SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells.

It is urgent to understand the infection mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for the prevention and treatment of COVID-19. The infection of SARS-CoV-2 starts when the receptor-binding domain (RBD) of viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) of the host cell, but the endocytosis details after this binding are not clear. Here, RBD and ACE2 were genetically coded and labeled with organic dyes to track RBD endocytosis in living cells. The photostable dyes enable long-term structured illumination microscopy (SIM) imaging and to quantify RBD-ACE2 binding (RAB) by the intensity ratio of RBD/ACE2 fluorescence. We resolved RAB endocytosis in living cells, including RBD-ACE2 recognition, cofactor-regulated membrane internalization, RAB-bearing vesicle formation and transport, RAB degradation, and downregulation of ACE2. The RAB was found to activate the RBD internalization. After vesicles were transported and matured within cells, RAB was finally degraded after being taken up by lysosomes. This strategy is a promising tool to understand the infection mechanism of SARS-CoV-2.

Computational design of stapled peptide inhibitor against SARS-CoV-2 receptor binding domain.

Since its first detection in 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been the cause of millions of deaths worldwide. Despite the development and administration of different vaccines, the situation is still worrisome as the virus is constantly mutating to produce newer variants some of which are highly infectious. This raises an urgent requirement to understand the infection mechanism and thereby design therapeutic-based treatment for COVID-19. The gateway of the virus to the host cell is mediated by the binding of the receptor binding domain (RBD) of the virus spike protein to the angiotensin-converting enzyme 2 (ACE2) of the human cell. Therefore, the RBD of SARS-CoV-2 can be used as a target to design therapeutics. The α1 helix of ACE2, which forms direct contact with the RBD surface, has been used as a template in the current study to design stapled peptide therapeutics. Using computer simulation, the mechanism and thermodynamics of the binding of six stapled peptides with RBD have been estimated. Among these, the one with two lactam stapling agents has shown binding affinity, sufficient to overcome RBD-ACE2 binding. Analyses of the mechanistic detail reveal that a reorganization of amino acids at the RBD-ACE2 interface produces favorable enthalpy of binding whereas conformational restriction of the free peptide reduces the loss in entropy to result higher binding affinity. The understanding of the relation of the nature of the stapling agent with their binding affinity opens up the avenue to explore stapled peptides as therapeutic against SARS-CoV-2.

Aspects of the physiochemical properties of SARS-CoV-2 to prevent S-protein receptor binding using Arabic gum

With the number of people infected with the new coronavirus exceeding millions of confirmed infections, the world is turning to scientists and researchers, everyone is waiting - impatiently - for the results of the research that is being carried out in full swing to find an effective treatment for the virus. The recent development of the virus has witnessed at least 17 mutations that may affect its external shape, especially on the S-protein receptor-binding domain (RBD), which helps it attach to human cells' receptor angiotensin-converting enzyme-2 (ACE2) to make RBD-ACE2 interaction and entry to host cell. This interaction becomes stronger in the new strains of the coronavirus due to a mutation that occurs in the S-proteins that attach to human cells. For researchers and scientists to be able to confront this pandemic that has spread in the world like wildfire, they must be armed with accurate understanding and clear knowledge about coronavirus. This study focuses on polysaccharides, specifically negatively polysaccharides, that can interfere with the positive charge of the surface of the SARS-CoV-2 and ACE2, thus inhibiting the virus's infectivity and destroying it. In addition, polysaccharides will boost the immune function of the vaccine, thereby fostering nonspecific immunity of the body and specific immunity of the body, cellular immunity, mucosal immunity, humoral immunity, and decreased pro-inflammatory expression. This research aims to reduce the attachment power and modify the pulling apart of the RBD and the angiotensin-converting enzyme 2 (ACE2) by polysaccharide molecules such as Arabic gum (AG) and carrageenan. The adapted fluorometric assay is used to investigate the probability of Arabic gum and ACE2 interactions. The obtained results confirmed that the interaction could take place between Arabic gum and ACE2. Several literature studies promote the use of the urchin egg as antiviral, especially for SARS-CoV-2, because it has sulfated fucan polysaccharide molecules that prevent interaction of SARS-CoV-2 with a host cell. But, to the best of our knowledge, we found that the effect of urchin egg as antiviral, especially for SARS-CoV-2 is very difficult due to the presence of immunoglobulin G (IgG) in the human cells containing sugars that terminate with N-glycolylneuraminic (Neu5Ac) as found in the sperm of sea urchin. So, s most probably an interaction has occurred between Neu5Ac in IgG of human cells and sulfated fucan polysaccharide molecules of urchin egg.

Systemic effects of missense mutations on SARS-CoV-2 spike glycoprotein stability and receptor-binding affinity.

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18 354 mutations in S protein were analyzed, and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3705 mutations in SARS-CoV-2 RBD and 11 324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.