Bing Sun

Coronavirus disease 2019 (COVID-19), caused by the novel human coronavirus SARS-CoV-2, is currently a major threat to public health worldwide. The viral spike protein binds the host receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD), and thus is believed to be a major target to block viral entry. Both SARS-CoV-2 and SARS-CoV share this mechanism. Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies. The in vivo experiments showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice; however, the cross-neutralizing activity was much weaker, indicating that there are distinct antigenic features in the RBDs of the two viruses. This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2. It is worth noting that a newly developed SARS-CoV-2 human antibody, HA001, was able to neutralize SARS-CoV-2, but failed to recognize SARS-CoV. Moreover, the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD, representing new binding sites for neutralizing antibodies. Overall, our study has revealed the presence of different key epitopes between SARS-CoV and SARS-CoV-2, which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.

Inflammasomes are multi-protein complexes that trigger the activation of caspase-1 and the maturation of interleukin-1β (IL-1β), yet the regulation of these complexes remains poorly characterized. Here we show that nitric oxide (NO) inhibited the NLRP3-mediated ASC pyroptosome formation, caspase-1 activation and IL-1β secretion in myeloid cells from both mice and humans. Meanwhile, endogenous NO derived from iNOS (inducible form of NO synthase) also negatively regulated NLRP3 inflammasome activation. Depletion of iNOS resulted in increased accumulation of dysfunctional mitochondria in response to LPS and ATP, which was responsible for the increased IL-1β production and caspase-1 activation. iNOS deficiency or pharmacological inhibition of NO production enhanced NLRP3-dependent cytokine production in vivo, thus increasing mortality from LPS-induced sepsis in mice, which was prevented by NLRP3 deficiency. Our results thus identify NO as a critical negative regulator of the NLRP3 inflammasome via the stabilization of mitochondria. This study has important implications for the design of new strategies to control NLRP3-related diseases.

Fourteen ORFs have been identified in the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome. ORF 3a of SARS-CoV codes for a recently identified transmembrane protein, but its function remains unknown. In this study we confirmed the 3a protein expression and investigated its localization at the surface of SARS-CoV-infected or 3a-cDNA-transfected cells. Our experiments showed that recombinant 3a protein can form a homotetramer complex through interprotein disulfide bridges in 3a-cDNA-transfected cells, providing a clue to ion channel function. The putative ion channel activity of this protein was assessed in 3a-complement RNA-injected Xenopus oocytes by two-electrode voltage clamp. The results suggest that 3a protein forms a potassium sensitive channel, which can be efficiently inhibited by barium. After FRhK-4 cells were transfected with an siRNA, which is known to suppress 3a expression, followed by infection with SARS-CoV, the released virus was significantly decreased, whereas the replication of the virus in the infected cells was not changed. Our observation suggests that SARS-CoV ORF 3a functions as an ion channel that may promote virus release. This finding will help to explain the highly pathogenic nature of SARS-CoV and to develop new strategies for treatment of SARS infection.