key: cord-0799796-2r427d98
authors: Zeng, Hongxiang; Gao, Xiang; Xu, Gang; Zhang, Shengyuan; Cheng, Lin; Xiao, Tongyang; Zu, Wenhong; Zhang, Zheng
title: SARS-CoV-2 helicase NSP13 hijacks the host protein EWSR1 to promote viral replication by enhancing RNA unwinding activity
date: 2022-02-24
journal: Infectious Medicine
DOI: 10.1016/j.imj.2021.12.004
sha: 5e52f06055cf4bc463f53b6ae255a98040541895
doc_id: 799796
cord_uid: 2r427d98

Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has led to a global coronavirus disease 2019 (COVID-19) pandemic. Currently, incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs. Results In this study, we found that SARS-CoV-2 hijacks the host protein EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) to promote the activity of its helicase NSP13 to facilitate viral propagation. NSP13 is highly conserved among coronaviruses and is crucial for virus replication, providing chemical energy to unwind viral RNA replication intermediates. Treatment with different SARS-CoV-2 NSP13 inhibitors in multiple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection. Using affinity-purification mass spectrometry, the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13. Furthermore, silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels. Mechanistically, EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability. Conclusion In conclusion, our results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) outbreak that has caused over 364 million infections, resulting in a serious global pandemic since December 2019 [1] [2] [3] [4] [5] .

Currently, there is an urgent need to better understand the molecular mechanisms of SARS-CoV-2 replication because this information will provide vital insights for the design of better drugs and vaccines. SARS-CoV-2 belongs to a large family of single-stranded, positive-sense RNA viruses containing large RNA genomes of [26] [27] [28] [29] [30] [31] kb, and encoding two large open reading frames (ORFs) called ORF1a and ORF1b [6] .

These ORFs are translated into two polyproteins, pp1a and pp1ab, which are proteolytically cleaved into the four structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, along with 16 nonstructural proteins (NSP1-16) and several accessory proteins named by ORF, which have been reported to modulate host immune responses [7] [8] [9] [10] . NSP12 and NSP13 reside in the functional center for virus replication: NSP12 is an RNA-dependent RNA polymerase (RdRp) required for genome replication [11] [12] [13] , while NSP13 possesses helicase activity for unwinding double stranded RNA (dsRNA) during the replication process [14] [15] [16] [17] [18] [19] .

Most RNA viruses, including coronaviruses (CoV-NL63, SARS-CoV, and Middle East respiratory syndrome coronavirus) and Flaviviridae (Zika virus and Dengue virus) produce dsRNA during viral replication, and so the RNA genome must be unwound for the next round of viral RNA replication to occur [20, 21] . Therefore, the viral helicase responsible for this unwinding activity represents an important drug target for inhibiting viral replication. Ivermectin has been found to be a potent inhibitor of flavivirus replication, specifically targeting the NS3 helicase activity of the Kunjin virus (an Australian variant of West Nile virus) [22] . ASP2151, a helicase-primase inhibitor has been found to inhibit replication of thymidine kinase-deficient herpes simplex virus 2 [23] . A clinical trial of amenamevir has been completed and licensed successfully, holding great promise for treating herpes zoster [24] . Thus, we believe that there is important potential in exploring host dependencies of the SARS-CoV-2 helicase NSP13 to identify other host proteins that are already targeted by existing drugs. EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) is ubiquitously expressed in most cell types and has many roles in distinct cellular processes and organ development [25] [26] [27] [28] [29] . EWSR1 has been reported to interact with heterogeneous RNA-binding proteins (hnRNPs), such as RBM38 and RBM39, which are involved in alternative RNA splicing [30] . It is noteworthy that EWSR1 protein can directly bind RNA in vitro, which is achieved by its C-terminal domain containing an RNA-recognition motif (RRM) and three RGG boxes [31] . It has been reported to interact with the cis-acting replication element (CRE) of hepatitis C virus, acting as a potential cofactor for virus replication [32] . A recent study also demonstrated that EWSR1 potentially binds to the SARS-CoV-2 genome directly [33] . However, it is not yet known whether EWSR1 regulates SARS-CoV-2 replication, or the specific mechanism by which this may occur.

In the current study, using mass spectrometry, we identified EWSR1 as a potent binding partner of SARS-CoV-2 helicase NSP13. Domain-mapping analyses revealed that the NTPase domain of NSP13 associates with the RNA binding domain (RBD) of EWSR1. Knockdown of EWSR1 can efficiently block SARS-CoV-2 replication, while supplementing the expression of EWSR1 can rescue viral replication. Finally, an in vitro dsRNA unwinding assay revealed that EWSR1 promotes the unwinding ability of NSP13. Thus, our findings identify a novel host factor for SARS-CoV-2 replication that could potentially be targeted for therapy.

For the eukaryotic expression vectors, the pcDNA6B-NSP13-flag plasmid was kindly provided by Prof. Peihui Wang from Shandong University, and the CMV-EWSR1-myc expression vector was purchased from Sino Biological Inc. GFP-tagged EWSR1 and mcherry-tagged NSP13 were constructed using a C113 ClonExpress MultiS One Step cloning kit (Vazyme).

For the prokaryotic expression vectors, the pET28a-6xHis-NSP13 plasmid was provided by Prof. Peihui Wang. The full-length human EWSR1 CDS was amplified from the CMV-EWSR1-myc template plasmid and cloned into the pET28a vector flanked by NdeI and XhoI. Truncated fragments TAD and RBD were generated by PCR from the full-length EWSR1 template and cloned into the pET28a vector.

HEK-293T, Huh7, and HeLa-ACE2 cell lines were cultured using DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). The Calu-3 cell line was cultured using alpha-MEM (Gibco) supplemented with non-essential amino acids, sodium pyruvate, and FBS (10%). The plasmids were adjusted to 3 μg for six well plates and 6 μg for 60 mm dishes, then mixed with polyethylenimine (PEI) (Sigma) transfection reagent in a 1μg:3μl ratio of plasmid:transfection reagent following the manufacturer's recommendations. After more than 30-36 h, cells were subsequently washed twice with PBS. Each step was performed by centrifugation at 600 ×g, 4 °C for 5 min. Cell pellets were lysed with 2× SDS sampling buffer and boiled at 95°C for 15-30 min.

Flag, c-myc, GAPDH, tublin, and actin antibodies were purchased from Transgene; EWSR1 antibody was purchased from Abcam; NP antibody was purchased from Sino Biological Inc; strep-tag II antibody was purchased from GenScript.

For the NSP13 inhibitor experiment, approximately 2×10 5 Huh7, VeroE6, Calu-3, thermocycler. For protein analysis, whole cell lysates were obtained by lysing in 2×SDS sampling buffer and proteins were separated by SDS-PAGE.

To identify proteins associated with NSP13, HEK-293T, and HeLa cells plated in 

Cells Proteins were quantified by the Pierce™ BCA protein assay kit (Thermo fisher Scientific) and stored at -80°C in aliquots. Proteins were separated by 10%

SDS-PAGE and visualized by Coomassie blue staining.

The dsRNA helix substrates consisted of two complementary RNA strands. One Table S1 .

The dsRNA with 5′-protrusions were generated by annealing the HEX-labeled and unlabeled strands in a 1:1 ratio in a 50 μL reaction mixture containing 25 mM HEPES-KOH (pH7.5) and 25 mM NaCl, followed by heating to 95°C and cooling down gradually to room temperature.

The standard dsRNA unwinding assay was performed as previously described. 

Data were analyzed using GraphPad Prism software version 8.0 (GraphPad software, San Diego, CA). All results were considered significant for P values <0.05.

Considering the high homology (99%) of NSP13 in SARS-like coronaviruses, we sought to explore whether we could inhibit SARS-CoV-2 replication by using levels in a dose-dependent manner in Vero cells (Fig. 1A) while it effectively inhibited NP expression at 100 μmol/L in Huh-7 cells (Fig. 1A) . Scutellarein also inhibited NP expression in a dose-dependent manner in HeLa-ACE2 cells but had only mild effects on Calu-3 cells (Fig. 1A) . Bismuth inhibited NP levels in HeLa-ACE2 cells (Fig. 1B) . In addition, SSYA10-001 also exhibited effective inhibition of NP expression in a dose-dependent manner in Huh-7 and Vero cells, but the effective concentration was 100 μmol/L in HeLa-ACE2 and Calu-3 cells (Fig.   1C ).

These results indicate that the three inhibitors the inhibitors had similar effects against SARS-CoV and SARS-CoV-2 and therefore may be applicable for use in treating future coronavirus outbreaks; however, more preclinical/clinical studies are needed to confirm whether this is effective in the clinic.

To explore the role of NSP13 in virus-host interactions, we used a flag-tagged affinity purification mass spectrometry approach to identify the human proteins that physically interacted with NSP13 in both 293T and HeLa cell lines. We also used NSP16 as a non-specific control to compare with the NSP13 interactome.

Immunoprecipitated protein complexes were captured by flag antibody and protein A beads, washed, separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie blue. The heavy and light IgG bands were excised and the remainder of the gel was analyzed by matrix-assisted laser desorption ionization-mass spectrometry to identify the associating proteins ( Fig.   2A ). The dotted arrows show immunoprecipitated NSP13 and NSP16. The bands in the closed boxes are specific binding partners of either NSP13 or NSP16 (Fig. 2C) .

We identified several differential binding proteins including an RNA-binding protein called EWSR1. We found that EWSR1 was present in two distinct batches of experiments among all the identified interactions (Fig. 2B) . To validate this interaction, we performed co-immunoprecipitation (co-IP) assays and found that EWSR1-myc co-precipitated with NSP13-flag when pulled-down with flag antibody (Fig. 3A) . Consistently, NSP13-strepII could be co-precipitated with EWSR1-myc when pulled-down with myc antibody (Fig. 3B) . We also confirmed this interaction by performing an IP assay with endogenous EWSR1. EWSR1 co-precipitated with NSP13-flag when pulled-down with flag antibody (Fig. S1) . Furthermore, green fluorescent protein (GFP)-EWSR1 was co-localized with mcherry-NSP13 in immunofluorescence analysis when co-expressed in 293T cells (Fig. 3C) . It is noteworthy that there is no high quality commercially available NSP13 antibody to perform endogenous immunofluorescence during viral infection (data not shown).

EWSR1 contains an N-terminal serine-tyrosine-glycine-glutamine (SYGQ)-rich domain that acts as a transcriptional activating domain (TAD), an RRM, and a zinc finger domain involved in RNA and DNA binding, as well as multiple Arg-Gly-Gly (RRG)-rich regions in the C-terminus that affect RNA binding (the RNA binding domain, RBD), and a proline-tyrosine (PY) region that functions as a nuclear localization signal [37] . To further pinpoint the region of EWSR1 that is required for the interaction with NSP13, EWSR1 was truncated into its TAD and RBD domains.

We found that only the RBD domain of EWSR1 co-precipitated with flag-tagged NSP13 and this interaction was comparable with that of the full length EWSR1 (Fig.   3D ).

Conversely, to identify the region of NSP13 that is essential for the interaction with EWSR1, we truncated NSP13 into the N-terminal zinc binding domain (ZBD) and C-terminal NTP/ATP hydrolysis-dependent unwinding activity domain (NTPase domain) [38] . We found that only the NTPase domain of NSP13 co-precipitated with myc-tagged EWSR1 and this interaction was comparable with that of the full length NSP13 (Fig. 3E) , suggesting that EWSR1 may promote virus replication by associating and enhancing the ATP hydrolysis process, thus promoting NSP13-mediated unwinding of dsRNA.

To further determine the biological role of EWSR1 in the SARS-CoV-2 viral life cycle, we investigated the effects of EWSR1 silencing on transcription and translation of NP. First, we designed three short hairpin RNAs targeting the 5′ untranslated region (UTR), 3′ UTR, and the coding sequence (CDS) of EWSR1. Lentivirus was packaged to transduce Huh7 cells endogenously expressing ACE2. Knockdown efficiency was confirmed using an anti-EWSR1 antibody. Compared with an irrelevant scramble,

shRNA-1 and shRNA-3 targeting ESWR1 resulted in a dramatic reduction of endogenous EWSR1 protein, indicating effective silencing (Fig. 4A) . We then chose shRNA-1 and shRNA-3 for further SARS-CoV-2 virus infection experiments. We found that viral NP expression in EWSR1-silenced cells was significantly decreased compared with that in wild-type (WT) controls at 48 h post-infection (Fig. 4B) . We also observed that NP mRNA expression in EWSR1-silenced cells as well as that in the supernatant were dramatically decreased compared with that in WT controls ( Fig.   4C and 4D). Furthermore, we noticed a time-dependent decrease in NP levels (Fig. 4E) and mRNA expression both in cells and the supernatant (Fig. 4F and 4G) suggesting that the RBD of EWSR1 was essential for binding to NSP13 and enhancing its activity, thus promoting dsRNA unwinding and virus replication.

Finally, we sought to examine whether EWSR1 binds to NSP13 and enhances its RNA helix unwinding activity. We expressed and purified His-tagged NSP13 and 16 EWSR1 FL/TAD/RBD protein in a BL21-DE3 expression system (Fig. 5A) . We also performed western blotting to confirm protein expression (Fig. S1A and 1B and 1C) .

We then constructed an RNA helix substrate with five protrusions by annealing a 42-nucleotide HEX-labeled RNA with a 24-nucleotide unlabeled RNA. This dsRNA helix was used to measure the helix unwinding efficiency of RNA helicases as reported by others [19] . The unwinding assay was performed by incubating the RNA helix substrate with NSP13 or other proteins of interest in a standard reaction mix containing Mg 2+ and ATP, followed by separation of the RNA strands by RNA electrophoresis. dsRNA without any protein was the negative control and boiled dsRNA substrates were used as the positive control. The HEX-labeled RNA strand was released from the RNA helix substrate in the presence of NSP13 protein and either full-length EWSR1 or the RBD fragment, dramatically increasing the unwinding efficiency, but not in the presence of TAD (Fig. 5B) , suggesting that EWSR1 promoted the unwinding activity of NSP13 by potentially binding to its

NTPase domain in vitro. The RBD of ESWR1 is the critical binding domain for the interaction with NSP13 in vitro.

By taking an unbiased approach to identifying host proteins that associate with SARS-CoV-2 helicase NSP13, we have for the first time defined EWSR1 as a host factor that shows enhancement of NSP13-mediated dsRNA unwinding. We also revealed that the RBD domain of EWSR1 specifically interacts with the NTPase domain of NSP13 and may enhance its unwinding activity. This binding was also confirmed by the in vitro dsRNA unwinding assay. We further employed loss of function approaches in SARS-CoV-2 infection to demonstrate that EWSR1 could promote viral replication and translation by physically binding to NSP13. Here, we uncovered a previously uncharacterized interaction between NSP13 and host mRNA binding protein EWSR1. This interaction was shown by mass spectrometry and confirmed by co-immunoprecipitation assay. EWSR1 was first discovered as one of the chromosomal translocations that drives the majority of childhood and adolescent Ewing Sarcoma (EWS) cases. While its function in tumorigenesis is well established, little is known about its role during virus infection.

A recent study reported that EWSR1 could directly bind to the hepatitis C virus genome, promoting efficient viral replication. EWSR1 was also reported to directly bind to the pan-viral genome as a host factor. It would be intriguing to elucidate whether EWSR1 and NSP13 binds on the same sites/regions of the SARS-CoV-2 RNA genomes or they binds the SARS-CoV-2 RNA genomes in a proximal distance., thus providing a spatial advantage for viral replication. Further studies will be needed to address this question.

The interaction between virus and host is an evolutionarily mutual interaction; the virus needs a variety of host resources and materials to establish and maintain an effective and persistent infection. We have demonstrated that SARS-CoV-2 NSP13

binds to EWSR1 and may hijack this host factor for building its own replication factory, but further studies are required to address how EWSR1 is recruited and binds to NSP13 during SARS-CoV-2 infection. Furthermore, rational design of small inhibitors to prevent this binding process may represent a promising strategy for drug development. Figure S1 . Endogenous EWSR1 co-immunoprecipitated with exogenous NSP13-flag.

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Fig 2. Determination of the host factors associated with SARS-CoV-2 NSP13. (A) Schematic of the mass spectrometry analysis of NSP13 immunoprecipitates

we overexpressed pCMV-NSP13-flag plasmid in 293T cells (empty vector and pCMV-NSP16-flag as control groups) followed by immunoprecipitation with flag antibody. Associated proteins were eluted with SDS buffer and then subjected to 10%

After Coomassie brilliant blue staining, IgG-heavy (52 kDa) and

Mass spectra were extracted, deconvolved, and deisotoped using Proteome Discoverer 1.4.1.14 (Thermo) and searched through the database (Homo sapiens, UniprotKB/Swissprot) for quantification analysis. (B) The number of EWSR1 peptides detected by mass spectrometry are shown in two separate experiments using HEK-293T cells. (C) Immunoprecipitates were subjected to SDS-PAGE and stained with Coomassie blue

This work was supported by grants from the National Science Fund for Distinguished Young Scholars (82025022), the Central Charity Fund of Chinese Academy of Medical Science (2020-PT310-009), and the Science and Technology Innovation Committee of Shenzhen Municipality (2020A1111350032). We thank Prof. Pei-hui Wang from Shandong University for kindly providing us with the NSP13 plasmids.

The authors declare that no conflict of interest exists.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: