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The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5. The presentation of viral antigens to T cells via the MHC molecules is a critical component of the host response to viral infection. Here the authors suggest SARS-CoV-2 possesses the immune evasion strategy against the MHC class I pathway by targeting key transcriptional regulators.

Efficient immune responses against viral infection are determined by sufficient activation of nucleic acid sensor–mediated innate immunity 1 , 2 . Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains an ongoing global pandemic. It is an urgent challenge to clarify the innate recognition mechanism to control this virus. Here we show that retinoic acid–inducible gene-I (RIG-I) sufficiently restrains SARS-CoV-2 replication in human lung cells in a type I/III interferon (IFN)-independent manner. RIG-I recognizes the 3′ untranslated region of the SARS-CoV-2 RNA genome via the helicase domains, but not the C-terminal domain. This new mode of RIG-I recognition does not stimulate its ATPase, thereby aborting the activation of the conventional mitochondrial antiviral-signaling protein-dependent pathways, which is in accordance with lack of cytokine induction. Nevertheless, the interaction of RIG-I with the viral genome directly abrogates viral RNA-dependent RNA polymerase mediation of the first step of replication. Consistently, genetic ablation of RIG-I allows lung cells to produce viral particles that expressed the viral spike protein. By contrast, the anti-SARS-CoV-2 activity was restored by all- trans retinoic acid treatment through upregulation of RIG-I protein expression in primary lung cells derived from patients with chronic obstructive pulmonary disease. Thus, our findings demonstrate the distinctive role of RIG-I as a restraining factor in the early phase of SARS-CoV-2 infection in human lung cells. RIG-I is a cytosolic nucleic acid sensor triggering type I IFN production. Takaoka and colleagues find that RIG-I recognizes SARS-CoV-2 RNA in a noncanonical manner and fails to activate type I IFN, but it directly restricts viral replication.

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.

Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes host proteases, including a plasma membrane-associated transmembrane protease, serine 2 (TMPRSS2) to cleave and activate the virus spike protein to facilitate cellular entry. Although TMPRSS2 is a well-characterized type II transmembrane serine protease (TTSP), the role of other TTSPs on the replication of SARS-CoV-2 remains to be elucidated. Here, we have screened 12 TTSPs using human angiotensin-converting enzyme 2-expressing HEK293T (293T-ACE2) cells and Vero E6 cells and demonstrated that exogenous expression of TMPRSS11D and TMPRSS13 enhanced cellular uptake and subsequent replication of SARS-CoV-2. In addition, SARS-CoV-1 and SARS-CoV-2 share the same TTSPs in the viral entry process. Our study demonstrates the impact of host TTSPs on infection of SARS-CoV-2, which may have implications for cell and tissue tropism, for pathogenicity, and potentially for vaccine development.

Cetylpyridinium chloride (CPC), a quaternary ammonium compound, which is present in mouthwash, is effective against bacteria, fungi, and enveloped viruses. This study was conducted to explore the antiviral effect of CPC on SARS-CoV-2. There are few reports on the effect of CPC against wild-type SARS-CoV-2 at low concentrations such as 0.001%–0.005% (10–50 µg/mL). Interestingly, we found that low concentrations of CPC suppressed the infectivity of human isolated SARS-CoV-2 strains (Wuhan, Alpha, Beta, and Gamma) even in saliva. Furthermore, we demonstrated that CPC shows anti-SARS-CoV-2 effects without disrupting the virus envelope, using sucrose density analysis and electron microscopic examination. In conclusion, this study provided experimental evidence that CPC may inhibit SARS-CoV-2 infection even at lower concentrations.

Emerging and reemerging tick-borne virus infections caused by orthonairoviruses (family Nairoviridae ), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129 mice) both for a better understanding of the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel orthonairovirus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.

SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection.

Although the SARS-CoV-2 epidemic worldwide has gradually decreased, in some areas, the situation has not yet been stamped and has become a global health emergency. It is quite possible that we could again be threatened by a new coronavirus. Therefore, new nucleotide analog drugs and vaccines or using drug repositioning for SARS-CoV-2 still has been developed, yet their safety and efficacy against COVID‐19 remains underexplored. Malabaricone C is 2,6-dihydroxyphenyl acylphenol found in edible plants such as the mace spice of nutmeg derived from the seeds of Myristica fragrans . In this study, we identified malabaricone C as the first inhibitor of SARS-CoV-2 from natural food with a safe alternative for drugs. Malabaricone C and its chemical derivatives showed EC 50 values of 1–1.5 μM against SARS-CoV-2 (WK-521, ancestral strain) and its variant strains in mammalian cells (HEK293T and Vero E6). In addition, we have successfully established novel evaluation system for the inhibition of SARS virus cell fusion by visualization for providing a versatile tool for study SARS-CoV-2 mediated fusion. Furthermore, our experiments suggested that malabaricone C could affect the distribution of sphingomyelin on the plasma membrane, which involves in viral infections. Thus, in light of the beneficial effect of malabaricone C on viral infection, the nontoxic malabaricone C is a suitable candidate as a drug that can be employed in the treatment and prevention of COVID-19. Moreover, it may potentially be used to treat acute infections of other enveloped viruses.

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains.