key: cord-0967476-snegzrj7
authors: Kato, Koki; Ikliptikawati, Dini Kurnia; Kobayashi, Akiko; Kondo, Hiroya; Lim, Keesiang; Hazawa, Masaharu; Wong, Richard W.
title: Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex
date: 2020-12-13
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.11.115
sha: dc9568fa48e8866a3caa2ff83d28580e0edfbef9
doc_id: 967476
cord_uid: snegzrj7

The novel human betacoronavirus SARS-CoV-2 has caused an unprecedented pandemic in the 21st century. Several studies have revealed interactions between SARS-CoV-2 viral proteins and host nucleoporins, yet their functions are largely unknown. Here, we demonstrate that the open-reading frame 6 (ORF6) of SARS-CoV-2 can directly manipulate localization and functions of nucleoporins. We found that ORF6 protein disrupted nuclear rim staining of nucleoporins RAE1 and NUP98. Consequently, this disruption caused aberrant nucleocytoplasmic trafficking and led to nuclear accumulation of mRNA transporters such as hnRNPA1. Ultimately, host cell nucleus size was reduced and cell growth was halted.

Since late 2019, the overwhelming effects of a novel coronavirus disease (named COVID-19 for "coronavirus disease 2019") have spread rapidly and developed into a global pandemic. An overriding exponential rate of new cases and deaths has urged scientists across the world to develop vaccines and antiviral drugs against the causative pathogen of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

However, many unknowns about the molecular invasion and replication pathways of SARS-CoV-2 components present major obstacles for COVID-19 studies. The SARS-CoV-2 ~30-kb positive-strand RNA genome contains 14 potential open reading frames (ORFs) and encodes replicase (ORF1a and ORF1ab), structural proteins (spike, nucleocapsid, membrane, and envelope), and accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b, and ORF9b) of poorly understood function. These accessory proteins may be important for virus replication [1, 2] .

Recently, two efforts utilizing immunoprecipitation coupled with mass spectrometry (IP-MS) of epitope-tagged viral proteins to map SARS-CoV-2-host protein-protein interactions (PPIs) identified >1000 putative virus-host PPIs in HEK293T cells [3, 4] .

Remarkably, the SARS-CoV-2 ORF6 interactome is uniquely enriched in nuclear pore complex (NPC) components, especially nucleoporins RNA export factor 1 (RAE1) and 98 (NUP98) [3, 5] . RAE1 (also known as Gle2 or Mnrp41), initially identified as an mRNA export protein [6, 7, 8] , is a homologue of spindle checkpoint protein of BUB3 [9, 10] .

We and others showed that RAE1 binds to the GLEBS motif of NUP98, allowing them to interact and function together in mRNA export or spindle assembly to prevent chromosome mis-segregation [10, 11, 12, 13, 14, 15] . RAE1 and NUP98, which are kinetically partitioned among other components in both cytoplasmic and nuclear compartments, also act as regulators for BMAL1 shuttling between the cytoplasm and nucleus, as well as maintenance of the correct pace of the circadian clock [16] . In addition, RAE1 was found to mediate ZEB1 expression by promoting epithelial-mesenchymal transition in breast cancer [17] .

Notably, RAE1 and NUP98 are the key host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV) [18, 19] . As a canonical seven-bladed β-propeller, RAE1 is a likely binding platform for other proteins [20] . Unsurprisingly, VSV promotes heterogeneous ribonucleoprotein A1 (hnRNPA1) relocalization in a RAE1-dependent manner; hnRNPs are thought to serve as mRNA export factors [21] . Similar to VSV, ORF6 also contains a methionine in its C-terminal region, which may facilitate interactions with J o u r n a l P r e -p r o o f RAE1 and NUP98 3. However, unlike HIV capsid entering into the nuclear pore [23, 24] it remains largely unknown why SARS-CoV2 ORF6 needs to interact with nucleoporins after infection because SARS-CoV2 hijacks the protein-making machinery of the host cell to translate its RNA directly into new copies of the virus.

Here, we found that overexpression of GFP-ORF6 induced mislocalization of RAE1 and NUP98. We also found that the nuclear size of ORF6-transfected cells was severely reduced, potentially due to hnRNPA1 nuclear accumulation and subsequent deferment of mRNA export. Finally, we showed that overexpression of RAE1 partially rescued this phenomenon.

J o u r n a l P r e -p r o o f 2. Methods

Constructs for overexpressing SARS-CoV-2 ORF6 were generated by ligation of synthetic DNA into the pEGFP-N1 vector (Clontech #6085-1) using EcoRI (New England Biolabs, R0101) and BamHI (TOYOBO, BAH-111) restriction sites. The FLAG-RAE1 plasmid was previously described [15] . For RAE1 siRNA, stealth RNAi targeting RAE1 was customarily synthesized (Invitrogen) to target the sequence corresponding to nucleotides 343-367 of human RAE1 mRNA (GenBank Accession No. AK292247). Universal negative control siRNA (SIC001) was purchased from Sigma-Aldrich.

Primary antibodies used for immunocytofluorescence, immunoprecipitation, and 

HEK293T cells were propagated in Dulbecco's Modified Eagle's Medium (Wako, 043-30085) supplemented with 10% (v:v) fetal bovine serum (Life Technologies, 10082147) and 50 U/mL penicillin-streptomycin (Nacalai Tesque, 26253-84), as previously described [25] . Cells were cultured at 37°C and 5% CO 2 .

DNA transfection was performed in six-well plates using Lipofectamine 2000 (Invitrogen, 11668019) with 1000 ng of plasmid DNA or 50 nM siRNA per well according to the manufacturer's protocol, as previously described [26] . 

For protein extraction, 6 × 10 5 cells were solubilized in 100 µL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Nonidet P-40, 2 mM EDTA, and 10% glycerol) supplemented with EDTA-free protease inhibitor cocktail (Nacalai Tesque, 03969). After centrifugation at 14,000 × g for 10 min at 4°C, supernatants were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore, IPVH00010). Membranes were incubated with primary antibodies and exposed to secondary HRP-conjugated antibodies. Proteins were visualized by chemiluminescence using Immobilon Western Chemilum HRP Substrate (Millipore, WBKLS0500). Images were acquired with a LAS-4000 image analyzer (Fujifilm).

Nuclear/cytoplasmic fractionation analysis was performed using nuclear and cytoplasmic extraction reagents according to the manufacturer's protocol (Thermo Scientific, 78840), as previously described [25, 27] . Cytoplasmic and nuclear extracts were then subjected to immunoblotting analysis.

Cells were transiently transfected with pEGFP-ORF6 and/or FLAG-RAE1 plasmids, and harvested 48 h after transfection. Cells were centrifuged at 500 × g for 5 min, lysed in lysis buffer supplemented with EDTA-free protease inhibitor cocktail. Next, cell lysates were centrifuged at 14,000 × g for 10 min at 4°C. The resulting supernatants were mixed with 5 µg of the indicated antibodies and Dynabeads protein A/G (Veritus, J o u r n a l P r e -p r o o f DB10001/10003), and rotated at 4°C overnight. The following day, beads were washed three times with lysis buffer. Whole-cell lysates and immunoprecipitates were analyzed by immunoblotting, as previously described [27] .

Cells were seeded into 96-well plates at a density of 3,000 cells per well and cultured for the indicated times. as previously described [28] . Samples were mixed thoroughly and measured at 570 nm for absorbance.

Nuclear area and intensity measurements were normalized to controls. Averaging and statistical analyses were performed for independently repeated experiments [29] . Twotailed Student's t test assuming equal variances was performed using GraphPad PRISM 7 software to evaluate statistical significance. P-values, number of independent experiments, number of nuclei quantified, and error bars are denoted in the figure legends.

Statistical analysis was performed using PRISM 7 software. Data are presented as mean ± standard deviation. Statistically significant differences in mean or median values between respective groups were tested by Student's t-test or one-way ANOVA analysis. P values < 0.05 were considered to indicate a statistically significant difference.

J o u r n a l P r e -p r o o f 3. Results

Given that RAE1 localizes around the nuclear rim and nucleoplasm, as well as in the cytoplasm, we hypothesized that there might be manifold forms of RAE1 and that ORF6

protein spatiotemporally interacts with RAE1 to form several ORF6-RAE1 protein complexes involved in reticence of host gene expression. To confirm their interaction, we first generated a fluorescent GFP-ORF6 construct and transfected it into HEK293T cells, using GFP vector as control (Fig. 1A ).

Immunoprecipitation analysis of HEK293T cells co-transfected with GFP-ORF6 and FLAG-RAE1 suggested that ORF6 unambiguously interacts with RAE1, but not other nucleoporins such as NUP153 or TPR (Fig. 1B) . These results are consistent with recent IP-MS and BioID-based interactome data [3, 5] . To examine RAE1 localization in ORF6-overexpressing cells, we performed confocal microscopy. As shown in Fig. 1C -D, ORF6 overexpression induced marked displacement of RAE1 from the nuclear rim to the cytoplasm in HEK293T cells and the human lung carcinoma cell line PC9 (Fig. 1E-F) .

These results suggest that disturbance of mRNA nucleocytoplasmic transport proteins (Fig. 2E) . Further double staining with Lamin B1, a nuclear membrane marker, indicated that ORF6 overexpression had no effect on nuclear rim staining, as it retained its usual punctuate distribution (Fig. 2F) . These results provide cellular evidence that ORF6 expression alone is sufficient to disturb the nucleocytoplasmic mRNA transport factor RAE1 and NUP98, contributing to mislocalization of host endogenous mRNA export machinery and ultimately driving nuclear size reduction.

Export of mRNAs is a multistep process involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through NPCs, and mRNP remodeling events prior to translation [22] . Thus, we next investigated hnRNPA1 expression and localization. Normally, hnRNPA1 is bound to RNAs during their processing in the nucleus, as well as some cytoplasmic mRNAs. hnRNPA1-containing mRNPs are thought to chaperone mRNAs through the NPC, which undergoes dynamic changes in structure and composition during transport. Importantly, relocalization of hnRNPA1 following VSV infection was previously correlated with inhibition of gene expression by VSV, and was found to be dependent on the host factor RAE1 [22] .

We next sought to determine whether ORF6 spatiotemporally regulated hnRNPA1 subcellular distribution by manipulating RAE1 localization. HEK293T cells transfected with GFP-ORF6 were labeled with an antibody to hnRNPA1 protein and stained with DAPI, which labels DNA to define the nuclear compartment. In Figure 3A , mock-transfected GFP vector control samples are shown in the top row for each protein, while GFP-ORF6-transfected cells are shown in the bottom row. In control cells, the signal for hnRNPA1 was coincident with DNA staining, indicating that these proteins were in the nucleus. We found that overexpression of SARS-CoV-2 ORF6 alone did not redistribute hnRNPA1 to the cytoplasm. Interestingly, the density of hnRNPA1 was significantly increased and consistently remained inside the nuclear region of GFP-ORF6-transfected cells. This result also suggests that ORF6 overexpression induced nuclear accumulation of hnRNPA1 (Fig. 3A) . Moreover, if the imaging results described above are correct, overexpression of RAE1 siRNA would partially mimick the GFP-ORF6 phenotype, including reappearance of abnormal hnRNPA1 nuclear accumulation. Indeed, confocal microscopy ( Fig. 3B ) and biochemical fractionation assay (Fig. 3C-D) of siRNA-mediated J o u r n a l P r e -p r o o f RAE1-knockdown HEK293T cells revealed that hnRNPA1 protein levels were largely unaltered and hnRNPA1 was consistently accumulated inside nuclei, respectively.

would partially relieve hnRNPA1 stacking through sequestration of endogenous nuclear hnRNPA1. Consistent with our prediction, we found that hnRNPA1 nuclear accumulation was partially relieved by hnRNPA1 nuclear stacking (Fig. 3C-D) , and inhibition of cell proliferation was also partially rescued (Supplemental Fig. 2) . Thus, a highly plausible interpretation of these results is that FLAG-RAE1 binds to and sequesters endogenous hnRNPA1.

J o u r n a l P r e -p r o o f

Here, we provide critical insights supporting the notion that SARS-CoV-2 ORF6 could be important for virus replication via dislocation of nuclear pore proteins RAE1 and NUP98.

Our results delineate a cascade of molecular and cellular events initiated by the inflow of ORF6 into cells. We speculate that SARS-CoV-2 ORF6 hijacks and subverts the host [30, 31] . It will be useful to visualize ORF6 and NPC protein dynamics in real time in the near future.

SARS-CoV2 encodes several interferon antagonists that delay host cell recognition of infection, innate immune sensing, and signaling pathways, as well as interferon-stimulated gene expression [32] . ORF6 also reportedly has critical roles in host innate immunity [33, 34] . Li et al. screened the viral proteins of SARS-CoV-2 and found that viral ORF6, ORF8, and nucleocapsid proteins were potential inhibitors of the type I interferon signaling pathway, a key component for antiviral responses of host innate immunity. ORF6 strongly inhibited interferon beta (IFN-β) and nuclear factor κB-responsive promoters, and was able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus; notably, only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with IFN-β [35] . Lei et al. showed that ORF6 hinders both type I interferon production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect [36] .

To increase viral replication and transmission between hosts, many pathogenic RNA viruses encode proteins that specifically antagonize nuclear import to inhibit host innate immunity and other critical cellular macromolecular processes [37] . For instance, some cardiovirus L proteins directly interact with Ran-GTPases, which are required for the export of new nuclear mRNA [38] . The Nipah virus W protein prevents both virus-and Toll-like receptor 3-triggered signaling in infected cells by inhibiting phosphorylation and J o u r n a l P r e -p r o o f activation of STAT1, as well as subsequent downstream interferon-stimulated gene induction [39] . Similarly, Ebola virus VP24 binds karyopherin α1 and blocks STAT1 nuclear import [40] . However, whether inhibition of host innate immunity by ORF6 is related to RAE1 and NUP98 nuclear displacement and nuclear size reduction remains to be investigated.

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. 

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We thank all members of the Wong laboratory. We also thank Edanz Group