key: cord-0869303-j285wwv8
authors: Khatun, Oyahida; Sharma, Mansi; Narayan, Rohan; Tripathi, Shashank
title: ORF6 protein of SARS-CoV-2 inhibits TRIM25 mediated RIG-I ubiquitination to mitigate type I IFN induction
date: 2022-05-06
journal: bioRxiv
DOI: 10.1101/2022.05.05.490850
sha: 1aad82b2974057e468190d26591312f4cee3ff80
doc_id: 869303
cord_uid: j285wwv8

Evasion and antagonism of host cellular immunity upon SARS-CoV-2 infection confers a profound replication advantage on the virus and contributes to COVID-19 pathogenesis. We explored the ability of different SARS-CoV-2 proteins to antagonize the host innate immune system and found that the ORF6 protein mitigated type-I IFN (interferon) induction and downstream IFN signaling. Our findings also corroborated previous reports that ORF6 blocks the nuclear import of IRF3 and STAT1 to inhibit IFN induction and signaling. Here we show that ORF6 directly interacts with RIG-I and blocks downstream type-I IFN induction and signaling by inhibiting K-63 linked ubiquitination of RIG-I by the E3 Ligase TRIM25. This involves ORF6-mediated targeting of TRIM25 for degradation, also observed during SARS-CoV-2 infection. The type-I IFN antagonistic activity of ORF6 was mapped to its C-terminal cytoplasmic tail, specifically to amino acid residues 52-61. Overall, we provide new insights into how the SARS-CoV-2 ORF6 protein inhibits type I-IFN induction and signaling through distinct mechanisms.

While the COVID-19 pandemic continues for the third year, its aetiological agent SARS- 33 CoV-2 stays in the focus of intense scientific investigation by researchers worldwide. 34 Tremendous progress has been made on the front of vaccine and antiviral development 35 of innate and adaptive immunity (Darnell et al, 1994) . Viruses have evolved a plethora of 23 mechanisms to inhibit IFN induction and subsequent signaling events to dampen host 24 immunity (Garcia-Sastre, 2017), and SARS-CoV-2 is no exception (Lei et al, 2020) , (Xia et (Li et al., 2021) ). 28 In this study, we screened the IFN-antagonistic ability of SARS-CoV-2 proteins and found 29 ORF6, among others, to be the most potent inhibitor of both IFN induction and signaling. We 30 mapped these activities to the cytoplasmic tail of ORF6, specifically the residues 52-61, 31 which are highly conserved. Our data were coherent with earlier studies where ORF6 was 32 shown to inhibit IFN response by blocking the nuclear import of key transcription factors 33 involved in IFN response. However, these are downstream events, and the molecular basis of 1 the highly efficient shutdown of IFN induction by SARS-CoV-2 ORF6 protein was not clear. 2 We show that ORF6 directly interacts with RIG-I and blocks its ubiquitination by E3 ligase 3 TRIM25. More specifically, the K63-linked ubiquitination of RIG-I, required for its stability 4 and activation, is blocked by SARS-CoV-2 ORF6 protein. SARS-CoV-2 infection and ORF6 5 expression alone lead to TRIM25 degradation, which was reversed upon inhibition of 6 proteasomes and autophagy. Overall, our data shed information on the molecular mechanisms 7 of IFN-antagonism mediated by SARS-CoV-2 ORF-6 protein. Multiple SARS-CoV-2 proteins antagonize Type-I IFN induction and signaling. 10 SARS-CoV-2 has a 29.7 kb genome, 2/3 rd of which from the 5' end encodes ORF1a/1b, 11 which in turn produces 16 NSPs after proteolytic processing of polyprotein 1a and 1ab (pp1a 12 and pp1ab); the 3' end of the genome encodes multiple sub-genomic RNAs that encode 4 13 structural proteins [Spike (S); Envelope (E); Membrane (M); and Nucleocapsid (N)] and at 14 least 9 accessory proteins (ORF3a; 3b; 6; 7a; 7b; 8; 9b; 9c and 10) (Wu et al, 2020a) , 15 (V'Kovski et al, 2021) . To identify the SARS-CoV-2 proteins which may interfere with type- 16 I IFN induction, plasmids expressing SARS-CoV-2 proteins were co-transfected with an 17 interferon-beta (IFNβ) promoter-driven Firefly luciferase reporter plasmid (pIFNβ-FFLuc), 18 3), and a control plasmid constitutively expressing renilla luciferase gene (pRL-TK). The 19 cells were treated with poly (I:C) to stimulate the type-I IFN induction pathway, and relative 20 luciferase units were calculated. We observed that 4 proteins, namely NSP1, NSP13, NSP14, 21 and ORF6, reduced IFN reporter induction to less than 25% of control ( Fig 1A) . Similarly, to 22 identify the SARS-CoV-2 proteins which may interfere with type-I IFN signaling and ISG 23 induction, we co-transfected SARS-CoV-2 plasmids with an ISRE promoter-driven Firefly 24 luciferase reporter plasmid (pIFNβ-FFLuc), 3), along with control plasmid constitutively 25 expressing renilla luciferase gene (pGL4). Cells were treated with universal interferon to 26 stimulate type I-IFN-signalling and ISG induction. Relative Luciferase Units (RLUs) were 27 calculated, and as before, we observed that the previous 4 proteins inhibited ISG induction to 28 less than 25% of control ( Fig 1B) . We then performed western blotting to ensure that these 29 effects on IFN induction and signaling were consistent with SARS-CoV-2 protein expression.

While all constructs were expressed at variable levels, the NSP11, Orf3b, and Orf7b 31 constructs did not produce a detectable band by western blot (Sup Figure 1A) . Apart from the 32 earlier 4 proteins, a few additional proteins also inhibited IFN induction (NSP5) or signaling 1 (NSP7, NSP9, ORF9b), though less effectively (Sup Fig 1B) . To further substantiate our 2 results, we compiled and compared the data from previous studies where similar reporter-3 based approaches were utilized to screen for IFN antagonists of SARS-CoV-2 ( Fig 1C, D) 4 (Lei et al., 2020) , , (Xia et al., 2020) , (Yuen et al, 2020) , , (Vazquez et al, 2021) , (Hayn et al., 2021) , (Stukalov et al., 2021) . Hence, we 10 decided to further explore the mechanistic aspects of IFN-antagonism by SARS-CoV-2 11 ORF6 protein. 13 through distinct mechanisms. 14 To further confirm the IFN-antagonistic activity of SARS-CoV-2 ORF6 protein, we 15 performed dose-response experiments and found that it can inhibit IFNβ and ISRE promoter- IFNβ induced by RIG-I, MAVS, TBK1, IKK , IRF3-5D, and IRF7-CA to varying degrees,   23 with the most prominent effect seen at the level of RIG-I (Fig 2B-G) . This was confirmed in 24 an experiment where ORF6 inhibited RIG-I 2CARD-induced IFNβ reporter activity in a 25 dose-dependent manner (Sup Fig 2D) . To further investigate this, we examined the 26 interaction of ORF6 with key mediators of IFN induction and signaling, including RIG-I, 27 MAVS, STAT1, and STAT2, and found that ORF6 interacted with all of them except STAT1 28 ( Fig 2H) . Immunofluorescence assays further confirmed this, which showed significant 29 ORF6 colocalization with RIG-I and to a limited extent with MAVS ( Fig 2I, Sup Fig 2E) .

Earlier studies have reported that ORF6 interferes with the nuclear translocation of nuclear translocation of IRF3 and STAT1 (Fig 2J, K) . Overall, these data indicated that 1 ORF6 inhibits both type-I IFN and downstream ISG induction by acting upon different 2 cellular components of these pathways.

The cytoplasmic domain of SARS-CoV-2 ORF6 is critical for Type I-IFN antagonism. 4 SARS-CoV-2 ORF6 protein is a 61 amino acid long accessory protein (Yuen et al., 2020) .

Its ortholog is present in SARS-CoV but absent in the MERS virus (Chan et al, 2020) . The C- 6 terminus of the SARS-CoV ORF6 is critical for the innate immune antagonism (Frieman et 7 al, 2007) . We compared the IFN antagonistic activity of ORF6 proteins from SARS-CoV-2 8 and SARS-CoV and found them to be similar (Sup Fig 3A) . SARS-CoV-2 ORF6 shares a 9 69% sequence similarity with its SARS CoV counterpart (Yuen et al., 2020) . Sequence 10 alignment of SARS-CoV-2 with other closely related coronavirus showed that N-terminus 11 cytoplasmic part (M1-Q8) and C-terminus cytoplasmic part (N47-D61) residues are relatively 12 conserved (Sup Fig 3C) . This prompted us to map the domains and amino acid residue of Cyto) of the protein ( Fig 3A) . Next, we tested the ability of the deletion construct to inhibit 20 IFN and ISG induction using the Luciferase reporter assay. We observed that ΔC constructs 21 lost the ability to inhibit IFN induction and downstream signaling (Fig 3B, C) . When 22 compared, ΔN was still effective in inhibiting IFN induction, but was less effective in 23 restricting ISRE activity than full-length (FL) ORF6 (Fig 3B, C) . The importance of the C 24 terminal domain in inhibiting IFN induction was further validated using RIG-I 2CARD and 25 IRF3-5D as inducers; here also ΔC construct was significantly less effective than full-length 26 ORF6 (Fig 3D, E) . We also tested the effect of ORF6 domain deletions on RIG-I interaction 27 by co-immunoprecipitation and IRF3 nuclear translocation by immunofluorescence. 28 Although we did not see the loss of interaction with RIG-I with ORF6 upon domain deletion, 29 there was overall reduced expression of RIG-I in the presence of ORF6 (Fig 3F) . In western 30 blotting experiments, the C-Cyto construct of ORF6 consisting of only the cytoplasmic tail 31 did not produce a detectable band (Fig 3 F) . Furthermore, while the full length and ΔN ORF6 32 were still effective in restricting IRF3 to the cytoplasm in SeV infected cells, ΔC ORF6 lost 33 6 this ability. (Fig 3G) . Overall, these data established that the C-terminal cytoplasmic domain 1 of the SARS-CoV-2 ORF6 protein is crucial for its ability to restrict both type-I IFN 2 induction and downstream ISG induction.

The amino acids 52-61 of SARS-CoV-2 ORF6 protein are essential for its type-I IFN 4 antagonistic activity.

The SARS-CoV-2 ORF6 protein has a conserved amino acid stretch from 52-61 aa in the C 6 terminal tail, implicated in IFN antagonism ( Fig 4A) (Lei et al., 2020) . To validate that, we So far, we have observed that SARS-CoV-2 protein could very potently inhibit RIG-I 23 mediated type-I IFN induction, and its C terminus tail was necessary for this activity. 24 However, deletion of the cytoplasmic tail had no impact on direct interaction between RIG-I 25 and ORF6. Hence, we speculated that perhaps ORF6 interferes with the post-translational 26 modification of RIG-I, which is important for its activity and stability. RIG-I is known to 27 undergo ubiquitination, which can regulate its activity and stability depending on the nature 28 of the linkage (Rehwinkel & Gack, 2020) . To this end, we examined wild type, K48 and K63 29 linked ubiquitination of RIG-I, in the presence or absence of ORF6 in an 30 immunoprecipitation experiment. We observed that the presence of ORF6 reduced overall 31 ubiquitination of RIG-I by wild-type ubiquitin; however, its inhibitory effect was much more 32 7 prominent against K63-linked ubiquitination than K48 (Fig 5A, B) . We also observed 1 reduced expression of RIG-I, in the K63 Ubiquitin overexpression condition in the presence 2 of ORF6 ( Fig 5B) . Further, we performed a similar experiment to test the ability of SARS-3 CoV-2 ORF6 deletion constructs to interfere with K63-inked RIG-I ubiquitination. We 4 observed that both full length and ΔN ORF6 were effective; however, ΔC ORF6 lost its 5 ability to reduce K63-linked RIG-I ubiquitination (Fig 5C, D) . These data confirmed that the presence of TRIM25. We observed that not only did TRIM25 mediated K63-linked RIG-I 10 ubiquitination reduce in the presence of ORF6, but the overall TRIM25 expression was also 11 drastically reduced (Fig 6 A, B ). This indicated that ORF6 was targeting TRIM25 expression 12 to inhibit its function of RIG-I ubiquitination. Viruses often co-opt cellular proteasome and 13 autophagy machinery to target innate immune signaling mediators for degradation (Choi et 14 al, 2018), (Gao & Luo, 2006) . To test whether the same applied in the case of ORF6 and 15 TRIM25, we examined the effect of proteasome inhibitor (MG132) and Bafilomycin A1 16 (BafA) (autophagy inhibitor) on ORF6 mediated reduction of Trim 25 expression. We 17 observed that full-length ORF6 reduced TRIM25 levels, which were partially rescued by both 18 MG132 and BafA (Fig 6C) . We also observed that ΔC ORF6 was less effective in reducing 19 TRIM25 expression, which is likely responsible for its loss of type-I IFN antagonism. The 20 proteasomal degradation was also observed in the case of SARS-CoV-2 infected cells and 21 was rescued significantly by MG132 treatment (Fig 6D) . Taken together, these data provide a 22 novel mechanism by which SARS-CoV-2 ORF6 inhibits RIG-I mediated type-I IFN 23 induction. study started with experimentally cataloging the SARS-CoV-2 proteins that either inhibit 5 type-I IFN induction or downstream signaling to produce ISGs or inhibit both. We observed 6 that 4 SARS-CoV-2 proteins (NSP1, NSP13, NSP14, and ORF6) were able to potently 7 inhibit both type-I IFN induction and signaling (Fig 1A, B) , which was in accordance with 8 multiple independent studies (Fig 1C, D) . The NSP1 protein has been shown to directly 9 interact with ribosomes and inflict general cellular mRNA translation shutdown. This also 10 results in inhibiting the production of IFN and ISGs during viral infection (Thoms et al, 11 2020). The NSP14 protein has been reported to shut down host translation, whereas NSP13 12 hijacks deubiquitinase USP13 to restrict IFN induction (Hsu et al, 2021) ; (Guo et al, 2021) . 13 In our experiments, the ORF6 protein was most effective in inhibiting both type-I IFN 14 induction and signaling. Several other research groups have also reported such activity of 15 SARS-CoV-2 ORF6. The mechanism of SARS-CoV-2 ORF6 activity has been mapped to 16 inhibition of nuclear import of crucial transcription factors (STAT1, IRF3) required for IFN 17 response. It does so by associating with the nuclear import co-factor Karyopherin alpha and In our study, ORF6 was found to exert a strong inhibitory action on RIG-I 2CARD 23 mediated type-I IFN induction, which is a very early step of RLR signaling upstream of 24 nuclear translocation of IRF3 or expression of ISGs. Direct action of ORF6 on RIG-I was not 25 studied before; hence we decided to explore this in more detail. We found that ORF6 directly 26 interacts with RIG-I and leads to its reduced expression. The C-terminal cytoplasmic tail of 27 ORF6 has been reported to be essential for its IFN antagonism (Lei et al., 2020) . We tested 28 the importance of the same in inhibition of RIG-I function and found that the C-terminal 29 region, especially residues 52-61 of ORF6, were crucial for restricting RIG-I mediated IFN York) and have been described before (Versteeg et al, 2013) . For constructing the deletion 32 mutants, the desired sequence was PCR amplified from pLVX-EF1alpha-SARS-CoV-2-1 ORF6-2xStrep-IRES-Puro plasmid, followed by cloning in the pCAGGS backbone using 2 EcoRI (ER0271, Thermo scientific) and XhoI (ER0691, Thermo scientific) restriction 3 enzyme including the full-length ORF6. Delta C plasmid was constructed using overlap 4 extension PCR. ORF6 variant mutants were constructed by subcloning the construct in a TA 5 backbone, followed by substituting the residues as described before (Edelheit et al, 2009 ) and 6 cloning in the pCAGGS backbone using EcoRI and XhoI. All numerical data of luciferase assays and qRt-PCR were analysed and plotted using 4 GraphPad Prism v8.0.2. Statistical significance was calculated using t-test with Bonferroni 5 corrections for multiple comparisons wherever necessary). The P values were indicated as *P 6 < 0.05; **P < 0.01; ***P < 0.001; ns = not significant. Error bars represent mean + standard 7 error. The model diagram of ORF6 action (Fig 7) and 3-D structure (Sup Fig 3C) were made 

ST conceived the study, designed the experiments, analyzed the data, and wrote the draft. 20 OK, MS and RN performed the experiments, analyzed the data, and revised the draft.

The authors declare no competing interests. 29 14 A. The graph depicts the quantification of the IFNβ dual luciferase assay. HEK293T cells 1 were co-transfected with IFNβ promoter-driven Firefly luciferase reporter plasmid, Renilla 2 luciferase reporter plasmid, and viral protein-expressing plasmid or empty vector. 24 hours 3 post-transfection, cells were induced with poly I: C, followed by assaying the cells for dual 4 luciferase activity (n=4). B. Quantification of ISRE dual luciferase assay from HEK293T 5 cells which were co-transfected with ISRE promoter-driven Firefly luciferase reporter 6 plasmid, Renilla luciferase reporter plasmid, and viral protein-expressing plasmid or empty 7 vector, followed by induction with Universal type I IFN (1000U/ml) for 12 hours (n=4). C-D. 8 Heatmap of studies that have tested the role of multiple SARS-CoV-2 proteins on IFN 9 induction (C) and ISRE induction (D) pathway using Luciferase assay. Here red and blue 10 color depicts the protein which antagonizes IFN and ISRE induction, respectively. Statistical 11 significance of the data is represented as *P < 0.05; **P < 0.01; ***P < 0.001; ns: not 12 significant. Error bars represent mean + standard error. universal IFN (1000 U/ml) for 1 hour, followed by fixation and staining. Scale bar 20 μM. 31 Statistical significance of the data is represented as *P < 0.05; **P < 0.01; ***P < 0.001; ns: 32 not significant. Error bars represent mean + standard error. 24 Quantification of ISRE promoter activation of ORF6 variants. The principle of the assay is 25 previously described in Fig 1B. Statistical significance of the data is represented as *P < 0.05; 26 **P < 0.01; ***P < 0.001; ns: not significant. Error bars represent mean + standard error. 

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