Phase-separated nucleocapsid protein of SARS-CoV-2 suppresses cGAS-DNA recognition by disrupting cGAS-G3BP1 complex.

Currently, the incidence and fatality rate of SARS-CoV-2 remain continually high worldwide. COVID-19 patients infected with SARS-CoV-2 exhibited decreased type I interferon (IFN-I) signal, along with limited activation of antiviral immune responses as well as enhanced viral infectivity. Dramatic progresses have been made in revealing the multiple strategies employed by SARS-CoV-2 in impairing canonical RNA sensing pathways. However, it remains to be determined about the SARS-CoV-2 antagonism of cGAS-mediated activation of IFN responses during infection. In the current study, we figure out that SARS-CoV-2 infection leads to the accumulation of released mitochondria DNA (mtDNA), which in turn triggers cGAS to activate IFN-I signaling. As countermeasures, SARS-CoV-2 nucleocapsid (N) protein restricts the DNA recognition capacity of cGAS to impair cGAS-induced IFN-I signaling. Mechanically, N protein disrupts the assembly of cGAS with its co-factor G3BP1 by undergoing DNA-induced liquid-liquid phase separation (LLPS), subsequently impairs the double-strand DNA (dsDNA) detection ability of cGAS. Taken together, our findings unravel a novel antagonistic strategy by which SARS-CoV-2 reduces DNA-triggered IFN-I pathway through interfering with cGAS-DNA phase separation.

Modulation of NBAS-Related Functions in the Early Response to SARS-CoV-2 Infection.

Upon infection, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is predicted to interact with diverse cellular functions, such as the nonsense-mediated decay (NMD) pathway, as suggested by the identification of the core NMD factor upframeshift-1 (UPF1) in the SARS-CoV-2 interactome, and the retrograde transport from the Golgi to the endoplasmic reticulum (ER) through the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where coronavirus assembly occurs. Here, we investigated the expression and localization of the neuroblastoma-amplified sequence (NBAS) protein, a UPF1 partner for the NMD at the ER, participating also in retrograde transport, and of its functional partners, at early time points after SARS-CoV-2 infection of the human lung epithelial cell line Calu3. We found a significant decrease of DExH-Box Helicase 34 (DHX34), suppressor with morphogenetic effect on genitalia 5 (SMG5), and SMG7 expression at 6 h post-infection, followed by a significant increase of these genes and also UPF1 and UPF2 at 9 h post-infection. Conversely, NBAS and other genes coding for NMD factors were not modulated. Known NMD substrates related to cell stress (Growth Arrest Specific 5, GAS5; transducin beta-like 2, TBL2; and DNA damage-inducible transcript 3, DDIT3) were increased in infected cells, possibly as a result of alterations in the NMD pathway and of a direct effect of the infection. We also found that the expression of unconventional SNARE in the ER 1, USE1 (p31) and Zeste White 10 homolog, ZW10, partners of NBAS in the retrograde transport function, significantly increased over time in infected cells. Co-localization of NBAS and UPF1 proteins did not change within 24 h of infection nor did it differ in infected versus non-infected cells at 1 and 24 h after infection; similarly, the co-localization of NBAS and p31 proteins was not altered by infection in this short time frame. Finally, both NBAS and UPF1 were found to co-localize with SARS-CoV-2 S and N proteins. Overall, these data are preliminary evidence of an interaction between NBAS and NBAS-related functions and SARS-CoV-2 in infected cells, deserving further investigation.

Coronavirus RNA-dependent RNA polymerase interacts with the p50 regulatory subunit of host DNA polymerase delta and plays a synergistic role with RNA helicase in the induction of DNA damage response and cell cycle arrest in the S phase.

Disruption of the cell cycle is a common strategy shared by many viruses to create a conducible cellular microenvironment for their efficient replication. We have previously shown that infection of cells with gammacoronavirus infectious bronchitis virus (IBV) activated the theataxia-telangiectasia mutated (ATM) Rad3-related (ATR)/checkpoint kinase 1 (Chk1) pathway and induced cell cycle arrest in S and G2/M phases, partially through the interaction of nonstructural protein 13 (nsp13) with the p125 catalytic subunit of DNA polymerase delta (pol δ). In this study, we show, by GST pulldown, co-immunoprecipitation and immunofluorescent staining, that IBV nsp12 directly interacts with the p50 regulatory subunit of pol δ in vitro and in cells overexpressing the two proteins as well as in cells infected with a recombinant IBV harbouring an HA-tagged nsp12. Furthermore, nsp12 from severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 was also able to interact with p50. These interactions play a synergistic role with nsp13 in the induction of S phase arrest. The fact that subunits of an essential cellular DNA replication machinery physically associate with two core replication enzymes from three different coronaviruses highlights the importance of these associations in coronavirus replication and virus-host interaction, and reveals the potential of targeting these subunits for antiviral intervention.

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation.

Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus-infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the pandemic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and SG formation induced by exogenous stress. Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2α phosphorylation, and the Nsp1 of SARS-CoV2 alone was sufficient to cause a decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear accumulation of the SG-nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in virus replication compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.

The stalk domain of SARS-CoV-2 NSP13 is essential for its helicase activity.

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.

Host Cellular RNA Helicases Regulate SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the largest RNA genome, approximately 30 kb, among RNA viruses. The DDX DEAD box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such a large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. In addition, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX1, DDX3, DDX5, DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to carry out viral replication. Conversely, DDX21 and MOV10 restricted SARS-CoV-2 infection through an interaction of SARS-CoV-2 N with host cellular RNA helicases. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has a large RNA genome, of approximately 30 kb. To regulate and maintain such a large viral RNA genome, host RNA helicases may be involved in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5, and DDX6 are required for SARS-CoV-2 infection. Interestingly, SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and by suppressing the host innate immune system.

The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

Architecture of a SARS-CoV-2 mini replication and transcription complex.

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.

Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase.

The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but nonstructural protein 13 (nsp13) stands out as a highly conserved and yet understudied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP-dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound, which presents a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 µs of a Gaussian-accelerated molecular dynamics simulation (at least 6 µs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A Gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key structural states of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inchworm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV, and V and suggest a power stroke-like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.

ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads.

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as "non-positive" (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses.

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA Helicase Activities That Can Be Inhibited by Bismuth Salts.

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.

COVID-19, cilia, and smell.

The novel coronavirus SARS-CoV-2 is the causative agent of the global coronavirus disease 2019 (COVID-19) outbreak. In addition to pneumonia, other COVID-19-associated symptoms have been reported, including loss of smell (anosmia). However, the connection between infection with coronavirus and anosmia remains enigmatic. It has been reported that defects in olfactory cilia lead to anosmia. In this Viewpoint, we summarize transmission electron microscopic studies of cilia in virus-infected cells. In the human nasal epithelium, coronavirus infects the ciliated cells and causes deciliation. Research has shown that viruses such as influenza and Sendai attach to the ciliary membrane. The Sendai virus enters cilia by fusing its viral membrane with the ciliary membrane. A recent study on SARS-CoV-2-human protein-protein interactions revealed that the viral nonstructural protein Nsp13 interacts with the centrosome components, providing a potential molecular link. The mucociliary escalator removes inhaled pathogenic particles and functions as the first line of protection mechanism against viral infection in the human airway. Thus, future investigation into the virus-cilium interface will help further the battle against COVID-19.

Global analysis of more than 50,000 SARS-CoV-2 genomes reveals epistasis between eight viral genes.

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.

Role of Chikungunya nsP3 in Regulating G3BP1 Activity, Stress Granule Formation and Drug Efficacy.

BACKGROUND: Ras-GTPase activating protein SH3-domain-binding proteins (G3BP) are a small family of RNA-binding proteins implicated in regulating gene expression. Changes in expression of G3BPs are correlated to several cancers including thyroid, colon, pancreatic and breast cancer. G3BPs are important regulators of stress granule (SG) formation and function. SG are ribonucleoprotein (RNP) particles that respond to cellular stresses to triage mRNA resulting in transcripts being selectively degraded, stored or translated resulting in a change of gene expression which confers a survival response to the cell. These changes in gene expression contribute to the development of drug resistance. Many RNA viruses, including Chikungunya (and potentially Coronavirus), dismantle SG so that the cell cannot respond to the viral infection. Non-structural protein 3 (nsP3), from the Chikungunya virus, has been shown to translocate G3BP away from SG. Interestingly in cancer cells, the formation of SG is correlated to drug-resistance and blocking SG formation has been shown to reestablish the efficacy of the anticancer drug bortezomib. METHODS: Chikungunya nsP3 was transfected into breast cancer cell lines T47D and MCF7 to disrupt SG formation. Changes in the cytotoxicity of bortezomib were measured. RESULTS: Bortezomib cytotoxicity in breast cancer cell lines changed with a 22 fold decrease in its IC50 for T47D and a 7 fold decrease for MCF7 cells. CONCLUSIONS: Chikungunya nsP3 disrupts SG formation. As a result, it increases the cytotoxicity of the FDA approved drug, bortezomib. In addition, the increased cytotoxicity appears to correlate to improved bortezomib selectivity when compared to control cell lines.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Evaluation of the potency of FDA-approved drugs on wild type and mutant SARS-CoV-2 helicase (Nsp13).

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.

SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists.

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.