key: cord-0829639-lrjrx0n2
authors: Xu, Duo; Biswal, Mahamaya; Neal, Arrmund; Hai, Rong
title: Review Devil's tools: SARS-CoV-2 antagonists against innate immunity
date: 2021-11-18
journal: Curr Res Virol Sci
DOI: 10.1016/j.crviro.2021.100013
sha: ed3597c724ba097ba7e8fa355f240c3ed9d0abb8
doc_id: 829639
cord_uid: lrjrx0n2

The unprecedented Coronavirus pandemic of 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Like other coronaviruses, to establish its infection, SARS-CoV-2 is required to overcome the innate interferon (IFN) response, which is the first line of host defense. SARS-CoV-2 has also developed complex antagonism approaches involving almost all its encoding viral proteins. Here, we summarize our current understanding of these different viral factors and their roles in suppressing IFN responses. Some of them are conserved IFN evasion strategies used by SARS-CoV; others are novel countermeasures only employed by SARS-CoV-2. The filling of gaps in understanding these underlying mechanisms will provide rationale guidance for applying IFN treatment against SARS-CoV-2 infection.

1 NSP1 (PDB: 7K7P) is a known potent IFN suppressor in other coronaviruses (16). In SARS-CoV,NSP1 has been 2 shown to inhibit IFN-I production through inhibiting expression of host genes (likely due to mRNA degradation 3 and/or direct translation inhibition) and reducing the phosphorylation of STAT1 (17) (18) (19) . Since SARS-CoV-2 4 NSP1 is over 80% amino acid sequence identical to its SARS-CoV counterpart, it is not surprising to see that 5

SARS-CoV-2 NSP1 can also inhibit . It can inhibit host gene expression through 6 blocking the mRNA export machinery (21, 22) or by directly binding to the 40S ribosomal subunit (23). Nsp1-7

bound 40S ribosomal complexes have been revealed by cryo-electron microscopy (cryo-EM) (23). Intriguingly, 8 SARS-CoV-2 NSP1 uses additional approaches to antagonize host IFN-I responses. It can prevent IFN induction 9 through blocking IRF3 phosphorylation, and lessen interferon-stimulated gene (ISG) induction by triggering the 10 depletion of TYK2 and STAT2, components of the IFN-I signaling pathway (24). 11 12 NSP3 13 NSP3 is the largest coronaviral protein (25, 26) with 3 three tandem MacroD-like macrodomains (Mac1, Mac2, 14 and Mac3). NSP3 binds to and removes ADP-ribose from proteins in a dynamic posttranslational process (25) . 15

This putative ADP-ribosylation function was further supported by the crystal structure of the Mac1 domain (PDB: 16 6WOJ) (25). Additionally, NSP3 was shown to be involved with the disruption of innate host immunity through 17 multiple functions, such as its papain-like protease (PLpro) being required for the separation of . It 18 also contains a potential deubiquitinase (DUB) domain based on sequence analysis, which suggests its role in 19

deubiquitinating host polyubiquitinated proteins (27, 28) . Experimental evidence has been obtained to show that 20 SARS-CoV-2 NSP3 inhibits IFN-I production through 1) cleaving the ubiquitin-like protein, ISG15, 2) decreasing 21 IRF3 phosphorylation, or 3) directly cleaving IRF3 (29-31 Both SARS-CoV-2 NSP14 and NSP15 significantly suppress the IFN-β promoter driven luciferase expression 47

and 40) . NSP14 contains two domains, 3'-to-5' exoribonuclease (ExoN) and guanine-N7-48 methyltransferase (N7-MTase). ExoN is required to maintain a certain degree of fidelity, and N7-MTase is 49 involved in mRNA capping (43). Further evidence was provided in a recent crystal structure of the ExoN region 50

(44). The conformation reveals that its ExoN (PDB: 7DIY) maintains a complete exoribonuclease fold and an 51 active configuration in the catalytic center. NSP14 was also shown to suppress host protein synthesis (45). 52

NSP15 is a uridine-specific endoribonuclease with a C-terminal EndoU like catalytic domain, conserved among 53

coronaviruses. Its nucleotide-bound crystal structure (PDB: 6X4I) suggests a second base binding site, which 54 J o u r n a l P r e -p r o o f can accommodate any other base (46). Even though endoribonuclease activity seems to be responsible for the 1 interference with the innate immune response, current evidence suggests that NSP15 is likely to suppress IFN-2 I induction through binding to RNF41, which is an E3 ligase involved in activation of IRF3 (41). Further studies 3 are still required to understand the molecular mechanisms underlying their suppression of IFN. 4 5 Accessory Proteins 6

Some of the eight SARS-CoV-2 accessory proteins have also been shown to be involved in modulating IFN 7 responses. 8 9

ORF3a 10 Along with two other viral proteins, E and ORF8a, ORF3a is a SARS-CoV-2 putative ion channel, which is located 11

at the plasma membrane of the ER and Golgi (47, 48 ORF3b is a viral protein, that travels between nucleus and cytoplasm during infection (54). Specifically, it 20

constantly translocates between the nucleus and the outer membrane of mitochondria (54). It is a potent 21 interferon suppressor for both SARS-CoV and SARS-CoV-2 viruses. Even though SARS-CoV-2 is more 22

sensitive to IFN-I treatment compared to SARS-CoV (55), SARS-CoV-2 ORF3b exhibited more efficient inhibition 23 of IFN-I induction than its SARS-CoV counterpart (54). This enhancement of suppression by SARS-CoV-2 24

ORF3b was achieved through inhibiting the nuclear localization of IRF3 (54). Its virulence role is further 25 supported by two natural variants, which encoded a similar elongated form of ORF3b due to the loss of its original 26 first stop codon (54). The variants were isolated from patients with particularly severe COVID-19 symptoms from 27 a family cluster in Quito, Ecuador (54). They exhibited even more potent inhibition on type I interferon responses 28 compared to the wild type form (54). Interestingly, when fully restoring the SARS-CoV ORF3b-like protein in 29 SARS-CoV-2 virus through reverting those stop codons, there was a decrease in anti-IFN activity (54). This 30

suggests that the C-terminal region of ORF3b has a negative impact on its immune suppression activity. To 31 better understand the underlying mechanism, structural information is needed. 32 33 ORF6 34 SARS-CoV-2 ORF6 is located at the nuclear pore through binding to Nup98-Rae1 complex (56, 57). This results 35

in blocking nuclear translocation of STAT1 and STAT2 causing compromised induction of ISGs (57). This 36 conclusion was further supported by the observation that there is no ISG inhibition when ORF6 was mutated to 37 prevent its binding to the nuclear pore complex (57). Through blocking of the nuclear pore, ORF6 can also cause 38 nuclear retention of host mRNAs, resulting in a reduction in expression of host genes (56) SARS-CoV-2 ORF8 is a secreted protein, containing an N-terminal signal sequence followed by an Ig-like 50 structure revealed in its crystal structure (PDB: 7JTL) (61, 62). Its N-terminal signal sequence guides its entry 51 into the lumen of the ER. There, SARS-CoV-2 ORF8 interacts with several host proteins, some of which have 52 been shown to be involved in ER-associated degradation. Besides its inhibitory role in suppressing IFN-β 53 production and signaling (63, 64), SARS-CoV-2 ORF8 was shown to down-regulate MHC-Ι and protect virus-54 infected cells against Cytotoxic T lymphocytes (CTLs) (65), even though the underlying mechanism remains 1 elusive. 2 3

ORF9b 4

Among the current known coronaviruses, ORF9b is only present in both SARS-CoV-2 and SARS-CoV, sized at 5 about 98-amino acid long, and encoded by an alternative reading frame within the N gene (66). It suppresses 6 IFN-I responses through targeting mitochondria adapter TOM70 (67, 68), which is further supported by the 7 crystal structure of ORF9b in complex with the cytosolic domain of TOM70 (PDB: 7DHG) (67). Additionally, it 8

can target TRIF or STING to block the activation of types I and III IFNs (69). In the same study, ORF9b was 9

shown to further compromise nuclear translocation of IRF3 by blocking IRF3 phosphorylation. Additionally, more 10

ORF9b-associated factors were revealed, such as RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 (69). It is 11

intriguing that such a small protein has so many binding partners. 12 13

Structural proteins 14

Among the four structure proteins encoded by SARS-CoV-2, two of them, M and N proteins, have also been 15

reported to suppress IFN-I production and signaling, just like their SARS-CoV counterparts. pathway through a different mechanism by inhibiting the phosphorylation of STAT1 and STAT2, resulting in their 30 retention in the cytoplasm (68). It is fascinating that there are such differences in their functions even though the 31 N proteins from both SARS viruses are highly conserved, with only 5 substitutions in their corresponding 32 consensus sequences (39). 33 34

Conclusions: 35 It is crucial to understand how SARS-CoV-2 compromises the host IFN-I responses, and understanding this will 36

shed light on the pathogenesis of SARS-CoV-2. Like other betacoronaviruses, SARS-CoV-2 possesses multiple 37 mechanisms inhibiting various innate immune responses, which results in a failure of fine-tuned innate immune 38 activation. Therefore, the skewed host defense causes enhanced disease severity and mortality rather than 39

protection. This knowledge will also provide a rationale for developing novel therapeutics targeting the distorted 40 immune escape mechanisms of SARS-CoV-2 and their role in compromising infection. Furthermore, studies 41 have already shown that SARS-CoV-2 is sensitive to IFN-I inhibition (55, 75). A more detailed understanding of 42 the interplay between SARS-CoV-2 and the host will warrant a reliable and practical IFN-based medical 43 intervention for COVID-19 treatment, particularly during the early stage of infection (55, 76, 77). 44 45

With the accumulating evidence from more studies on SARS-CoV-2 proteins that antagonize IFN-I response, we 46 are also aware that there are discrepancies for some of these factors. For example, some reports found that 47 NSP12 inhibits the IFN-I response, whereas some did not (32, 36, 40, 78, 79) . The differences might be caused 48 by the different experimental conditions used in these studies. Therefore, it is important to validate these results 49 in the context of viral replication. Using reverse genetic systems, recombinant viruses should be generated to 50 carry the specific mutations and examined for their abilities to suppress IFN-I signaling. These discrepancies 51 also reflect that we are still at the early stages of understanding the underlying mechanisms of host-virus 52

interactions during SARS-CoV-2 infection. Follow-up research is still required to reveal the authentic picture of 53 this virus-host arms race. It is even more important to identify mutations located in the domains of the 54 J o u r n a l P r e -p r o o f corresponding viral IFN antagonists so that we will be better prepared for the ongoing emergence of SARS-CoV- 

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J o u r n a l P r e -p r o o f Highlight:The innate interferon (IFN) response is our first lines of defense to contain viral infection. Evidence has shown that SARS-CoV-2 is more sensitive to IFN-I inhibition compared to SARS-CoV. However, studies have also revealed that SARS-CoV-2 devoted multiple viral factors to build up a complex system to inhibit host IFN responses. Therefore, the understanding will have direct clinical implication which will provide rationale base to treat SARS-CoV-2 infection at the early stage.

Declaration of interests ☒ 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:The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work rep J o u r n a l P r e -p r o o f