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Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation [1]

['Jibin Sadasivan', 'Department Of Biochemistry', 'Molecular Biology', 'Life Sciences Institute', 'University Of British Columbia', 'Vancouver', 'British Columbia', 'Marli Vlok', 'Xinying Wang', 'Arabinda Nayak']

Date: 2022-12 

Abstract Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is the formation of stress granules (SG), which are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket paralysis virus (CrPV), is a multifunctional protein that can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and CrPV infection in both Drosophila S2 cells and adult flies. Here, we uncoupled CrPV-1A’s functions and provide insight into its underlying mechanism for SG inhibition. CrPV-1A mediated inhibition of SGs requires the E3 ubiquitin-ligase binding domain and the R146 residue, but not the Ago-2 binding domain. Wild-type but not mutant CrPV-1A R146A localizes to the nuclear membrane which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome changes in CrPV-infected cells are dependent on the R146 residue. Finally, Nup358/RanBP2 is targeted and degraded in CrPV-infected cells in an R146-dependent manner and the depletion of Nup358 blocks SG formation. We propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with a nuclear event that contributes to SG inhibition in order to promote infection.

Author summary Viruses often inhibit a cellular stress response that leads to the accumulation of RNA and protein condensates called stress granules. How this occurs and why this would benefit virus infection are not fully understood. Here, we reveal a viral protein that can block stress granules and identify a key amino acid residue in the protein that inactivates this function. We demonstrate that this viral protein has multiple functions to modulate nuclear events including mRNA export and transcription to regulate stress granule formation. We identify a key host protein that is important for viral protein-mediated stress granule inhibition, thus providing mechanistic insights. This study reveals a novel viral strategy in modulating stress granule formation to promote virus infection.

Citation: Sadasivan J, Vlok M, Wang X, Nayak A, Andino R, Jan E (2022) Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation. PLoS Pathog 18(12): e1010598. https://doi.org/10.1371/journal.ppat.1010598 Editor: Jeffrey Wilusz, Colorado State U., UNITED STATES Received: May 19, 2022; Accepted: November 17, 2022; Published: December 1, 2022 Copyright: © 2022 Sadasivan 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 relevant data are within the manuscript and its Supporting Information files. Funding: This study was supported by Canadian Institute of Health Research operating grant (PJT-178342) https://cihr-irsc.gc.ca/e/193.html to EJ; Natural Sciences and Engineering Research Council of Canada Discovery grant (RGPIN-2017-04515) https://www.nserc-crsng.gc.ca/index_eng.asp to EJ; National Institutes of Health (A132131 - R01AI137471) https://www.nih.gov/ to RA and SERB-UBC Doctoral scholarship to JS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Stress granules (SGs) are dynamic, non-membranous, cytosolic aggregates of ribonucleoprotein (RNP) complexes that assemble following cellular stress [1]. Typically, overall translational inhibition resulting from a cellular stress response promotes SG formation, but is not necessary under certain cellular contexts [2–4]. SGs contain non-translating mRNAs, translation initiation factors, and ribonucleoproteins [5]. The assembly of SGs is mediated through the interactions of proteins with non-translating RNAs, resulting in liquid-liquid phase separation, where in part the RNA component serves as scaffolds for recruitment of RNA binding proteins. SG assembly is proposed to be a multistep process in which the assembly of a stable dense core of mRNA and proteins is held together by a surrounding shell of less concentrated RNPs [6]. Common SG protein markers include RasGAP-SH3-binding protein (G3BP1), T-cell intracellular antigen 1 (TIA-1), TIA-1 related protein (TIAR), and Poly-A binding protein (PABP), however, hundreds of other proteins have been identified that are enriched in SGs [7,8]. Moreover, relatively long mRNAs are enriched in SGs, possibly to promote concentration of proteins for liquid-liquid phase separation [9,10]. SGs are dynamic and reversible structures that continuously sort and route messenger RNP (mRNP) components. SGs affect mRNP localization, functions and signaling pathways that can have significant impacts on biological processes [11]. As such, the dysregulation of SG assembly/disassembly is implicated in neurodegenerative diseases, autoimmune diseases, cancers and virus infections [12]. Over the past decade, significant progress has been made in unraveling the SG composition and assembly pathways. However, the molecular mechanism and underlying signaling pathways that regulate SG dynamics, and the consequences of SG assembly are not completely understood. Classical induction of SG assembly is initiated by the activation of one or more stress-sensing eIF2α kinases, that phosphorylate serine-51 of the α subunit of eukaryotic translation initiation factor 2 (eIF2), which is the main factor that delivers initiator Met-tRNA to the 40S pre-initiation complex [13]. In mammals, there are four eIF2α kinases, Protein kinase R (PKR), Protein kinase RNA-like endoplasmic reticulum kinase (PERK), Gene control nonderepressible 2 (GCN2) and Heme-regulated inhibitor kinase (HRI) [14–17]; whereas in insects, there are only two, PERK and GCN2 [18]. Phosphorylation of eIF2α results in inhibition of overall translation in the cell which can lead to robust SG formation. As a result, besides hallmark SG protein markers and poly(A)+ RNA, several eukaryotic translation initiation factors and the 40S subunit are often found in SG foci [19]. Although it is often thought that translation inhibition is a pre-requisite for SG formation, this is not strictly necessary under certain cellular contexts [20]. Virus infection, in general, leads to modulation and inhibition of SG formation [21,22], which is observed across different classes of RNA and DNA viruses and across species suggesting a fundamental viral strategy to modulate SGs for productive infection. For example, RNA viruses such as poliovirus and hepatitis c virus (HCV) infection leads to a depletion of G3BP1 and TIA-1 foci formation [23,24]. The disruption of SG assembly can be attributed to one or more viral proteins, which has revealed distinct mechanisms that affect SG. One such mechanism is to counter SG assembly by modulating PKR activation. For instance, Middle East Respiratory Syndrome (MERS) Coronavirus accessory protein 4a, Influenza virus NS1 protein and Vaccinia virus E3L inhibits SG formation by sequestering dsRNA to block PKR activation [25–28]. Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 protein binds to PKR and PKR activating protein (PACT) to inhibit PKR activation and SG formation [29]. Besides modulating PKR activity, some viruses act directly on SG through virally-encoded proteases that cleave key SG proteins to facilitate SG disassembly. Poliovirus 3C protease and Foot-and-mouth disease virus (FMDV) 3C and Leader proteases cleave G3BP to inhibit SG formation [23,30,31]. Viruses also co-opt SG components to facilitate infection. Flaviviruses such as West Nile virus and Zika virus hijack SG-nucleating proteins TIA-1,TIAR and G3BP and subvert them to viral replication complexes [32,33] whereas Human Immunodeficiency virus-1 (HIV-1) sequesters the SG protein Staufen-1 to RNPs containing viral RNA and gag protein [34]. Murine norovirus utilizes the NS3 protein to redistribute G3BP to the site of viral replication [35]. In addition, the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) nucleocapsid protein phase separates with G3BPs and rewires the G3BP interactome to disassemble SGs [36–38]. The distinct mechanisms and utilization of viral proteins to disassemble SGs across different virus classes highlight the importance of SG modulation during virus infection. Although it is apparent that viruses modulate SGs, the reasons underlying this event are not fully understood. SG formation may sequester viral protein or RNA, as observed with flavivirus infection [39], thus inhibition of SG may be a general viral strategy to allow viral protein synthesis and replication. Alternatively, antiviral RNA sensors and factors such as PKR, Retinoic acid inducible gene I (RIG-I), Melanoma differentiation-associated protein 5 (MDA5), oligoadenylate synthetase (OAS), ribonuclease L (RNase L), Tripartite motif containing 5 (Trim5), RNA-specific adenosine deaminase 1 (ADAR1) and cyclic GMP-AMP synthase (cGAS) have been found in SGs, termed antiviral SGs (avSGs), which may act as an antiviral hub to co-ordinate immune responses to limit viral replication [40–43]. Influenza A virus RNA and RIG-1 have been found in avSGs during infection, which is thought to trigger the RIG-I-dependent interferon response [42]. Finally, studies have implicated SG formation in apoptosis, thus blocking SG during infection may delay this process to allow completion of the viral life cycle [44]. The functional consequences of SG formation and its causal relationship to virus infection remains to be clarified. Dicistroviruses are single stranded positive sense RNA viruses that primarily infect arthropods [45,46]. Members of the family Dicistroviridae include the honeybee dicistroviruses, Israeli acute paralysis virus, Kashmiri bee virus and Black queen cell virus, that have been linked to honeybee disease, and Taura syndrome virus, which has led to panaeid shrimp outbreaks [47]. The dicistrovirus RNA genome consists of two main open reading frames (ORF) (Fig 1A). ORF1 encodes the viral non-structural proteins, such as the RNA helicase, protease and RNA-dependent RNA polymerase and ORF2 encodes the viral structural proteins, which mediate virion assembly [45]. Both ORFs are driven by distinct internal ribosome entry sites (IRES) that have been studied extensively [48–51]. The intergenic IRES utilizes a streamlined translation initiation mechanism whereby the IRES mediate direct assembly of ribosomes and starts translation at a non-AUG codon [45,52]. The dicistrovirus Cricket paralysis virus (CrPV) and Rhopalosiphum padi virus (RhPV) 5’UTR IRES resembles an IRES similar to the mechanism used by HCV, requiring translation initiation factors, eIF2, eIF3 and initiator Met-tRNA i to start translation [53–55]. Studies using model dicistroviruses CrPV and Drosophila C virus (DCV) have uncovered fundamental virus host interactions in insects. Dicistrovirus infections can lead to transcriptional and translational shutdown, evasion of the insect antiviral RNAi response and SG inhibition [56–59]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. CrPV-1A expression inhibits stress granules in response to arsenite treatment. (A) Depiction of the CrPV genome with the structure of CrPV-1A protein (PDB 6C3R) (below) highlighting the domains selected for mutagenesis. (B) Schematic of CrPV-1A-2A-GFP RNA containing the CrPV 5’ and 3’UTRs. (C) Confocal immunofluorescence images of S2 cells transfected with control 5’cap-GFP-poly (A)+, wild type or R146A mutant CrPV-1A-2A-GFP RNAs (16 hours) followed by one-hour treatment in the presence or absence of 500 μM sodium arsenite. The arrows show transfected cells. Shown are representative transfected cells detecting GFP fluorescence (green), Rin antibody staining (red), Hoechst dye staining for nucleus (blue) and merged images. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (D) Box plot showing the number Rin foci per cell. At least 50 cells were counted for each condition from three independent experiments. Data are mean ± SD. P > 0.05 (ns) p < 0.0001(****) by a one-way ANOVA (nonparametric) with a Bonferroni’s post hoc-test. https://doi.org/10.1371/journal.ppat.1010598.g001 The CrPV and DCV 1A proteins are viral suppressors of RNAi (VSR) that suppress the insect antiviral RNAi pathway [58,59]. The 1A protein is the first viral non-structural protein translated within ORF1. Immediately downstream of the 1A protein is a 2A peptide, which mediates a "stop-go" translation mechanism that leads to release of the mature 1A protein [60]. DCV-1A, a 99 amino acid protein, is a double-stranded RNA (dsRNA) binding protein that sequesters dsRNA intermediates from Dicer-2 mediated processing by the RNAi machinery. CrPV-1A, a 166 amino acid protein, employs a dual mechanism by which it binds to and inhibits Argonaute-2 (Ago-2) activity and stability [58,59,61]. Ago-2 mutant Drosophila are more susceptible to dicistrovirus infection, demonstrating the importance of the antiviral effects of Ago-2 [61,62]. CrPV-1A binding to Ago-2 inhibits its activity and also leads to Ago-2 degradation via an E3 ubiquitin ligase-dependent pathway [59]. Biochemical and single molecule studies showed that CrPV-1A inhibits the initial seed base-pairing targeting by Ago-2-RISC (RNA induced silencing complex) [63]. Structural and biochemical analyses have mapped distinct functions to specific domains on CrPV-1A. Specifically, CrPV-1A interacts with Ago-2 through a flexible loop containing a TALOS (targeting argonaute for loss of silencing) element and recruits the host ubiquitin complex, Cul2-Rbx1-EloBC through a BC box domain (Fig 1A) [59]. The F114 residue within TALOS is critical for Ago-2 binding and the L17 and A21 residues in the BC box domain are required for recruitment of the ubiquitin ligase complex [59]. We previously showed that the CrPV-1A protein inhibits SG foci formation and transcription [64]. CrPV-1A’s ability to inhibit SG and transcription is mapped to a single R146 residue at the C terminus. Mutant CrPV (R146A) virus infection is attenuated which is correlated with an increase in SG formation, strongly implicating potential antiviral properties of SG formation. Moreover, blocking transcription inhibited SG formation and restored CrPV (R146A) virus infection, suggesting that the SG modulation is linked to a nuclear event(s) [64]. In summary, CrPV-1A is a multifunctional protein that modulates several host cell processes to promote infection. Whether the specific functions of CrPV-1A are mutually exclusive or interdependent have yet to be examined. In this study, we use overexpression and mutagenesis approaches to uncouple the relationship between the multiple functions of CrPV-1A. We show that CrPV-1A’s ability to inhibit SGs is dependent on the BC Box ubiquitin complex-interacting domain and independent of the Ago-2 binding TALOS element. We also demonstrate that CrPV-1A localizes to the nuclear periphery which correlates with nuclear poly(A)+ RNA enrichment. Transcriptome analysis and gene depletion studies suggest that CrPV-1A modulates host steady state RNA levels and mRNA export. Finally, productive CrPV infection requires the nuclear pore complex protein Nup358/RanBP2 in a CrPV-1A R146-dependent manner. We propose that CrPV-1A mediated SG inhibition is linked to nuclear events including transcriptional shutoff and nuclear mRNA accumulation to promote infection.

Discussion Inhibition of SGs is a general strategy employed by many viruses to facilitate infection [21]. The mechanism and consequences of SG inhibition during virus infection are not fully understood. In this study, we uncoupled the functions of the multifunctional CrPV-1A protein and reveal specific domains important for SG inhibition and virus infection. Specifically, we demonstrated that SG inhibition is dependent on the BC box domain of CrPV-1A, which recruits the Cul2-Rbx1-EloBC complex, and acts in concert with an essential R146 residue to promote infection. We provided insights into this mechanism by showing that the wild-type CrPV-1A but not mutant CrPV-1A(R146A) protein, localizes to the nuclear periphery, induces nuclear poly(A)+ RNA accumulation, and modulates global transcriptome changes. Finally, we showed that Nup358 is targeted for degradation by CrPV-1A in a R146-dependent manner. Together, we propose a novel viral strategy whereby the viral protein CrPV-1A targets Nup358 for degradation via its R146-containing C-terminal tail and recruitment of the Cul2-Rbx1-EloBC complex inhibiting SG formation and RNA transport, consequently leading to poly (A)+ mRNA in the nucleus that further contributes to SG inhibition and facilitate productive virus infection. The effects of the R146A mutation on CrPV-1A’s function are illuminating that point to a nuclear event(s) controlled by CrPV-1A that are likely interdependent. Besides disrupting CrPV-1A’s ability to block SG assembly and Ago-2 activity, the CrPV-1A protein localizes to the nuclear periphery and mediates poly (A)+ mRNA nuclear enrichment and global transcriptome changes under infection (Figs 4–6). As mRNAs act as scaffolds for SG assembly [18,64,69,75], the enrichment of poly (A)+ mRNA in the nucleus in CrPV-1A expressing cells may serve two purposes: 1) to block global host mRNA translation and antiviral responses and 2) to deplete mRNA from the cytoplasm leading to SG inhibition. This viral strategy is reminiscent of other viral proteins that modulate nuclear events to facilitate SG formation and infection. As examples, picornavirus 2A protease expression regulates SG assembly and RNA export [76,77] and influenza virus polymerase-acidic protein-X (PA-X) protein inhibits SG formation concomitant with cytoplasmic depletion of poly(A) RNA and accumulation of poly(A) binging protein (PABP) in the nucleus [78]. There is also precedent that modulation of mRNA export can affect SG formation. A recent study showed that blocking mRNA export pathways with Tubercidin, an adenosine analog, induces SG formation, likely indirectly regulating cytoplasmic protein synthesis [79]. Conversely, sequestering mRNA export factors into SGs can inhibit nucleocytoplasmic transport [80]. In this study, we present a new paradigm of SG inhibition by a viral protein that directly modulates nuclear mRNA export. How does CrPV-1A regulate multiple cellular processes? Even though CrPV-1A is only 166 amino acids in length, there are multiple domains that mediate specific cellular functions. One of the best-known functions of CrPV-1A is its ability to bind to Ago-2 via its TALOS domain and degrade Ago-2 by recruiting the Cul2-Rbx1-EloBC via its BC Box domain [59]. By systematically uncoupling the functions of CrPV-1A via specific mutations singly or in combination with R146A, we demonstrate that CrPV-1A’s ability to block SG formation is not dependent on its ability to bind to Ago-2 (F114A mutation) (Fig 3). It is also clear that the TALOS domain does not contribute to CrPV-1A’s effects on enrichment of nuclear poly(A)+ mRNA (Fig 4). However, our results point to a role of the BC Box domain as mutations within this domain resulted in a deficit in SG inhibition by CrPV-1A. These results strongly suggest that recruitment of Cul2-Rbx1-EloBC ubiquitin ligase complex is required for CrPV-1A’s effects on SG inhibition. Further, our results identified Nup358 as a key component in inhibiting SG formation by CrPV-1A and promoting CrPV infection. Nup358 (also known as RanBP2) is an integral component of the cytoplasmic filaments of the nuclear pore complex that mediates nucleocytoplasmic transport of mRNA and protein [73,81]. Indeed, depletion of Nup358 in Drosophila cells blocks mRNA export from the nucleus (Fig 8) [73]. Recent studies have shown that Nup358 localizes to SGs [80,82]. Moreover, Nup358 plays a prominent role in virus infections. In HIV infected cells, Nup358 facilitates transport of the viral genome into the nucleus [83]. Additionally, vaccinia virus recruits Nup358 to the viral factories to enhance virus infection [84]. In this study, our model posits that CrPV-1A recruitment of the Cul2-Rbx1-EloBC ubiquitin ligase complex targets Nup358 for proteosome-dependent degradation to inhibit mRNA export and subsequently, to block SG formation (Fig 10). In support of this model, Nup358 levels, which are decreased in CrPV-infected cells, are recovered in MG132-treated cells and depletion of Nup358 results in inhibition of stress-induced SG formation (Figs 7 and 8). We note that MG132 treatment decreases CrPV infection in general, which is similar to that observed with other plus strand RNA virus infections [85]. It is likely that proteasome activity is required for CrPV infection. For example, Ago-2 degradation by CrPV-1A requires proteasome activity [59]. Moreover, all of these effects are dependent on the R146 residue within the C-terminal tail of CrPV-1A. The R146A mutation may disrupt CrPV-1A/Nup358 interactions (Fig 9) or may alter CrPV-1A subcellular localization and thereby sequester it from interacting with Nup358. Finally, the effects of the R146A mutation may alter protein conformations that mediate these effects. Our model showed that the CrPV-1A C-terminal tail interacts directly or indirectly with Nup358 to mediate degradation by the Cul2-Rbx1-EloBC ubiquitin ligase complex. CrPV-1A acts as a hub that interacts with multiple partners and through recruitment of the Cul2-Rbx1-EloBC ubiquitin ligase complex leads to proteosome-dependent degradation [59]. It will be of interest to investigate in more detail of the CrPV-1A/Nup358 interactions and whether ubiquitination of Nup358 is required for degradation or inactivation. Although the mammalian Nup358 is also a small ubiquitin-like modifier (SUMO) E3 ligase, that can SUMOylate Ago-2 and is linked to SG dynamics [86–90], however the Drosophila Nup358 lacks an obvious sumoylation domain [73]. PPT PowerPoint slide

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TIFF original image Download: Fig 10. Model of stress granule inhibition by CrPV-1A. During CrPV infection, CrPV-1A localizes to the nuclear membrane in an R146-dependent manner and recruits Cul2-Rbx1-EloBC complex to ubiquitinate Nup358 leading to its degradation. The degradation of Nup358 results in a block in mRNA export, resulting in the enrichment of poly (A)+ mRNAs in the nucleus, and together inhibits stress granule formation which, facilitates virus infection. https://doi.org/10.1371/journal.ppat.1010598.g010 Besides affecting the above cellular processes, the CrPV-1A R146A mutation also modulates the CrPV-2A peptide stop-go activity (Fig 2). 2A peptide activity relies primarily on a conserved DxExNPGP sequence whereby the stop-go peptidyl-tRNA hydrolysis occurs between the last G and P [60]. However, sequences upstream of this conserved region also contributes to 2A activity [91,92]. The R146A mutation, which is 20 amino acids upstream of the "stop-go" cleavage, would still be within the ribosome exit tunnel during translation and thus, specific peptide-ribosome exit tunnel interactions likely affects CrPV-2A activity. Although the inhibitory effects on 2A activity is modest (~2%), it is possible that the expression of the fusion CrPV-1A-2A-2B protein may act in a dominant manner to the effects observed by CrPV-1A(R146A) expression including mRNA export, Nup358 degradation and SG formation, ideas that needs to be examined further. The small CrPV-1A protein employs a multi-prong ‘Swiss-army knife’ approach to block the insect antiviral response, transcription, RNA metabolism and SG formation, all of which facilitate infection [64]. SGs are "sinks" of RNA and protein that may sequester viral proteins and RNA that may delay virus infection. For example, the CrPV 3C protease can localize to SGs [69]. SG inhibition is likely a key viral strategy to ensure viral proteins and RNA are available to promote the viral life cycle. Our previous study also showed that CrPV-1A can also block SGs in human cells, thus it will be interesting to determine whether there are common mechanisms for SG inhibition by CrPV-1A across species [64]. Although sequence analysis of the other dicistrovirus 1A proteins do not show any obvious conservation, it has been shown that some may have similar functions; the related DCV-1A protein can inhibit the antiviral RNAi pathway through a distinct mechanism by binding to dsRNA [58]. It will be of interest to determine whether these other dicistrovirus 1A proteins act similarly as CrPV-1A, which may shed light into the diversity of protein domains that target specific host factors for productive virus infection. Given the growing global health concerns of arthropod-borne viruses such as Zika virus, Dengue virus and Chikungunya virus, it will be of importance to further understand the underlying fundamental virus-host interactions such as stress granule inhibition in insects in order to develop novel antiviral strategies.

Materials and methods Cell culture and virus infection Drosophila S2 cells (Invitrogen) derived from a primary culture of late-stage Drosophila melanogaster embryo were maintained and passaged in either Shields and Sang medium (Sigma) or Schneider’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco) and 1X Penicillin-Streptomycin at 25°C. The wild-type and mutant CrPV clones [93] were used to prepare virus stocks and the stock was expanded by reinfecting naïve cells as described previously [56]. S2 cells were infected with wild type or mutant virus at the desired multiplicity of infection in phosphate buffer saline (PBS) at 25°C. After 30 mins of absorption, complete medium was added, and cells were harvested at desired time points. Virus titers were determined by Fluorescence Foci Forming Unit assay using immunofluorescence (anti-VP2) as previously described [56]. Plasmids The Drosophila expression vector pAc 5.1/V5-His B containing CrPV 5’UTR-GFP-3’ UTR, CrPV 5’UTR-1A-GFP-3’ UTR was generated using Gibson assembly (NEB Gibson assembly). The respective mutants were generated using Site directed mutagenesis. dsRNA targets were selected chosen from Updated Targets of RNAi Reagents Fly (Flybase) Fragments of candidate genes NXF1 (Accession number: AJ318090.1;position:2056–2362), GP210 (Accession number: AF322889.1; position: 2077–2576), mTor (Accession number: NM_057719.4; position:462–961), Rae1 (Accession number: NM_CP023332.1; position:17547164–17546605), Nup88 (Accession number:AY004880.1; Position: 116–714), Nup214 (Accession number: NM_143782.3; Position: 1412–1853) and Nup358 (Accession number: NM_143104.3; position: 1768–2338) targeting all isoforms with no off targets were used for dsRNA mediated knockdown. The amplicons were synthesized as gene fragments (Twist Bioscience) containing a T7 polymerase promoter flanking either side of the amplicon and directly cloned into a pTOPO plasmid using EcoR1 (NEB). FLuc plasmid was described previously [59,64]. The plasmids were digested with EcoRI, and the digested and purified products were directly used for in vitro transcription reactions. Gene encoding the Drosophila Nup358 transcript variant B (Accession number NM_001260373.2) with 3X-N terminal FLAG tag was cloned between BamHI and XhoI sites in pAc/His B vector (Genscript). All plasmids were sequence confirmed by Sanger sequencing (Genewiz). In vitro transcription and translation T7 polymerase reactions were performed as described previously [94]. Briefly 5 μg pAc CrPV 5’UTR-1A-GFP-3’ or pCrPV-3 plasmids were linearized with Eco53KI (NEB) or 5 μg pTOPO dsRNA plasmids with EcoR1 (NEB) in reaction containing 1X T7 buffer (50mM Tris-HCl, 15mM MgCl 2, 2 μM Spermidine Trihydrochloride, 5 μM DTT), 10 mm NTP mix (NEB), Ribolock (Thermo scientific), 2 units of yeast inorganic pyrophosphate (NEB) and T7 Polymerase for 4–6 hours. DNAse I (NEB) treated samples were cleaned up using RNAeasy cleanup kit (Qiagen). GFP RNA was capped and polyadenylated (Cellscript) and then purified (RNAeasy kit, Qiagen). The integrity of RNA was verified by denaturing RNA agarose gel electrophoresis. The quantity of RNA was determined using Nanodrop (Thermo Scientific). In vitro translation assays of the wild type or mutant CrPV-1A RNAs were performed in Spodoptera frugiperda 21 (sf-21) insect cell extract (Promega). Briefly, 2 μg RNA was incubated with sf-21 extract in the presence of [35S]-Methinone-Cysteine (Perkin-Elmer >1000Ci/mmol) and F buffer (40mM KOAc. 0.5 mM MgCl 2 ) for 2 hrs at 30°C. The resulting translated proteins were resolved by a sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) gel and analyzed by phosphoimager analysis (Typhoon, Amersham, GE life sciences). Transfections For DNA transfections, 1.5 million S2 cells were transfected with 2 μg of plasmid using Xtreme-GENE HP DNA transfection reagent (Roche) according to the manufacturer’s protocol. Transfected cells were incubated in complete Shields and Sang medium for 16–24 hours. Transfection of in vitro transcribed RNAs in S2 cells performed using Lipofectamine 2000 (Invitrogen) as described by manufacturers protocol for 16–24 hours. RNA interference For dsRNA mediated gene knockdown, 3 million cells were incubated with serum free medium containing 60 μg dsRNA per well of a 6 well plate for 1 hour at 25°C. The soaked cells were supplemented with complete medium containing FBS and incubated for 4 days at 25°C. Cell viability of silenced cells were monitored by Trypan Blue dye exclusion assay. Immunofluorescence and in situ hybridization Transfected S2 cells transferred to coverslips precoated with 0.5 mg/mL Concanavalin A (Calbiochem) in 12 well plates for 2 hours. 16–24 hours post transfection the cells were fixed in 3% w/v paraformaldehyde in PBS, then permeabilized in PBS containing 0.1% Triton X-100 for 30 minutes and blocked with 2% BSA for 30 minutes. For in situ hybridization, the cells were incubated in hybridization buffer (2X SSC, 20% formamide, 0.2% BSA, 1 μg/μL yeast tRNA) for 15 mins at 37°C and subsequently, the cells were incubated with 1 mg/mL oligo(dT) conjugated to Cy5 (IDT) overnight at 46°C in hybridization buffer. The next day, the cells were washed with 2X SSC with 20% formamide twice for 5 min each at 37°C, 2X SSC for 5 min at 37°C, 1X SSC once for 5 min and 1X PBS for 5 min prior to staining with the primary antibodies. The primary antibodies and the dilutions used were as follows: α-CrPV-1A (1:200), α-Lamin A (1:1000, DGRC), α-Rin (1:500, generous gift from Eric Lecuyer). Cells were washed three times with PBS and then incubated with secondary antibody (1:1000 goat anti-rabbit antibody or goat anti-mouse antibody conjugated to Texas Red and 1:1000 goat anti-mouse antibody conjugated to Alexa Fluor 647 (Life Technologies) and 1:1000 donkey anti-goat antibody conjugate to Texas Red (Thermo Fisher Scientific) and Hoechst dye (1:20,000 in PBS, Invitrogen) to stain for nuclei. Coverslips were mounted on slides with Prolong gold antifade reagent (Invitrogen). The cells were imaged and analyzed using a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) with a 63x objective. Representative images are shown and were analyzed in ImageJ. Rin granules were counted using a quantitatively measured threshold intensity and defined circularity using Image J. Intensity measurements were done using Image J [95]. Box plots and graphs generated using GraphPad Prism is used to represent the data. cDNA synthesis and quantitative real time PCR Total RNA was extracted from cells using Monarch total RNA Miniprep kit (NEB). cDNA synthesis was performed using Lunascript RT Supermix Kit (NEB) as per manufacturer’s protocol. qRT PCR was performed using Luna Universal qPCR master mix (NEB) as per manufacturer’s protocol. CrPV genome was amplified using; 5’-CAGTGCCTTACATTGCCA-3’ and 5’-AACTTCTACTCGCACTATTC-3’ and Rps6 was amplified using primers 5’-CGATATCCTCGGTGACGAGT-3’ and 5’ -CCCTTCTTCAAGACGACCAG-3’ Western Blot analysis S2 cells were washed with PBS and harvested in RIPA buffer (150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 50 mM Tris-HCl, pH 8.0 and protease inhibitor cocktail (Roche)). Protein samples that were freeze thawed three times,spun down at 13,000 rpm for 15 minutes at 4°C and the supernatants were collected as the total protein extracts. Protein concentration was determined by Bradford assay (Biorad). Equal amounts (in micrograms) of lysates were separated on 4–15% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). Subsequently, the membranes were blocked for 30 mins in 5% skim milk and TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween-20) and probed with primary antibody for 1 hour. The dilutions and primary antibodies used were as follows: α-CrPV-1A (1:1000), α-GFP (1:1000, Roche). α-CrPV-VP2 (1:1000, Genscript), α-CrPV-3C (1:1000, (raised against CrPV-3C peptide sequence NH 2 -CTDMFDYESESYTQR-C), Genscript), α-CrPV-Nup358 (1:1000. raised against Nup358 peptide sequence NH 2- CGSTDKSEPGKDAGP-C), Genscript), α-Tubulin (1:1000, DSHB). Membranes were washed with TBS-T three times and incubated with secondary antibodies for 1 hour at room temperature. Following secondary antibodies were used: IRDye 800CW goat-anti-rabbit IgG or IRDye 680CW goat anti-mouse at 1:5000 (LI-COR Biosciences). Membranes were washed with TBS-T three times and protein bands were detected and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). Alternatively, 1:5,000 dilution of donkey anti-rabbit IgG-horseradish peroxidase (Amersham) or a 1:5,000 dilution of goat anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnology) was used to detect proteins by enhanced chemiluminescence (Thermo Scientific). Co-immunoprecipitation Drosophila S2 cells were transfected with 3X-FLAG-Nup358 DNA for 24 hours, followed by transfection with in vitro transcribed RNA encoding GFP, CrPV-1A or CrPV-1A(R146A) for 16 hours. Cells were washed with PBS, lysed in the Pierce-MS Compatible Magnetic IP Kit lysis buffer, and freeze thawed three times to extract the total protein. After centrifugation, the supernatant was collected, and protein concentrations were determined by Bradford assay (Biorad). Co-immunoprecipitation was performed using Pierce-MS Compatible Magnetic IP Kit (Thermofisher) according to the manufacturer’s protocol with minor modifications. Briefly 25 μL magnetic beads were incubated with 5 μg mouse monoclonal FLAG antibody (Sigma) at 4°C overnight. The beads were washed 2X times with an IP Lysis buffer and incubated with 1 mg total protein for 2 hours at 4°C overnight. Unbound proteins were washed off using wash buffers and the beads complexed with immunoprecipitated proteins were resuspended in the Pierce-MS Compatible Magnetic IP Kit elution buffer and processed for western blotting analysis. RNA seq: sample preparation, library generation and analysis S2 cells (1.5 X 107) were infected with wild-type or R146A mutant CrPV at an MOI 3. Total RNA was extracted from mock or virus infected cells using Trizol reagent (Invitrogen) at 2 and 4 hours post infection. The samples were treated with DNAse I for 1 hour at 37°C and were re-extracted using Trizol. The RNA integrity was verified by denaturing gel analysis. Polyadenylated RNA was isolated using NEBNext poly(A) mRNA isolation module and the quality and quantity of RNA were determined by electrophoresis on the bioanalyzer (Agilent). NEBNext Ultra II DNA library prep kit was used to generate libraries. Size selection was performed on adaptor ligated libraries using agarose gel, generating cDNA libraries size ranging from 150–275 nucleotides. The enriched libraries were purified using QlAquick purification column. Sequencing of a pool of multiplexes libraries were performed on an Illumina HiSeq 4000 PE100 Platform (Génome Québec). At least 19 million reads were generated from each sample. Libraries, sequencing, and quality control of the sequencing were performed by the Nanq facility at Génome Québec. Reads were trimmed based on quality using the default parameters of Trimmomatic and assessed using FastQC as part of Unipro UGENE v1.29 [96,97]. and mapped to the Drosophila melanogaster genome using default paired-end parameters of Bowtie2 as part of UGENE. Reads mapping to the CrPV genome were removed for downstream analysis to maintain normalization based only on total host gene transcript numbers. Reads were mapped to the D. melanogaster transcriptome and quantified using the quasi-mapper Salmon 1.8.0 [98]. Differentially expressed genes were identified using iDEP 0.92 (http://bioinformatics.sdstate.edu/idep92/) and the Bioconductor package DESeq2. used for heatmap visualization with Integrated differential expression and pathway analysis (iDEP) [99]. The raw sequencing data was submitted under Gene expression accession number PRJNA771107. Venn diagrams for the comparison of different gene expression data were generated using InteractiveVenn [100]. Network analysis of gene ontologies was performed using ClueGo v2.5.6 [101]as part of Cytoscape v3.7.2 [102] using the EBI-UniPRot-GOA Molecular Function database (17.02.2020). Anti-CrPV-1A Polyclonal antibody DNA fragment encoding the full length CrPV-1A gene was cloned into pet28b vector using Nd1 and Xho1 enzymes and the resultant construct with C terminal His-tag was used for protein expression in E.coli BL21DE3 cells (modified from [63]). Expression of CrPV-1A protein was carried out in E coli (BL21DE3) cells grown in Terrific broth medium at 16°C overnight. induced with 0.5 mM Isopropyl-β-D-thiogalactoside (IPTG). The soluble protein was purified using Ni-NTA Agarose beads (Qiagen) in a buffer containing 30 mM HEPES-KOH pH 7.4, 100 mM KOAc, 2 mM Mg(Ac) 2 , 300 mM Imidazole, 10% glycerol, 1 mM DTT with complete mini EDTA free protease inhibitor tablet. The purified samples were dialyzed and further analyzed over a superdex 50 gel filtration column equilibrated with exchange buffer (30 mM HEPES-KOH pH 7.4, 100 mM KOAc, 2 mM Mg(OAc) 2, 10% Glycerol, 1 mM DTT). All purified proteins were flash-frozen in liquid nitrogen and stored at -80°C. The polyclonal antibody against CrPV-1A in rabbits was generated by Genscript. USA.

Acknowledgments We thank Eric Lecuyer for generously providing the Rin antibody. We acknowledge the UBC Life Science Institute core imaging facility for use of the Leica Sp5 microscope and Cellomics microscope. We thank Génome Québec for the support with RNA sequencing, Irvin Wason for helping with the gel filtration chromatography, and the Jan lab (Jodi Chien, Rachel DaSilva, Reid Warsaba and Christina Young) for discussions and critical reading of the paper.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010598

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