Factor Xa cleaves SARS-CoV-2 spike protein to block viral entry and infection.

Serine proteases (SP), including furin, trypsin, and TMPRSS2 cleave the SARS-CoV-2 spike (S) protein, enabling the virus to enter cells. Here, we show that factor (F) Xa, an SP involved in blood coagulation, is upregulated in COVID-19 patients. In contrast to other SPs, FXa exerts antiviral activity. Mechanistically, FXa cleaves S protein, preventing its binding to ACE2, and thus blocking viral entry and infection. However, FXa is less effective against variants carrying the D614G mutation common in all pandemic variants. The anticoagulant rivaroxaban, a direct FXa inhibitor, inhibits FXa-mediated S protein cleavage and facilitates viral entry, whereas the indirect FXa inhibitor fondaparinux does not. In the lethal SARS-CoV-2 K18-hACE2 model, FXa prolongs survival yet its combination with rivaroxaban but not fondaparinux abrogates that protection. These results identify both a previously unknown function for FXa and an associated antiviral host defense mechanism against SARS-CoV-2 and suggest caution in considering direct FXa inhibitors for preventing or treating thrombotic complications in COVID-19 patients.

Potent NKT cell ligands overcome SARS-CoV-2 immune evasion to mitigate viral pathogenesis in mouse models.

One of the major pathogenesis mechanisms of SARS-CoV-2 is its potent suppression of innate immunity, including blocking the production of type I interferons. However, it is unknown whether and how the virus interacts with different innate-like T cells, including NKT, MAIT and γδ T cells. Here we reported that upon SARS-CoV-2 infection, invariant NKT (iNKT) cells rapidly trafficked to infected lung tissues from the periphery. We discovered that the envelope (E) protein of SARS-CoV-2 efficiently down-regulated the cell surface expression of the antigen-presenting molecule, CD1d, to suppress the function of iNKT cells. E protein is a small membrane protein and a viroporin that plays important roles in virion packaging and envelopment during viral morphogenesis. We showed that the transmembrane domain of E protein was responsible for suppressing CD1d expression by specifically reducing the level of mature, post-ER forms of CD1d, suggesting that it suppressed the trafficking of CD1d proteins and led to their degradation. Point mutations demonstrated that the putative ion channel function was required for suppression of CD1d expression and inhibition of the ion channel function using small chemicals rescued the CD1d expression. Importantly, we discovered that among seven human coronaviruses, only E proteins from highly pathogenic coronaviruses including SARS-CoV-2, SARS-CoV and MERS suppressed CD1d expression, whereas the E proteins of human common cold coronaviruses, HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1, did not. These results suggested that E protein-mediated evasion of NKT cell function was likely an important pathogenesis factor, enhancing the virulence of these highly pathogenic coronaviruses. Remarkably, activation of iNKT cells with their glycolipid ligands, both prophylactically and therapeutically, overcame the putative viral immune evasion, significantly mitigated viral pathogenesis and improved host survival in mice. Our results suggested a novel NKT cell-based anti-SARS-CoV-2 therapeutic approach.

The roles of APOBEC-mediated RNA editing in SARS-CoV-2 mutations, replication and fitness.

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.

SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS To Evade the Innate Immune Response.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

SARS-CoV-2: Mechanism of infection and emerging technologies for future prospects.

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally to over 200 countries with more than 23 million confirmed cases and at least 800,000 fatalities as of 23 August 2020. Declared a pandemic on March 11 by World Health Organization, the disease caused by SARS-CoV-2 infection, called coronavirus disease 2019 (COVID-19), has become a global public health crisis that challenged all national healthcare systems. This review summarized the current knowledge about virologic and pathogenic characteristics of SARS-CoV-2 with emphasis on potential immunomodulatory mechanism and drug development. With multiple emerging technologies and cross-disciplinary approaches proving to be crucial in our global response against COVID-19, the application of PROteolysis TArgeting Chimeras strategy, CRISPR-Cas9 gene editing technology, and Single-Nucleotide-Specific Programmable Riboregulators technology in developing antiviral drugs and detecting infectious diseases are proposed here. We also discussed the available but still limited epidemiology of COVID-19 as well as the ongoing efforts on vaccine development. In brief, we conducted an in-depth analysis of the pathogenesis of SARS-CoV-2 and reviewed the therapeutic options for COVID-19. We also proposed key research directions in the future that may help uncover more underlying molecular mechanisms governing the pathology of COVID-19.