SARS-CoV-2 N protein enhances the anti-apoptotic activity of MCL-1 to promote viral replication.

Viral infection in respiratory tract usually leads to cell death, impairing respiratory function to cause severe disease. However, the diversity of clinical manifestations of SARS-CoV-2 infection increases the complexity and difficulty of viral infection prevention, and especially the high-frequency asymptomatic infection increases the risk of virus transmission. Studying how SARS-CoV-2 affects apoptotic pathway may help to understand the pathological process of its infection. Here, we uncovered SARS-CoV-2 imployed a distinct anti-apoptotic mechanism via its N protein. We found SARS-CoV-2 virus-like particles (trVLP) suppressed cell apoptosis, but the trVLP lacking N protein didn't. Further study verified that N protein repressed cell apoptosis in cultured cells, human lung organoids and mice. Mechanistically, N protein specifically interacted with anti-apoptotic protein MCL-1, and recruited a deubiquitinating enzyme USP15 to remove the K63-linked ubiquitination of MCL-1, which stabilized this protein and promoted it to hijack Bak in mitochondria. Importantly, N protein promoted the replications of IAV, DENV and ZIKV, and exacerbated death of IAV-infected mice, all of which could be blocked by a MCL-1 specific inhibitor, S63845. Altogether, we identifed a distinct anti-apoptotic function of the N protein, through which it promoted viral replication. These may explain how SARS-CoV-2 effectively replicates in asymptomatic individuals without cuasing respiratory dysfunction, and indicate a risk of enhanced coinfection with other viruses. We anticipate that abrogating the N/MCL-1-dominated apoptosis repression is conducive to the treatments of SARS-CoV-2 infection as well as coinfections with other viruses.

The roles of critical pro-inflammatory cytokines in the drive of cytokine storm during SARS-CoV-2 infection.

In patients with severe COVID-19, acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS), and even mortality can result from cytokine storm, which is a hyperinflammatory medical condition caused by the excessive and uncontrolled release of pro-inflammatory cytokines. High levels of numerous crucial pro-inflammatory cytokines, such as interleukin-1 (IL-1), IL-2, IL-6, tumor necrosis factor-α, interferon (IFN)-γ, IFN-induced protein 10 kDa, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein-1, and IL-10 and so on, have been found in severe COVID-19. They participate in cascade amplification pathways of pro-inflammatory responses through complex inflammatory networks. Here, we review the involvements of these critical inflammatory cytokines in SARS-CoV-2 infection and discuss their potential roles in triggering or regulating cytokine storm, which can help to understand the pathogenesis of severe COVID-19. So far, there is rarely effective therapeutic strategy for patients with cytokine storm besides using glucocorticoids, which is proved to result in fatal side effects. Clarifying the roles of key involved cytokines in the complex inflammatory network of cytokine storm will help to develop an ideal therapeutic intervention, such as neutralizing antibody of certain cytokine or inhibitor of some inflammatory signal pathways.

A vaccine delivery system promotes strong immune responses against SARS-CoV-2 variants.

Global coronavirus disease 2019 (COVID-19) pandemics highlight the need of developing vaccines with universal and durable protection against emerging SARS-CoV-2 variants. Here we developed an extended-release vaccine delivery system (GP-diABZI-RBD), consisting the original SARS-CoV-2 WA1 strain receptor-binding domain (RBD) as the antigen and diABZI stimulator of interferon genes (STING) agonist in conjunction with yeast ß-glucan particles (GP-diABZI) as the platform. GP-diABZI-RBD could activate STING pathway and inhibit SARS-CoV-2 replication. Compared to diABZI-RBD, intraperitoneal injection of GP-diABZI-RBD elicited robust cellular and humoral immune responses in mice. Using SARS-CoV-2 GFP/ΔN transcription and replication-competent virus-like particle system (trVLP), we demonstrated that GP-diABZI-RBD-prototype vaccine exhibited the strongest and durable humoral immune responses and antiviral protection; whereas GP-diABZI-RBD-Omicron displayed minimum neutralization responses against trVLP. By using pseudotype virus (PsVs) neutralization assay, we found that GP-diABZI-RBD-Prototype, GP-diABZI-RBD-Delta, and GP-diABZI-RBD-Gamma immunized mice sera could efficiently neutralize Delta and Gamma PsVs, but had weak protection against Omicron PsVs. In contrast, GP-diABZI-RBD-Omicron immunized mice sera displayed the strongest neutralization response to Omicron PsVs. Taken together, the results suggest that GP-diABZI can serve as a promising vaccine delivery system for enhancing durable humoral and cellular immunity against broad SARS-CoV-2 variants. Our study provides important scientific basis for developing SARS-CoV-2 VOC-specific vaccines.

Advances in the development of therapeutic strategies against COVID-19 and perspectives in the drug design for emerging SARS-CoV-2 variants.

Since Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has challenged public health around the world. Currently, there is an urgent need to explore antiviral therapeutic targets and effective clinical drugs. In this study, we systematically summarized two main therapeutic strategies against COVID-19, namely drugs targeting the SARS-CoV-2 life cycle and SARS-CoV-2-induced inflammation in host cells. The development of above two strategies is implemented by repurposing drugs and exploring potential targets. A comprehensive summary of promising drugs, especially cytokine inhibitors, and traditional Chinese medicine (TCM), provides recommendations for clinicians as evidence-based medicine in the actual clinical COVID-19 treatment. Considering the emerging SARS-CoV-2 variants greatly impact the effectiveness of drugs and vaccines, we reviewed the appearance and details of SARS-CoV-2 variants for further perspectives in drug design, which brings updating clues to develop therapeutical agents against the variants. Based on this, the development of broadly antiviral drugs, combined with immunomodulatory, or holistic therapy in the host, is prior to being considered for therapeutic interventions on mutant strains of SARS-CoV-2. Therefore, it is highly acclaimed the requirements of the concerted efforts from multi-disciplinary basic studies and clinical trials, which improves the accurate treatment of COVID-19 and optimizes the contingency measures to emerging SARS-CoV-2 variants.

Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis.

Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 µL reaction at isothermal 58℃ within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.

HIF-1α promotes SARS-CoV-2 infection and aggravates inflammatory responses to COVID-19.

Cytokine storm induced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a major pathological feature of Coronavirus Disease 2019 (COVID-19) and a crucial determinant in COVID-19 prognosis. Understanding the mechanism underlying the SARS-CoV-2-induced cytokine storm is critical for COVID-19 control. Here, we identify that SARS-CoV-2 ORF3a and host hypoxia-inducible factor-1α (HIF-1α) play key roles in the virus infection and pro-inflammatory responses. RNA sequencing shows that HIF-1α signaling, immune response, and metabolism pathways are dysregulated in COVID-19 patients. Clinical analyses indicate that HIF-1α production, inflammatory responses, and high mortalities occurr in elderly patients. HIF-1α and pro-inflammatory cytokines are elicited in patients and infected cells. Interestingly, SARS-CoV-2 ORF3a induces mitochondrial damage and Mito-ROS production to promote HIF-1α expression, which subsequently facilitates SARS-CoV-2 infection and cytokines production. Notably, HIF-1α also broadly promotes the infection of other viruses. Collectively, during SARS-CoV-2 infection, ORF3a induces HIF-1α, which in turn aggravates viral infection and inflammatory responses. Therefore, HIF-1α plays an important role in promoting SARS-CoV-2 infection and inducing pro-inflammatory responses to COVID-19.

SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation.

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1ß and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.