with LMB or knocking down XPO1 level interfered with the inhibitory effect of DRB4 on CNV replication (Fig 4C and 4D). Altogether, we suggest that the antiviral function of DRB4 and its re-localization to the tombusvirus VROs depends on XPO1 nucleocytoplasmic export function.
(A) Recruitment of eGFP-DRB4 to VROs is reduced in XPO1 knockdown (KD) N. benthamiana infected with CNV. Left top panel: Confocal microscopic images show poor recruitment of eGFP-DRB4 into VROs (indicated by RFP-SKL) in XPO1 KD N. benthamiana. Bottom panel: Control experiment with N. benthamiana plants agroinfiltrated with the TRV vectors carrying partial GST sequence. Scale bars represent 10 μm. Right panel: Quantification of fluorescent intensity of eGFP-DRB4 in VRO regions was done with Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (*** represents P < 0.001). Error bars represent standard deviation (SD). (B) Recruitment of eGFP-DRB4 to VROs is reduced in N. benthamiana infected with CNV and treated with 400 nM LMB versus 0.5% EtOH. See further details in panel A. (** represents P < 0.01). (C) Transient expression of Flag-DRB4 in N. benthamiana inhibits CNV accumulation. Top panel: CNV RNA accumulation at 2 dpi was measured by northern blot analysis. N. benthamiana leaves were agroinfiltrated to express Flag-DRB4 (pGD vector as control) and the agroinfiltrated leaves were either infiltrated with 0.5% ethanol (EtOH) as a control or 400 nM of Leptomycin B (LMB). Middle panel: The 18S ribosomal RNA is shown in an agarose gel stained with ethidium-bromide as the loading control. Bottom panel: The expression of Flag-DRB4 was measured by western blot from the above samples using anti-flag antibody. Coomassie Brilliant Blue staining was used for the normalization of total proteins as loading control. (D) Top panel: Accumulation of CNV gRNA and sgRNAs at 2 dpi was measured by northern blot in XPO1-silenced plants expressing eGFP-DRB4. Agrobacteria infiltration to express eGFP-DRB4 (in the absence of p19 silencing suppressor) was performed 7 days after silencing of XPO1 in N. benthamiana, followed two days later by inoculation with CNV. Bottom panel: The ribosomal RNA is shown as the loading control in the agarose gel stained with ethidium-bromide. Each experiment was repeated three times.
The second component of the RNAi machinery tested was AGO2. AGO2 protein is a component of the antiviral RISC complex, which targets plant RNA viruses, including tombusviruses [61,62]. We transiently expressed eGFP-AGO2 in N. benthamiana plants infected with CNV (S10 Fig), TBSV (S11 Fig) or CIRV (S12 Fig). Confocal microscopy analysis showed the efficient re-localization of AGO2 and XPO1 to VROs, representing either the clustered peroxisomes or mitochondria. As expected, AGO2 overexpression inhibited CNV replication (S13C Fig). However, treatment of N. benthamiana plants overexpressing AGO2 with LMB (S13C Fig) or knocking down XPO1 via VIGS (S13D Fig) dampened the antiviral activity of AGO2. LMB treatment or knocking down XPO1 expression significantly reduced the re-localization of AGO2 to CNV (S13A and S13B Fig), TBSV (S14A and S14B Fig) or CIRV VROs (S14C Fig). Based on these data, we conclude that the antiviral function and re-localization of AGO2 to tombusvirus VROs depends on XPO1 nuclear export function.
XPO1 delivers cellular intrinsic restriction factors from the nucleus to VROs during viral infections
Tombusvirus replication is not only affected by the host powerful RNAi machinery, but cellular intrinsic restriction factors (CIRFs) limit tombusviruses in yeast and plants [14]. Several of the CIRFs are localized in the nucleus. Therefore, we tested if the recruitment of nuclear CIRFs is affected by XPO1 nucleocytoplasmic transport function. First, we studied the noncanonical function of CenH3 histone variant, which is recruited to tombusvirus VROs from the nucleus, resulting in inhibition of tombusvirus replication [63]. eGFP-CenH3 is co-localized with RFP-XPO1 in TBSV (S15 Fig), CNV (S16 Fig) and CIRV VROs (S17 Fig) and in the nucleus. Knocking down XPO1 level or treatment of N. benthamiana plants with LMB inhibited the re-localization of CenH3 into TBSV (Fig 5A and 5B) or CIRV VROs (S18 Fig) by ~2.5-to-4-fold. Treatment of N. benthamiana leaves with LMB interfered with the inhibitory effect of CenH3 on TBSV (Fig 5C) and CIRV replication (S18C Fig). Altogether, these data support the notion that the antiviral function and re-localization of CenH3 to the tombusvirus VROs depends on XPO1 nuclear export function.
(A) Recruitment of eGFP-CenH3 to VROs is reduced in XPO1 KD N. benthamiana infected with TBSV. Left top panel: Confocal microscopic images show poor recruitment of eGFP-CenH3 into VROs (indicated by RFP-SKL) in XPO1 KD N. benthamiana. Bottom panel: Control experiment with N. benthamiana plants agroinfiltrated with the TRV vector carrying partial GST sequence. Scale bars represent 10 μm. Right panel: Quantification of fluorescent intensity of eGFP-CenH3 in VRO regions with Olympus FV3000 FLUO-view software. See further details in Fig 4A. Fig 6A. (B) Recruitment of eGFP-CenH3 to VROs is reduced in N. benthamiana infected with TBSV and treated with 400 nM LMB versus 0.5% EtOH. Note that we show two independent sets of images to illustrate similar trends in different plants. See further details in Fig 4A. Fig 6A. (C) Transient expression of Flag-CenH3 in N. benthamiana does not inhibit TBSV accumulation in plants treated with 400 nM LMB. Top panel: TBSV RNA accumulation at 1.5 dpi was measured by northern blot analysis. N. benthamiana leaves were agroinfiltrated to express Flag-CenH3 (or pGD vector as control) and the agroinfiltrated leaves were either infiltrated with 0.5% ethanol (EtOH) as a control or 400 nM LMB. Middle panel: The 18S ribosomal RNA is shown in an agarose gel stained with ethidium-bromide as the loading control. Bottom panel: The expression of Flag-CenH3 was measured by western blot from the above samples using anti-flag antibody. Coomassie Brilliant Blue staining was used for the normalization of total proteins as loading control. Each experiment was repeated.
The second nuclear CIRF tested was Nuc-L1 nucleolin (Nsr1 in yeast) that was previously shown to inhibit TBSV replication in yeast [64]. Nuc-L1 interacts with both p33 replication protein (S19B Fig) and the TBSV RNA [64]. Nuc-L1 also interacts with XPO1 mostly in the nucleus in N. benthamiana (S19A Fig). Interestingly, LMB treatment strongly inhibited the BiFC signal between Nuc-L1 and XPO1 in the nucleus (S19A Fig, bottom panels). Nuc-L1 interacted with XPO1 in both the nucleus and TBSV VROs in TBSV-infected control plants (S19A Fig, top panel). However, LMB-treatment strongly inhibited Nuc-L1 - XPO1 interaction in TBSV VROs (S19A Fig, second panel). LMB-treatment neutralized the inhibitory effect of Nuc-L1 over-expression on CNV accumulation in N. benthamiana (S19C Fig). Western-blotting showed that the LMB-treatment did not inhibit the expression of Nuc-L1 (S19C Fig). These data support the role of XPO1 in delivering Nuc-L1 antiviral restriction factor into tombusvirus VROs. Based on the above data, we conclude that several host restriction factors (CIRF) are delivered from the nucleus into tombusvirus VROs in an XPO1-dependent fashion.
Critical role of the actin network in delivering XPO1 and antiviral cargos into tombusvirus VROs
The above data convincingly showed that XPO1 interacts with the tombusvirus replication proteins and delivers antiviral factors from the nucleus to VROs to restrict tombusvirus replication. How are XPO1 and the nuclear restriction factors targeted to VROs? One of the possible mechanisms is the contribution of the actin network. TBSV is known to hijack actin filaments to recruit cytosolic factors with pro-viral functions into VRO-associated vir-condensates [65–67]. TBSV p33 replication protein inhibits actin depolymerization function of Cof1/ADF1 actin filament disassembly protein, thus resulting in stable actin filaments and actin cables in TBSV-infected cells [54]. Therefore, we decided to test if the co-opted stabilized actin filaments could assist the movement of XPO1 and its cargos into VROs. Confocal imaging revealed the association of XPO1 with actin filaments within VROs and in the cytosol of TBSV- or CIRV-infected cells (Fig 6A and 6B). Moreover, BiFC experiments showed that TBSV p33 and CIRV p36 replication proteins in complex with XPO1 were associated with actin filaments (Fig 6D and 6E). We also observed that XPO1 cargos, such as DRB4 (S20A and S20B Fig), AGO2 (S20D–F Fig) and CenH3 (S21A and S21B Fig) were associated with actin filaments within VROs and in the cytosol of TBSV- or CIRV-infected cells. These observations highlighted the possibility that the co-opted actin filaments are used to deliver XPO1 and its nuclear cargos into VROs.
(A-B) Confocal microscopy images show co-localization of eGFP-XPO1, RFP-Actin filaments and VROs, indicated by BFP-SKL during TBSV replication or by CoxIV-BFP mitochondrial marker during CIRV replication in N. benthamiana. The actin filaments were visualized by agro-expression of Lifeact, which binds to filamentous actin (F-actin) in plant cells. The actin filaments co-localized with eGFP-XPO1 are indicated by yellow arrows. The scale bar represents 10 μm. The enlarged images from the red boxed areas contain VROs and nuclear region. Note that A&B panels are Z-stack projection images including 10 layers. (C) Localization of eGFP-XPO1, RFP-Actin, and CoxIV-BFP mitochondrial marker in mock-treated N. benthamiana. The scale bar represents 10 μm. (D-E) Confocal microscopy images show the co-localization of RFP-Actin and BiFC interaction signals of nYFP-XPO1 and TBSV p33-cYFP or CIRV p36-cYFP in N. benthamiana cells. TBSV VROs are marked with SKL-BFP peroxisomal marker, whereas CIRV VROs are marked with CoxIV-BFP mitochondrial marker. The scale bar represents 10 μm. Note that these panels are Z-stack projection images including 10 layers. Each experiment was repeated.
To further test this model, we destroyed the cytosolic actin network by transiently expressing RavK effector of Legionella bacterium in N. benthamiana cells (Fig 7C) [67–69]. Transient expression of RavK in cells infected with TBSV led to ~2.5-fold reduced recruitment of XPO1 into TBSV VROs (Fig 7A and 7B). Transient RavK expression also inhibited XPO1 recruitment into CIRV VROs formed by clustered mitochondria by ~3-fold (S22A and S22B Fig). We also transiently expressed VipA effector of Legionella bacterium in N. benthamiana cells, which is an actin nucleator and stabilizes actin filaments (Fig 7C) [70]. VipA expression is known to facilitate TBSV replication [67,68]. Interestingly, transient expression of VipA in cells infected with TBSV led to ~2-fold enhanced recruitment of XPO1 into TBSV VROs (Fig 7A and 7B). These data suggest that recruitment of XPO1 into VROs is facilitated by co-opted actin filaments in TBSV-or CIRV-infected cells.
(A) Confocal microscopy images show recruitment of eGFP-XPO1 into VROs is affected by actin filaments during TBSV replication. Top panel: Co-localization of eGFP-XPO1, RFP-Actin filaments and VROs, indicated by BFP-SKL during TBSV replication in N. benthamiana. See further details in Fig 8A. Middle two panels: Confocal microscopy images show the poor recruitment of eGFP-XPO1 into TBSV VROs when the actin filaments were destroyed by the transient expression of RavK effector of Legionella bacterium from a plasmid via agroinfiltration in N. benthamiana. Bottom three panels: Confocal microscopy images show efficient recruitment of eGFP-XPO1 into TBSV VROs when the actin filaments were induced and stabilized by the transient expression of VipA effector of Legionella bacterium from a plasmid via agroinfiltration in N. benthamiana. Scale bars represent 10 μm. Note that these panels are Z-stack projection images including 10 layers. (B) Quantification of eGFP-XPO1 within VROs based on experiments shown in panel A. The fluorescent intensity of eGFP-XPO1 in VRO regions is quantified by Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001, ** represents P < 0.01). Error bars represent standard deviation (SD). (C) Control experiments to show distribution patterns of actin filament network when RavK or VipA effectors are transiently expressed versus no protein expression in N. benthamiana. Scale bars represent 10 μm. Note that these panels are Z-stack projection images including 10 layers. Each experiment was repeated.
Transient expression of RavK inhibited the delivery of XPO1 cargos DRB4 (Fig 8A and 8B), CenH3 (Fig 8C and 8D) and AGO2 (Fig 8E and 8F) into TBSV VROs by ~2-to-4-fold. Similarly, disruption of actin filaments by RavK inhibited the recruitment of DRB4 (S22C and S22D Fig), AGO2 (S23C and S23D Fig) and CenH3 (S23A and S23B Fig) into CIRV VROs by ~2-to-4-fold. These data established that the co-opted actin network facilitates the delivery of XPO1 and its nuclear antiviral cargos into tombusvirus VROs to restrict tombusvirus replication in plants.
Confocal microscopy images show the poor recruitment of eGFP-DRB4 (A), eGFP-CenH3 (C), and eGFP-AGO2 (E) into TBSV VROs when the actin filaments were destroyed by the transient expression of RavK effector of Legionella bacterium from a plasmid via agroinfiltration in N. benthamiana. Top two panels in (A, C, and E) show the control confocal images without the expression of RavK effector in N. benthamiana. Scale bars represent 10 μm. Note that these panels are Z-stack projection images including 10 layers. Quantification of VRO-localized eGFP-DRB4 (B), eGFP-CenH3 (D) and eGFP-AGO2 (F) is shown in graphs. The fluorescent intensity in VROs is quantified by Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001). Error bars represent standard deviation (SD). Each experiment was repeated three times.
Regulatory co-factors of XPO1 are recruited into tombusvirus VROs
The disassembly of the XPO1-RanGTP-cargo ternary complex requires the conversion of RanGTP into a GDP-bound form (RanGDP) in the cytosol. This step is facilitated by RanGAP1 or RanGAP2 and RanBP1 co-factors [47,71]. We found that RanGAP1/2 are recruited into TBSV (S24A and S24B Fig) and CIRV VROs (S25 and S25B Fig). In addition, RanBP1 is also present in TBSV (S24C Fig) and CIRV VROs (S25C Fig). Colocalization experiments showed that the recruitment of RanGAP1/2 (S24D and S24E Fig) and RanBP1 factors (S24F Fig) into TBSV VROs takes place with the help of the actin network. Similarly, the actin network could play a role in the recruitment of RanGAP1/2 (S25D and S25E Fig) and RanBP1 factors (S25F Fig) to VROs consisting of aggregated mitochondria in CIRV-infected leaves. Recruitment of these cytosolic regulatory co-factors of XPO1 suggests that XPO1-RanGTP-cargo complex is likely disassembled in tombusvirus VROs, leading to the release of the antiviral cargos from the cargo complex in VROs. RanGAP1/2 and RanBP1 do not seem to interact with TBSV p33 or CIRV p36 replication proteins based on BiFC analysis in N. benthamiana (S26A and S26B Fig). These regulatory co-factors are likely recruited into VROs via interaction with XPO1.
Recruited XPO1 and its antiviral cargos are present in vir-condensates associated with membranous VROs
Tombusvirus VROs contain co-opted peroxisomal or mitochondrial membranes harboring the viral spherules, the sites of RNA replication, in association with vir-condensates formed via p33-induced liquid-liquid phase separation of sequestered cytosolic proteins [65]. The vir-condensate concentrates co-opted cytosolic factors, such as glycolytic and fermentation enzymes, to produce ATP locally in VROs to support robust TBSV replication [65]. The vir-condensate also stores key autophagy factors to dampen the antiviral activity of autophagy [72,73]. To test if XPO1 and the delivered antiviral cargos are present in vir-condensates, we performed FRAP experiments in N. benthamiana cells replicating TBSV. The slow recovery of the fluorescent signals after photobleaching of VROs suggested that XPO1 (Fig 9A) and the delivered DRB4 (Fig 9B), CenH3 (Fig 9C) or AGO2 (Fig 9D) cargos are all present in TBSV and CIRV (S27 Fig) vir-condensates within VROs.
(A) FRAP analysis shows partial fluorescence recovery of RFP-XPO1 after photobleaching in a single VRO in N. benthamiana during TBSV replication. Top panel: Co-localization of replication protein p33-BFP and RFP-XPO1 in VRO before photobleaching. Middle panels: Confocal microscopy images show partial fluorescence recovery of RFP-XPO1 at 0-, 40-, and 80-seconds post photobleaching during TBSV replication. Note that signals of p33-BFP did not recover in the FRAP assay because it is a membrane-bound protein. Scale bars represent 5 μm. Bottom panel: The graph shows time course analysis of FRAP data on RFP-XPO1 signal recovery in individual VROs during TBSV replication. Sample size n is annotated in the figure. Shaded area represents SD. Similar FRAP analysis of RFP-DRB4 (panel B), RFP-CenH3 (panel C), and RFP-AGO2 (panel D) are also shown. See further details in panel A. Each experiment was repeated three times.
To further characterize the interactions of the viral replication protein and the recruited antiviral proteins within the vir-condensates, we performed in vitro assays to study co-droplet formation. TBSV p33 was shown previously to form droplets in vitro due to weak interactions supported by its IDR1 region based on liquid-liquid phase separation [65,74]. Using purified C-terminal fragment of eGFP-tagged TBSV p33 (p33C), which is exposed to the cytosol [75,76], to aid protein solubility [77], together with purified mRFP-tagged DRB4 resulted in round co-droplets in the presence of 2.5% PEG crowding agent in vitro (S28A Fig). mRFP-DRB4 also formed a few droplets when applied in higher concentration (S28B Fig). Similarly, eGFP-p33C also formed co-droplets with purified mRFP-CenH3 (S28C Fig). Altogether, these data are consistent with the model that TBSV p33 and the recruited DRB4 and CenH3 antiviral proteins could partition in droplets formed via liquid-liquid phase separation.
In sum, these observations suggest that the vir-condensate is the location where the co-opted antiviral factors perform their inhibitory functions on tombusvirus replication. Thus, the host cells exploit the tombusvirus-induced vir-condensates to concentrate antiviral proteins in VROs as a virus-defense mechanism to restrict tombusvirus replication.
Discussion
Tombusviruses, similar to other (+)RNA viruses, replicate in the cytosol and exploit organellar membrane surfaces to build VROs. However, previous genome- and proteome-wide studies of TBSV in the yeast model host, which supports TBSV replication, also identified numerous host factors localized mainly in the nucleus in the absence of viral components [35,42,78]. These findings indicated a yet unknown contribution of the nucleus to tombusvirus replication. Among the nuclear factors identified, XPO1 exportin shuttle protein was intriguing as a restriction factor. Mutation of XPO1/Crm1 in yeast enhanced TBSV replication, thus revealing a new layer in antiviral mechanisms [38]. But how could a nuclear shuttle protein affect the replication of a cytosolic virus?
XPO1 is a major protein interaction hub, which is involved in exporting ~1,000 proteins and RNA-protein complexes out of the nucleus [43,46]. Therefore, viral hijacking of XPO1 seems to provide easy access to protein rich resources, such as RNA-binding proteins and helicases, which are otherwise hidden in the nucleus from a cytosolic virus. However, among the cargo proteins of XPO1 could be restriction factors, which present a challenge to a simple (+)RNA virus with limited protein coding capacity, such as tombusviruses. Accordingly, data presented in this paper show that XPO1 acts as a restriction factor (CIRF) of tombusvirus replication for both the peroxisome-associated TBSV and CNV and the mitochondria-associated CIRV in N. benthamiana plants. The in vitro replicase reconstitution experiment showed that XPO1 directly inhibits TBSV replication, likely via binding and sequestering of p33 replication protein, thus preventing p33 to perform its essential functions during viral replication. Interestingly, treatment with LMB inhibitor, which inhibits cargo binding by XPO1 in the nucleus [46], did not inhibit p33 and XPO1 interaction based on BiFC experiments. This suggests that XPO1-p33 interaction domain(s) is different from the canonical cargo-binding site blocked by LMB. Therefore, it is likely that cytosolic interaction between p33 and XPO1 promotes the recruitment of XPO1 into VROs. Further studies will be aimed to dissect the molecular details of noncanonical p33 and XPO1 interaction.
Based on in planta experiments with LMB inhibitor of XPO1 or knocking down XPO1 level via VIGS showed that the main tombusviral restriction function of XPO1 is the delivery of nuclear RNAi and other CIRF factors into VROs. We found that these nuclear host factors were poorly re-located into VROs when XPO1 function was inhibited with LMB or XPO1 level was knocked down. Additional evidence on the nucleocytosolic export function of XPO1 in antiviral activity is based on mutation in the yeast co-factor Srm1/Prp20 guanine nucleotide exchange factor (RanGEF, known as RCC1 in humans) of XPO1, which led to enhanced TBSV replication in yeast. RanGEF promotes cargo loading to XPO1 in the nucleus [79]. Altogether, these host restriction factors block the functions of viral RNAs and/or replication proteins in VROs, thus limiting the efficiency of TBSV replication. We suggest that re-localization and antiviral functions of DRB4 and AGO2 RNAi components and CenH3 and nucleolin Nuc-L1 CIRFs are dependent on XPO1 nucleocytoplasmic function in N. benthamiana plants. These are selected proteins based on their known restriction functions [60,61,63,64]. However, one can imagine that many more nuclear proteins, including those with antiviral or pro-viral functions, depend on XPO1 to be re-localized to the VROs. The emerging picture is that the co-opted XPO1 is a major protein interaction hub with a central role in deciding the outcome of tombusvirus infections by delivering a pool of restriction factors into VROs (Fig 10).
(#1) In the nucleus, high gradient of RanGTP is established with the contribution of guanine nucleotide exchange factor (RanGEF, also known as RCC1). RanGTP binding to XPO1 leads to conformational change opening the cargo loading groove in XPO1. Then, nuclear export signal (NES) sequence in the cargo is loaded onto XPO1, leading to the formation of cargo-XPO1-RanGTP ternary protein complex. (#2) Subsequently, this ternary protein complex docks at nuclear pore complex (NPC) in nuclear envelope, and then is exported to the cytosol through NPC. (#3) During replication of tombusviruses, the replication protein (TBSV p33 or CIRV p36) stabilizes the actin cytoskeleton by inhibiting the depolymerization of actin filaments by COF1/ADF1. TBSV p33 interacts with XPO1 ternary complex, resulting in recruitment of p33-cargo-XPO1-RanGTP to VROs along the stable actin filament networks. XPO1 regulatory cofactors, such as Ran GTPase-activating Protein1 (RanGAP1) and Ran Binding Protein1 (RanBP1), are also recruited into VROs represented by clustered peroxisomes (shown) or clustered mitochondria in case of CIRV infection. XPO1 might also be directly recruited by TBSV p33 to VROs via p33-XPO1 interaction in the cytosol. (#4 and the enlarged boxed area) The XPO1-complex dissembles in vir-condensate (induced by membrane-bound p33 or CIRV p36) due to the stimulatory activities of co-opted RanBP1 and RanGAP1, releasing RanGDP and cargos from the XPO1 complex. The released antiviral cargo performs inhibitory functions in vir-condensate associated with the membranous VROs. We propose that the host exploits XPO1 and the vir-condensate to concentrate and sequester antiviral cargos that enhances their effectiveness in inhibiting tombusvirus replication.
The direct binding of XPO1 to p33 replication protein in the cytosol raises the question if XPO1 could serve as a “sensor” of RNA virus infection. Plant cells might be able to detect the reduced availability of “free” XPO1 in the cytosol and/or the nucleus during infection. Indeed, the expression of XPO1 increases during TBSV replication, which could lead to robust mobilization of nuclear antiviral factors to suppress viral infection. Future work will address the proposed additional antiviral functions of XPO1 in more detail.
XPO1 was able to perform its antiviral function by binding to the viral p33 replication protein and using the co-opted actin network to target VROs. The recruitment of DRB4, AGO2 and CenH3 antiviral factors to VROs was dependent on both XPO1 and the co-opted functional actin filaments. The emerging theme on the roles of the co-opted actin network is that it facilitates the delivery of both cytosolic and nuclear proteins into VROs. These proteins provide either pro-viral or antiviral functions [66,67]. Moreover, actin filaments also form “fences” within/around the vir-condensate and VROs, providing structural components to VROs [65]. Thus, the current work further expands our current understanding of the essential, but complex roles played by the co-opted actin network in VRO assembly and tombusvirus replication. Due to pleiotropic effect of the actin network on VRO assembly, the balance or imbalance in delivering proviral (dependency) or restriction host factors into VROs could decide if the virus becomes successful or its host ultimately resists the infection [54,67].
Based on the recruitment of XPO1 and its antiviral cargos as well as XPO1 co-factors into tombusvirus VROs, we propose that XPO1 mobilizes the antiviral cargos from the nucleus to VROs (Fig 10). We showed that the XPO1 co-factor Srm1/Prp20 guanine nucleotide exchange factor (RanGEF, known as RCC1 in humans) inhibits TBSV replication in yeast. RanGEF promotes the conversion of nuclear RanGDP into RanGTP, which binds to XPO1, resulting in conformational change by opening the cargo loading groove in XPO1. Then, the cargos are loaded via nuclear export signal (NES) sequences onto XPO1 [43,46,79]. The cargo-XPO1-RanGTP ternary protein complex is exported to the cytosol through nuclear pore complex. In the cytosol, the XPO1 complex takes advantage of the actin cytoskeleton, which is co-opted and stabilized by viral replication protein (TBSV p33 or CIRV p36) by inhibiting the depolymerization of actin filaments by COF1/ADF1 [54]. We propose that TBSV p33 interacts with XPO1 ternary complex in the cytosol and the complex is delivered to VROs along the co-opted actin filament networks. We showed that XPO1 regulatory cofactors, such as Ran GTPase-activating Protein1/2 (RanGAP1/2) and Ran Binding Protein1 (RanBP1), are also recruited into VROs. It is likely that the XPO1/RanGTP/cargo ternary complex dissembles in vir-condensate due to the stimulatory activities of co-opted RanBP1 and RanGAP1/2, releasing RanGDP and cargos from the XPO1 complex [43,46]. FRAP analysis showed that the released antiviral cargos are present in vir-condensate substructure of the VROs. Therefore, the XPO1-delivered antiviral proteins could perform inhibitory functions within vir-condensate associated with VROs. In sum, we propose that XPO1 delivers and sequester antiviral cargos in vir-condensate (Fig 10). Overall, the high concentration of antiviral cargos in vir-condensates enhances their effectiveness in inhibiting tombusvirus replication.
The established core roles of VROs are to support viral replication and protect the viral RNAs from degradation by host antiviral mechanisms. However, a major finding of this work is the demonstration of the abundant presence of antiviral factors within the vir-condensate substructure within the membranous VROs. Previous work demonstrated that vir-condensate contains recruited pro-viral cytosolic host factors, such as glycolytic and fermentation enzymes, which produce ATP locally to supply the energy requirement of tombusvirus replication [65]. The vir-condensate also stores autophagy proteins, such as NBR1 autophagy receptor and ATG8f core autophagy protein, in associated vir-NBR1 bodies to dampen the antiviral roles of autophagic degradation [72,80]. However, based on the current work, the emerging picture on the role of the vir-condensate is becoming more complex: it seems to be a “central battleground” between the virus and the host for supremacy in controlling virus infection. Accordingly, we found that a large fraction of the nuclear antiviral proteins tested accumulate in vir-condensate associated with VROs (based on FRAP assay), not in the cytosol. Thus, their antiviral actions most likely occur in vir-condensates/VROs, not in the cytosol. In addition, our data showed that DRB4 and CenH3 are concentrated/partitioned in p33 droplets in vitro (S28 Fig), suggesting that DRB4 and CenH3 are present in p33-induced condensates associated with the co-opted membranes in VROs. Also, when XPO1 function is blocked by LMB, then DRB4 amount is low in VROs and DRB4 expression mostly lost its antiviral activity (Fig 4). Altogether, we propose that the balance between the co-opted pro-viral and antiviral factors within the vir-condensate could be a major determining factor of virus replication and host susceptibility. We predict that by tilting the balance toward antiviral factors within vir-condensates might lead to new efficient virus therapeutics or host resistance against viruses.
XPO1 as a viral restriction factor was described only in few cases, including Dengue virus NS5 protein, which enters the nucleus and interacts with XPO1 to modulate host antiviral responses [81,82]. XPO1-mediated export of DDX3X helicase is required to enhance immunity against human parainfluenza virus-3 [83]. Inhibition of XPO1 or its knock down inhibits coronavirus (mouse hepatitis virus) replication, whereas XPO1 effect on SARS-CoV-2 is tissue specific in humans [84]. Thus, XPO1 plays critical role in host immunity, suggesting that our understanding of XPO1 restriction function against tombusviruses might have broad relevance in RNA virus research.
The nucleocytoplasmic function of XPO1 is exploited by several viruses. For example, XPO1 is an important protein for HIV RNA transport out of the nucleus with the help of HIV Rev protein [85,86]. Other nuclear viruses or their proteins, such as influenza virus, Nipah virus, Rhinovirus and DNA viruses depend on XPO1 to exit out of the nucleus [87–90]. XPO1 facilitates the nuclear export of the RdRp protein of Turnip mosaic virus from the nucleus to the cytosol [91,92]. Thus, the emerging picture is that XPO1 is hugely important in viral diseases in humans and plants [87].
Summary
XPO1 exportin is a central interaction nod, which emerges as a major player in tombusvirus replication in plants. The emerging theme from our current studies is that XPO1 with the help of co-opted actin network provides nucleocytoplasmic transport of several viral restriction factors into the cytosolic VROs that restrict tombusviruses replication. The delivered restriction factors provide inhibitory functions within the vir-condensates associated with membranous VROs. Altogether, XPO1 is a critical protein interaction hub with major implications in viral replication.
Materials and methods
VIGS-based knockdown of XPO1 in N. benthamiana
To silence expression of XPO1 in N. benthamiana, we used a virus-induced gene silencing (VIGS) approach [48,93]. A 171-bp segment of NbXPO1 (Accession number: LC434544) was cloned and inserted into pTRV2 vector to obtain the silencing vector pTRV2::NbXPO1 according to [94]. The leaves of N. benthamiana were infiltrated with a mixture of agrobacterium carrying pTRV1 (OD600 0.2) and pTRV2::NbXPO1 (OD600 0.2) or pTRV1 (OD600 0.2) and pTRV2::cGFP (OD600 0.2) as control for tombusvirus replication assay. Note that for the confocal microscopy experiments, in which NbXPO1 was knocked down in H2b-CFP or H2b-RFP transgenic N. benthamiana, the leaves were infiltrated with a mixture of agrobacterium carrying pTRV1 (OD600 0.2) and pTRV::GST(OD600 0.2) plasmids as the control. For virus replication assay, the upper leaves were inoculated with sap preparations containing TBSV, CIRV or CNV 7 d later. Note that we used CNV20KSTOP not expressing the p20 suppressor of gene silencing protein [48]. The inoculated leaves were harvested at 1.5 dpi, 2 dpi and 2 dpi, respectively, to assay TBSV, CIRV and CNV RNA accumulation. For confocal laser microscopy analysis, the upper leaves were infiltrated with a mixture of agrobacterium expressing fluorescence protein fused to proteins of interest (OD600 0.3) and mitochondrial or peroxisome markers (OD600 0.3). The agroinfiltrated leaves were analyzed by confocal laser microscopy 2.5 days later.
Transient expression of XPO1 to study replication of tombusviruses in N. benthamiana
To transiently express AtXPO1 (Accession number: AT5G17020) in the leaves of N. benthamiana, the coding region was cloned and inserted into pGD-eGFP-MCS vector to obtain pGD-eGFP-XPO1 (S1 Table). The leaves of N. benthamiana were infiltrated with a mixture of agrobacterium carrying pGD-eGFP-XPO1 (OD600 0.5) and pGD-p19 (OD600 0.2) to express AtXPO1. The mixture of agrobacterium carrying empty vector pGD-eGFP-EV (OD600 0.5) and pGD-p19 (OD600 0.2) was infiltrated into the leaves as the control. To block the function of XPO1, the chemical inhibitor Leptomycin B (LMB) (Cell Signaling Technology, Inc.) was co-infiltrated with the agrobacterium mixture using the concentration of 400 nM, whereas 0.5% ethanol (EtOH) was co-infiltrated with the agrobacterium mixture as the control [95,96]. The agroinfiltrated leaves were inoculated with sap containing TBSV, CIRV or CNV 18 h later. The samples were harvested at 1.5 dpi for TBSV and 2 dpi for CIRV and CNV, respectively, for total RNA or total protein extraction in the virus replication assay. Note that we applied a second, reinforcement infiltration of 400 nM LMB and 0.5% EtOH into the leaves 24 h after the first infiltration.
Confocal microscopy imaging
To analyze the subcellular localization of XPO1 in healthy cells of H2b-CFP transgenic N. benthamiana, a mixture of agrobacterium carrying pGD-eGFP-XPO1 (OD600 0.3), mitochondrial marker pGD-CoxIV-RFP (OD600 0.3) or peroxisomal marker pGD-RFP-SKL (OD600 0.3) and pGD-p19 (OD600 0.2) were co-infiltrated into leaves. Subcellular localization of XPO1 during tombusvirus replication was done using leaves agroinfiltrated as above, followed by inoculation with sap containing TBSV, CNV or CIRV 12 h later. In protein co-localization experiments, we used a mixture of agrobacterium carrying pGD-p33-BFP or pGD-p36-BFP (OD600 0.3), pGD-eGFP-XPO1 (OD600 0.3), pGD-RFP-SKL or pGD-CoxIV-RFP (OD600 0.2) and pGD-p19 (OD600 0.2) for infiltration into the leaves of wild type N. benthamiana. The confocal microscopy imaging was performed at 2 or 2.5 post agrobacterium infiltration.
To analyze eGFP-XPO1 and its cargos in VROs during tombusvirus replication, a mixture of agrobacterium carrying pGD-eGFP-XPO1 or cargos (OD600 0.3), pGD-RFP-SKL (OD600 0.3), pGD-p19 (OD600 0.2) together with 400 nM LMB or 0.5% (V/V) EtOH was infiltrated into the leaves of H2b-CFP transgenic N. benthamiana, followed by inoculation with TBSV or CIRV sap 12 h later. The confocal microscopy imaging was performed at 2 days post virus inoculation.
To analyze the subcellular locations of actin filaments and eGFP-XPO1 or its cargos during tombusvirus replication, the leaves of N. benthamiana were infiltrated with a mixture of agrobacterium carrying pGD-eGFP-XPO1 or cargos (OD600 0.3), pGD-BFP-SKL (OD600 0.3), LifeAct-RFP(OD600 0.03) [97], and pGD-p19 (OD600 0.2). The agroinfiltrated leaves were inoculated with TBSV 12 h later. Leaf samples were analyzed by confocal microscopy 2 days after TBSV inoculation. For CIRV infections, pGD-BFP-SKL (OD600 0.3) was replaced by pGD-CoxIV-BFP.
To alter the actin filament network [98,99], plasmids pGD-Flag-RavK (OD600 = 0.3) or pGD-Flag-VipA (OD600 = 0.3) carrying Legionella effector genes, either RavK or VipA, were co-infiltrated with the combinations mentioned above, followed by inoculation with TBSV or CIRV sap. The confocal imaging was performed 2 d later [100].
During the confocal microscopy analysis, CFP and BFP fusion proteins were excited at 458 nm or 405 nm, respectively, and detected at 460–490 nm or 430–470 nm, respectively. eGFP and RFP fusion proteins were excited at 488 and 561 nm and detected at a range of 495–535 nm and 560–600 nm, respectively, using an Olympus FV3000 microscope.
BiFC (Bimolecular fluorescence complementation) assay
To test interaction between p33 and XPO1, a mixture of agrobacterium of pGD-nYFP-XPO1 (OD600 0.3), pGD-p33-cYFP (OD600 0.3), pGD-SKL-RFP (OD600 0.3) and pGD-p19 (OD600 0.1) was infiltrated into the leaves of N. benthamiana, followed by inoculation with TBSV sap 12 h later. To identify the interaction between p36 and XPO1, the leaves of N. benthamiana were co-infiltrated with agrobacterium carrying pGD-nYFP-XPO1 (OD600 0.3), pGD-p36-cYFP (OD600 0.3), pGD-CoxIV-RFP (OD600 0.3), and pGD-p19 (OD600 0.3), and were inoculated with CIRV sap 12 h later. The combination of pGD-nYFP-XPO1 and pGD-GST-cYFP was used as control. The leaf samples were analyzed with confocal laser microscopy at 2-2.5 dpi.
Fluorescence recovery after photobleaching (FRAP)
Agroinfiltration of N. benthamiana. with various combinations of plasmids was done as described above. Note that the leaves were treated with 10 μM Latrunculin B (Abcam) to avoid the movement of VROs at least 3 h before microscopic imaging [101]. Photo-bleaching was performed in the middle area of VROs. Photobleaching process lasted ~4–5 seconds with 405 nm laser at 80% intensity. And the confocal images were captured automatically by program for every 10 sec until 80 sec. For calculations, all the fluorescent intensity values were quantified using FLUO-view software installed on FV3000 Olympus confocal microscopy operation system. The calculation method for recovery rate was conducted according to [101]. The recovery curves including the recovery rate at different time points were plotted by GraphPad 9 software.
Analysis of TBSV replication with in vitro reconstituted TBSV replicase in yeast cell-free extract (CFE)
Yeast cell-free extract (CFE) that supports in vitro TBSV repRNA replication was prepared with yeast strain BY4741 as described [102,103]. Briefly, the in vitro reconstituted TBSV replicase assay was performed using a mixture of 2 μL of CFE, 0.5 μg DI-72 (+)repRNA, 0.2 μg affinity-purified maltose-binding protein (MBP)-p33 as well as MBP-p92pol (both recombinant proteins were purified from E. coli), 5 μl of buffer A [30mM HEPES-KOH (pH 7.4), 150 mM potassium acetate, 5 mM magnesium acetate, 0.13 M sorbitol], 2 μl of 150 mM creatine phosphate, 0.2 μL of 10 mg/ml creatine kinase, 0.4 μl actinomycin D (5mg/ml), 0.2 μl of 1 M dithiothreitol (DTT), 0.2 μl of RNase inhibitor, 2 μl a ribonucleotide (rNTP) mixture (10 mM of ATP, CTP, and GTP as well as 0.25 mM UTP) and 0.1 μL of 32PUTP in a total of 20 μl reaction volume. The reaction was performed at 25°C for 3h and then stopped by the addition of 5 volumes of 1% SDS and 5 mM EDTA, followed by phenol-chloroform extraction and RNA precipitation. Then the repRNA products and dsRNA replication intermediates were analyzed by electrophoresis in 0.5X Tris-borate-EDTA (TBE) buffer in a 5% polyacrylamide gel (PAGE) containing 8 M urea. Additional information is provided in S1 Text [104–109 ] and S1–S3 Tables.
Supporting information
S1 Fig. Knockdown of XPO1 gene expression leads to enhanced CNV and CIRV replication in N. benthamiana.
(A) Accumulation of CNV genomic (g)RNA and subgenomic (sg)RNAs at 2 days after CNV inoculation in XPO1-silenced (KD) plants was measured by northern blot analysis. The inoculation with CNV was performed 7 days after VIGS. Second panel: The ribosomal RNA is shown as the loading control in agarose gel stained with ethidium-bromide. CNV gRNA is visible in the gel. Right panel: Accelerated and more severe CNV-induced symptom development was observed in XPO1-silenced N. benthamiana. The symptoms were documented at 5 days post CNV inoculation. (B) Left panels: Accumulation of CIRV gRNA in XPO1-silenced N. benthamiana plants at 2 d post inoculation (dpi) was measured by northern blot analysis. See further details in panel A. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s001
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S2 Fig. Expression of XPO1 inhibits tombusvirus accumulation in N. benthamiana.
(A-B). Transient expression of XPO1 in N. benthamiana inhibits accumulation of CNV and CIRV, respectively. Top panels: Accumulation of CNV or CIRV RNAs at 2 dpi was measured by northern blot analysis. Bottom panel: The 18S ribosomal RNA is shown in an agarose gel stained with ethidium-bromide as the loading control. N. benthamiana leaves were agroinfiltrated for transient expression of XPO1 (pGD vector as control), in combination with 0.5% ethanol (EtOH) as a control or 400 nM of Leptomycin B (LMB), a chemical inhibitor of XPO1. The LMB and EtOH treatments were repeated 24 h later. (C) Expression of XPO1 was measured by western blot using anti-GFP antibody. Coomassie Brilliant Blue staining was used for the normalization of total proteins as loading control.
https://doi.org/10.1371/journal.ppat.1012841.s002
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S3 Fig. Recruitment of XPO1 by tombusviral replication proteins into VROs in N. benthamiana.
(A) Confocal laser microscopy images show co-localization of CIRV p36-BFP replication protein and eGFP-XPO1 during CIRV replication. Transgenic N. benthamiana expressing H2B-RFP as the nuclear marker was used. Scale bars represent 10 μm. (B) The subcellular localization of eGFP-XPO1 in the mock-treated N. benthamiana. Mitochondria are indicated by CoxIV-RFP. Scale bars represent 10 μm. (C) Note that eGFP-XPO1 partitioned between the nucleus and VROs during CIRV replication. Middle panel: The nuclear localization and relocation to VROs of eGFP-XPO1 were captured in the same plant cell. Top panel: Enlarged images of the nuclear area. Bottom panel: Enlarged images of the VRO region. Scale bars represent 10 μm. (D) Confocal laser microscopy images show partial re-localization of eGFP-XPO1 into CNV VROs decorated by RFP-SKL marker. The VRO is pointed at by a white arrow, whereas the nucleus is marked by a yellow arrow. Transgenic N. benthamiana expressing H2B-CFP as the nuclear marker was used in (B, C and D). Scale bars represent 10 μm. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s003
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S4 Fig. XPO1 interacts with CIRV p36 replication protein.
(A) Control BiFC experiments with the combination of GST-cYFP and nYFP-XPO1 was performed in H2B-CFP transgenic N. benthamiana infected with TBSV. The nucleus is marked by a yellow arrow (B) Interaction between CIRV p36-cYFP and nYFP-XPO1 was identified by BiFC during CIRV replication in H2B-CFP transgenic N. benthamiana. The VROs, consisting of clustered mitochondria, are visualized by CoxIV-RFP mitochondrial marker. The interaction signals in VROs are indicated by white arrows, whereas the nucleus is marked by a yellow arrow. Scale bars represent 10 μm. (C) Copurification of CIRV Flag-p36 and Flag-p95pol replication proteins with His6-XPO1 from subcellular membranes of yeast. Top two panels: Co-purified His6-XPO1 (lane 2) and Flag-affinity-purified Flag-p36 were detected using western blot analysis. The negative control yeasts expressed His6-p36 and His6-p95pol together with His6-XPO1. Middle three panels: Identification of protein expression in total samples from yeasts were detected by western blotting using the shown antibodies. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s004
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S5 Fig. LMB treatment does not prevent XPO1 interaction with TBSV p33 or CIRV p36 replication proteins in VROs.
(A-B) Interaction between TBSV p33-cYFP or CIRV p36-cYFP and nYFP-XPO1 was identified by BiFC in H2B-CFP transgenic N. benthamiana. The leaves were treated with either 400 nM LMB or 0.5% EtOH (control) twice via infiltration. The VROs, consisting of either clustered peroxisomes or clustered mitochondria, are visualized by either RFP-SKL or CoxIV-RFP mitochondrial marker. We show two sets of images to demonstrate that VROs localize either proximal to nucleus (top images) or, more frequently, at distal positions (bottom images). The interaction signals in VROs are indicated by white arrows, whereas the nucleus is marked by a yellow arrow.
https://doi.org/10.1371/journal.ppat.1012841.s005
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S6 Fig. DRB4 and XPO1 are recruited to VROs during TBSV replication.
(A) Confocal microscopy images show colocalization of eGFP-DRB4 and RFP-XPO1 in VROs marked with p33-BFP replication protein during TBSV replication in N. benthamiana. Scale bars represent 10 μm. (B) A fraction of eGFP-DRB4 is relocated to VRO marked by p33-BFP, which includes clustered peroxisomes (marked with by RFP-SKL) during TBSV replication in N. benthamiana. The nucleus is pointed at by a yellow arrow. Note that the VRO is localized at proximal position to nucleus. Scale bars represent 10 μm. (C) Top panel: The enlarged images (the red boxed area in the middle panel) show partial nuclear localization of eGFP-DRB4 during TBSV replication. Middle panel: Confocal images of a single cell from a H2B-CFP transgenic N. benthamiana infected with TBSV. Note that the VRO is localized at distal position from the nucleus. Bottom panel: The enlarged images (the red boxed area in the middle panel) show eGFP-DRB4 is present in a single VRO during TBSV replication. Scale bars represent 10 μm. (D) Confocal microscopy images show the subcellular localization of eGFP-DRB4 in healthy plant cells of H2B-CFP transgenic N. benthamiana. (E) LMB chemical inhibitor of XPO1 restricts eGFP-DRB4 shuttle protein mostly in the nucleus. Top panels: eGFP-DRB4 is restricted in the nucleus when 400 nM LMB is co-infiltrated with the agrobacterium carrying pGD-eGFP-DRB4 in the healthy leaves of N. benthamiana. Bottom panel: eGFP-DRB4 localizes in the nucleus and cytosol when cells were treated with 0.5% EtOH as the control. Scale bars represent 10 μm. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s006
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S7 Fig. DRB4 and XPO1 are recruited to VROs during CNV replication.
(A) Confocal microscopy images show colocalization of eGFP-DRB4 and RFP-XPO1 in VROs marked with p33-BFP replication protein during CNV replication in N. benthamiana. Scale bars represent 10 μm. (B) A fraction of eGFP-DRB4 is relocated to VROs represented by clustered peroxisomes (marked with by RFP-SKL) during CNV replication in H2B-CFP transgenic N. benthamiana. H2B-CFP is used as nuclear marker. Top panel: The enlarged images (the red boxed area in the middle panel) show eGFP-DRB4 is present in a single VRO during CNV replication. Middle panel: Confocal images of a single cell from a H2B-CFP transgenic N. benthamiana infected with CNV. Bottom panel: The enlarged images (the red boxed area in the middle panel) show partial nuclear localization of eGFP-DRB4 during CNV replication. Scale bars represent 10 μm. (C) Confocal microscopy images show the subcellular localization of eGFP-DRB4 in healthy plant cells of H2B-CFP transgenic N. benthamiana. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s007
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S8 Fig. DRB4 and XPO1 are recruited to VROs during CIRV replication.
(A-D) Confocal microscopy images show localization of eGFP-DRB4 in VROs in H2B-CFP transgenic N. benthamiana infected with CIRV. CoxIV-RFP marks the clustered mitochondria representing CIRV VROs. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. See further details in S6 Fig. (E) Confocal microscopic images show poor recruitment of eGFP-DBR4 into VROs (indicated by CoxIV-RFP) in XPO1 KD N. benthamiana infected with CIRV. Control experiment with N. benthamiana plants agroinfiltrated with the TRV vector carrying partial GST sequence. Scale bars represent 10 μm. Right panel: Quantification of fluorescent intensity of eGFP-DRB4 in CIRV VRO regions with Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (** represents P < 0.01).
https://doi.org/10.1371/journal.ppat.1012841.s008
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S9 Fig. Reduced recruitment of DRB4 into VROs during TBSV replication when XPO1 function is inhibited.
(A) Confocal microscopy images show re-localization of eGFP-DRB4 in VROs in H2B-CFP transgenic N. benthamiana infected with TBSV. XPO1 level was reduced by VIGS, whereas XPO1 was not targeted in control plants. RFP-SKL marks the clustered peroxisomes representing TBSV VROs, pointed at by white arrows, whereas the nucleus is pointed at by yellow arrow. Right panel: Quantification of fluorescent intensity of eGFP-DRB4 is shown in TBSV VRO regions in XPO1 KD leaves versus in controls. T-test is used for data analysis utilizing GraphPad Prism 9 (*** represents P < 0.001). See further details in S6 Fig. (B) Confocal microscopic images show poor recruitment of eGFP-DBR4 into VROs in 400 nM LMB-treated N. benthamiana infected with TBSV. Control experiment included 0.5% EtOH treatment of N. benthamiana leaves infected with TBSV. Scale bars represent 10 μm. Right panel: Quantification of fluorescent intensity of eGFP-DRB4 is shown in VRO regions in LMB-treated leaves versus in controls.
https://doi.org/10.1371/journal.ppat.1012841.s009
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S10 Fig. AGO2 and XPO1 are recruited to VROs during CNV replication.
(A-D) Confocal microscopy images show partial re-localization of eGFP-AGO2 in VROs in H2B-CFP transgenic N. benthamiana infected with CNV or mock treated. See further details in S7 Fig.
https://doi.org/10.1371/journal.ppat.1012841.s010
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S11 Fig. AGO2 and XPO1 are recruited to VROs during TBSV replication.
(A-C) Confocal microscopy images show partial re-localization of eGFP-AGO2 in VROs in H2B-CFP transgenic N. benthamiana infected with TBSV or mock treated. See further details in S6 Fig.
https://doi.org/10.1371/journal.ppat.1012841.s011
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S12 Fig. AGO2 and XPO1 are recruited to VROs during CIRV replication.
(A-D) Confocal microscopy images show partial re-localization of eGFP-AGO2 in VROs in H2B-CFP transgenic N. benthamiana infected with CIRV or mock treated. CoxIV-RFP marks the clustered mitochondria representing CIRV VROs. Scale bars represent 10 μm. See further details in S6 Fig.
https://doi.org/10.1371/journal.ppat.1012841.s012
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S13 Fig. XPO1 delivers nuclear AGO2 to VROs to inhibit CNV replication.
(A) Recruitment of eGFP-AGO2 to VROs is reduced in XPO1 knockdown (KD) N. benthamiana infected with CNV. Left top panel: Confocal microscopic images show poor recruitment of eGFP-AGO2 into VROs (indicated by RFP-SKL) in XPO1 KD N. benthamiana. Bottom panel: Control experiment with N. benthamiana plants agroinfiltrated with the TRV vectors carrying partial GST sequence. Scale bars represent 10 μm. Right panel: Quantification of fluorescent intensity of eGFP-AGO2 in VRO regions was done with Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (* represents P < 0.1). Error bars represent standard deviation (SD). (B) Recruitment of eGFP-AGO2 to VROs is reduced in N. benthamiana infected with CNV and treated with 400 nM LMB versus 0.5% EtOH. See further details in panel A. (** represents P < 0.01). (C) Transient expression of Flag-AGO2 in N. benthamiana does not inhibit CNV accumulation when leaves are treated with LMB. Note that CNV p20 was not expressed in plants. Top panel: CNV RNA accumulation at 2 dpi was measured by northern blot analysis. N. benthamiana leaves were agroinfiltrated to express Flag-AGO2 (pGD vector as control) and the agroinfiltrated leaves were either infiltrated twice with either 0.5% ethanol (EtOH) as a control or 400 nM of Leptomycin B (LMB). Middle panel: The 18S ribosomal RNA is showed in an agarose gel stained with ethidium-bromide as the loading control. Bottom panel: The expression of Flag-AGO2 was measured by western blot from the above samples using anti-flag antibody. Coomassie Brilliant Blue staining was used for the normalization of total proteins as loading control. (D) Top panel: Accumulation of CNV gRNA and sgRNAs at 2 dpi was measured by northern blot analysis in XPO1-silenced plants expressing eGFP-AGO2. Agroinfiltration to express eGFP-AGO2 (in the absence of p19 silencing suppressor) was performed 7 days after silencing of XPO1 in N. benthamiana, followed two days later by inoculation with CNV. Bottom panel: The ribosomal RNA is shown as the loading control in the agarose gel stained with ethidium-bromide. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s013
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S14 Fig. XPO1 delivers nuclear AGO2 to TBSV and CIRV VROs.
(A-B) Recruitment of eGFP-AGO2 to VROs is reduced in LMB-treated (A) or XPO1 knockdown (KD) (panel B) N. benthamiana infected with TBSV. Left top panel: Confocal microscopic images show poor recruitment of eGFP-AGO2 into VROs (indicated by RFP-SKL) in XPO1 KD H2B-CFP transgenic N. benthamiana. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. Scale bars represent 10 μm. Right panels: Quantification of fluorescent intensity of eGFP-AGO2 in VRO regions was done with Olympus FV3000 FLUO-view software. (C) Recruitment of eGFP-AGO2 to VROs is reduced in XPO1 KD H2B-CFP transgenic N. benthamiana infected with CIRV. CoxIV-RFP marks the clustered mitochondria representing CIRV VROs. See further details in panel A. (** represents P < 0.01).
https://doi.org/10.1371/journal.ppat.1012841.s014
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S15 Fig. CenH3 and XPO1 are recruited to VROs during TBSV replication.
(A-D) Confocal microscopy images show localization of eGFP-CenH3 in VROs in H2B-CFP transgenic N. benthamiana infected with TBSV or mock-treated. RFP-SKL marks the clustered peroxisomes representing TBSV VROs. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. Scale bars represent 10 μm. See further details in S6 Fig.
https://doi.org/10.1371/journal.ppat.1012841.s015
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S16 Fig. CenH3 and XPO1 are recruited to VROs during CNV replication.
(A-D) Confocal microscopy images show localization of eGFP-CenH3 in VROs in H2B-CFP transgenic N. benthamiana infected with CNV or mock-treated. RFP-SKL marks the clustered peroxisomes representing CNV VROs. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. Scale bars represent 10 μm. See further details in S7 Fig.
https://doi.org/10.1371/journal.ppat.1012841.s016
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S17 Fig. CenH3 and XPO1 are recruited to VROs during CIRV replication.
(A-D) Confocal microscopy images show localization of eGFP-CenH3 in VROs in H2B-CFP transgenic N. benthamiana infected with CIRV. CoxIV-RFP marks the clustered mitochondria representing CIRV VROs. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. Scale bars represent 10 μm. See further details in S8 Fig.
https://doi.org/10.1371/journal.ppat.1012841.s017
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S18 Fig. XPO1 delivers nuclear CenH3 to VROs to inhibit CIRV replication.
(A) Recruitment of eGFP-CenH3 to CIRV VROs is reduced in XPO1 knockdown (KD) N. benthamiana infected with CIRV. Left top panel: Confocal microscopic images show poor recruitment of eGFP-CenH3 into VROs (indicated by CoxIV-RFP) in XPO1 KD N. benthamiana. Bottom panel: Control experiment with N. benthamiana plants agroinfiltrated with the TRV vectors carrying partial GST sequence. Scale bars represent 10 μm. Right panel: Quantification of fluorescent intensity of eGFP-CenH3 in VRO regions was done with Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001). Error bars represent standard deviation (SD). (B) Recruitment of eGFP-CenH3 to VROs is reduced in N. benthamiana infected with CIRV and treated with 400 nM LMB versus 0.5% EtOH. See further details in panel A. (*** represents P < 0.001). (C) Transient expression of Flag- CenH3 in N. benthamiana does not inhibit CIRV accumulation when leaves were treated with LMB versus EtOH. Top panel: CIRV RNA accumulation at 2.5 dpi was measured by northern blot analysis. N. benthamiana leaves were agroinfiltrated to express Flag-CenH3 (pGD vector as control) and the agroinfiltrated leaves were either infiltrated with 0.5% ethanol (EtOH) as a control or 400 nM of Leptomycin B (LMB). Middle panel: The 18S ribosomal RNA is showed in an agarose gel stained with ethidium-bromide as the loading control. Bottom right panel: The expression of Flag-CenH3 was measured by western blot from the above samples using anti-flag antibody. Coomassie Brilliant Blue staining was used for the normalization of total proteins as loading control. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s018
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S19 Fig. XPO1 is needed to deliver nuclear NUCLEOLIN to VROs to inhibit CNV replication.
(A) Interaction between XPO1 and NUC-L1 (NUCLEOLIN) and recruitment of NUC-L1 to TBSV VROs is dampened by treatment of leaves with LMB versus EtOH. Top panel: Confocal microscopic images show interaction between XPO1 and NUC-L1 and recruitment of NUC-L1 to TBSV VROs based on BiFC assay. TBSV VROs are indicated by RFP-SKL in H2B-CFP transgenic N. benthamiana. Yellow arrows indicate the nucleus marked with H2B-CFP, while VROs are marked with white arrows. Second panel: Similar BiFC assay as in panel A, except leaves were treated with LMB or EtOH. Control experiments were done with H2B-CFP transgenic N. benthamiana plants treated with LMB or not treated. Scale bars represent 10 μm. (B) BiFC assay shows interaction between nYFP-NUC-L1 and p33-cYFP in VROs in H2B-CFP transgenic N. benthamiana infected with TBSV. (C) Transient expression of NUC-L1-GFP in N. benthamiana does not inhibit CNV accumulation when leaves were treated with LMB versus EtOH. Top panel: CNV RNA accumulation at 2 dpi was measured by northern blot analysis. N. benthamiana leaves were agroinfiltrated to express NUC-L1-GFP (GFP as control) and the agroinfiltrated leaves were either infiltrated with 0.5% ethanol (EtOH) as a control or 40 or 400 nM LMB. Middle panel: The 18S ribosomal RNA is showed in northern blot as the loading control. Bottom right panel: The expression of NUC-L1-GFP was measured by western blot from the above samples using anti-GFP antibody. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s019
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S20 Fig. Association of DRB4 or AGO2 with co-opted actin filaments during tombusvirus replication.
(A-B) Confocal microscopy images show co-localization of eGFP-DRB4 with RFP-Actin filaments and VROs, indicated by BFP-SKL during TBSV replication or by CoxIV-BFP mitochondrial marker during CIRV replication in N. benthamiana. The actin filaments were visualized by agro-expression of Lifeact, which binds to filamentous actin (F-actin) in plant cells. The actin filaments co-localized with eGFP-DRB4 are indicated by yellow arrows. The scale bar represents 10 μm. The enlarged images from the red boxed areas contain CIRV VROs and actin filaments in the vicinity of the nucleus. (C) Localization of eGFP-DRB4, RFP-Actin, and CoxIV-BFP mitochondrial marker in mock-treated N. benthamiana. The scale bar represents 10 μm. (D-E) Confocal microscopy images show the co-localization of eGFP-AGO2 with RFP-Actin and TBSV or CIRV VROs in N. benthamiana cells. TBSV VROs are marked with SKL-BFP peroxisomal marker, whereas CIRV VROs are marked with CoxIV-BFP mitochondrial marker. The scale bar represents 10 μm. (F) Localization of eGFP-AGO2, RFP-Actin, and CoxIV-BFP mitochondrial marker in mock-treated N. benthamiana. The scale bar represents 10 μm. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s020
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S21 Fig. Association of CenH3 with co-opted actin filaments during tombusvirus replication.
(A-B) Confocal microscopy images show co-localization of eGFP-CenH3 with RFP-Actin filaments and VROs, indicated by BFP-SKL during TBSV replication or by CoxIV-BFP mitochondrial marker during CIRV replication in N. benthamiana. The actin filaments were visualized by agro-expression of Lifeact, which binds to filamentous actin (F-actin) in plant cells. The actin filaments co-localized with eGFP-CenH3 are indicated by yellow arrows. The scale bar represents 10 μm. The enlarged images from the red boxed areas contain CIRV VROs and actin filaments. (C) Localization of eGFP-CenH3, RFP-Actin, and CoxIV-BFP mitochondrial marker in mock-treated N. benthamiana. The scale bar represents 10 μm.
https://doi.org/10.1371/journal.ppat.1012841.s021
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S22 Fig. Actin filament network plays a critical role in delivering XPO1 or DRB4 antiviral cargo to CIRV VROs.
Confocal microscopy images show the poor recruitment of eGFP-XPO1 (A), or eGFP-DRB4 (C) into CIRV VROs when the actin filaments were destroyed by the transient expression of RavK effector of Legionella bacterium from a plasmid via agroinfiltration in N. benthamiana. Top two panels in (A and C) show the control confocal images without the expression of RavK effector in N. benthamiana. Scale bars represent 10 μm. Quantification of VRO-localized eGFP-XPO1 (B) or eGFP-DRB4 (D) is shown in graphs. The fluorescent intensity in VROs is quantified by Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001). Error bars represent standard deviation (SD). Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s022
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S23 Fig. Actin filament network plays a critical role in delivering CenH3 or AGO2 antiviral cargos to CIRV VROs.
Confocal microscopy images show the poor recruitment of eGFP-CenH3 (A), or eGFP-AGO2 (C) into CIRV VROs when the actin filaments were destroyed by the transient expression of RavK effector of Legionella bacterium from a plasmid via agroinfiltration in N. benthamiana. See further details in S16 Fig. Scale bars represent 10 μm. Quantification of VRO-localized eGFP-CenH3 (B) or eGFP-AGO2 (D) is shown in graphs. The fluorescent intensity in VROs is quantified by Olympus FV3000 FLUO-view software. T-test is used for data analysis utilizing GraphPad Prism 9 (**** represents P < 0.0001). Error bars represent standard deviation (SD). Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s023
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S24 Fig. Regulatory co-factors of XPO1 are recruited into VROs during TBSV replication.
Confocal microscopy images show the recruitment of eGFP-RanGAP1 (A), eGFP-RanGAP2 (B), or eGFP-RanBP1-1b (C) into VROs during TBSV replication. The bottom images in panels A, B and C show the subcellular localizations of eGFP-RanGAP1, eGFP-RanGAP2, or eGFP-RanBP1-1b in mock inoculated control plant cells. Yellow arrows indicate the nucleus, while VROs are marked with white arrows. Note these images were taken using H2B-CFP transgenic N. benthamiana marking the nucleus. Confocal microscopy images also show the associations of eGFP-RanGAP1 (D), eGFP-RanGAP2 (E), eGFP-RanBP1-1b (F) and p33-BFP replication protein with actin filament networks during TBSV replication. Scale bars represent 10 μm.
https://doi.org/10.1371/journal.ppat.1012841.s024
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S25 Fig. Regulatory co-factors of XPO1 are recruited into VROs during CIRV replication.
Confocal microscopy images show the recruitment of eGFP-RanGAP1 (A), eGFP-RanGAP2 (B), or eGFP-RanBP1-1b (C) into VROs during CIRV replication. The bottom images in panel A, B and C show the subcellular localizations of eGFP-RanGAP1, eGFP-RanGAP2, or eGFP-RanBP1-1b in control plant cells. The CIRV VROs are labeled with either CoxIV-BFP mitochondrial marker or p36-BFP replication protein during CIRV replication. Note these images were taken using H2B-CFP transgenic N. benthamiana marking the nucleus. Confocal microscopy images also show the associations of eGFP-RanGAP1 (D), eGFP-RanGAP2 (E), eGFP-RanBP1-1b (F) and CIRV p36-BFP replication protein with actin filament networks during CIRV replication. Scale bars represent 10 μm. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s025
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S26 Fig. Tombusvirus replication proteins do not interact with regulatory co-factors of XPO1 in N. benthamiana.
(A) TBSV p33 replication protein does not interact with regulatory co-factors of XPO1. From the first to the third panels: BiFC-based interaction results of nYFP-tagged RanGAP1, RanGAP2, and RanBP1-1b with p33-cYFP are shown during TBSV replication. Bottom panel: BiFC assay shows that p33-cYFP interacts with nYFP-XPO1 during TBSV replication as the positive control. (B) CIRV p36 replication protein does not interact with regulatory co-factors of XPO1. From the first to the third panels: BiFC-based interaction results of nYFP-tagged RanGAP1, RanGAP2, and RanBP1-1b with p36-cYFP are shown during CIRV replication. Bottom panel: BiFC assay shows that p36-cYFP interacts with nYFP-XPO1 during CIRV replication as the positive control. Scale bars represent 10 μm.
https://doi.org/10.1371/journal.ppat.1012841.s026
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S27 Fig. XPO1 and the antiviral cargos are present in vir-condensates associated with membranous CIRV VROs.
(A) FRAP analysis shows partial fluorescence recovery of RFP-XPO1 after photobleaching in a single CIRV VRO in N. benthamiana during CIRV replication. Top panel: Co-localization of CIRV p36-BFP replication protein and RFP-XPO1 in VRO before photobleaching. Middle panels: Confocal microscopy images show partial fluorescence recovery of RFP-XPO1 at 0-, 40-, and 80-seconds post photobleaching during CIRV replication. Note that signals of CIRV p36-BFP did not recover in the FRAP assay because it is a membrane-bound protein. Bottom panel: The graph shows time course analysis of FRAP data on RFP-XPO1 signal recovery in individual CIRV VROs. Sample size n is annotated in the figure. Shaded area represents SD. Scale bars represent 5 μm. Similar FRAP analysis of RFP-DRB4 (panel B), RFP-CenH3 (panel C), and RFP-AGO2 (panel D) are also shown. See further details in panel A. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s027
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S28 Fig. TBSV p33 replication protein and either DRB4 or CenH3 co-partition in droplets in vitro.
(A) Confocal images show the co-partitioning of the purified mRFP-DRB4 and eGFP-p33C in droplets in the presence of 2.5% PEG8000. The top and bottom panels show droplets formed by different amounts of mRFP-DRB4 as indicated. Scale bars represent 10 µm. (B) Note that purified mRFP-DRB4 and eGFP-p33C form droplets under the same conditions as in panel A. (C) Confocal images show the co-partitioning of the purified mRFP-CenH3 and eGFP-p33C in droplets in the presence of 2.5% PEG8000. The top and bottom panels show droplets formed by different amounts of mRFP-CenH3 as indicated. Scale bars represent 10 µm. (D) Note that purified mRFP-CenH3 forms a few droplets under the same conditions as in panel C. Each experiment was repeated three times.
https://doi.org/10.1371/journal.ppat.1012841.s028
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S1 Text. Supplementary Materials and Methods [104–106].
https://doi.org/10.1371/journal.ppat.1012841.s029
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S1 Table. List of plasmids constructed during this work.
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S2 Table. List of plasmids from prior work [107–109].
https://doi.org/10.1371/journal.ppat.1012841.s031
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Acknowledgments
The authors thank Drs. W. Lin, and J. Pogany for technical assistance with experiments and valuable comments. We thank Dr. Boone (U. Toronto) for providing srm1 temperature-sensitive yeast mutant.
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