TL;DR
The phylogenetic relationship of the A222V substitution in the S protein relative to various global isolates is investigated, indicating high mutation rates in the S gene, characterised by diverse point mutations.
A genome engineering technology is used to change a single amino acid in the viruses’ main protease enzyme to match that of circulating Omicron isolates to demonstrate antiviral efficacy of approved drugs and uncover mutants with reduced drug sensitivity.
The results show the value of nanobody technology for identifying novel neutralising epitopes in the S2 region of beta-coronaviruses with potential for the development of new selective anti-viral agents.
The emergence of the SARS-CoV-2 pandemic led to the spread of highly transmissible variants, such as the Delta variant, which originated in India, underscoring the urgent need to develop new antivirals, therapeutics, and vaccines. In our previous study, we showed that Membrane-Envelope Virus-like Particles exhibit antigenicity and neutralization activity. Hence, our present study was conducted to evaluate whether the M protein alone can form VLPs that elicit an immune response. Using computational methods, we identified key interacting residues in M-protein that contribute to VLP formation and interact with other structural proteins, including Spike (S), Nucleocapsid (N), and Envelope (E). The SARS-CoV-2-M protein was expressed in Sf-21 insect cells, and the resulting VLPs were purified, analyzed for shape and size, and characterized using DLS, FESEM, and TEM. The purified VLPs were injected into BALB/c mice to evaluate their immune response compared with uninfected controls. The biophysical analysis confirms that the particles are round and have a size of ~ 180–200 nm. The serum levels of IgG, IgM, and IgA were found to be higher in immunized mice than in uninfected mice. Further qRT-PCR analysis demonstrated the levels of IFN-γ, IL-2, and IL-12, indicating a TH1-biased immune response against the M protein. Our study demonstrates that the highly conserved M protein can self-assemble into VLPs and elicit humoral and cellular immune response. Furthermore, our study indicates that while M-protein VLPs elicit significant antibodies and cytokine responses, they do not induce detectable neutralizing activity when given alone.
The starting point of this work was a SARS-CoV-2 neutralizing peptide (LW25.13), which binds to the receptor-binding domain of the viral spike protein and inhibits the attachment of the virus to its cellular receptor ACE2. As LW25.13 is unable to neutralize later SARS-CoV-2 variants, such as omicron, we have extended the neutralization breadth of LW25.13 through structural and bioinformatic analysis. This involved the systematic variation of a range of positions and yielded peptides neutralizing SARS-CoV-2 beta and omicron at low nanomolar concentrations, while preserving the strong neutralizing capacity against earlier virus variants (wild-type, alpha, delta), as well as the proteolytic stability and α-helical conformation of the peptide. This gain in neutralizing breadth illustrates the utility of the peptide as a scaffold that can be adapted to different virus variants, which may prove useful for the development of peptides against new coronavirus variants of concern in the future.
The co-circulation of multiple viruses can lead to distinct pathological outcomes, yet how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection influences other viral infections remains poorly understood, despite its documented high frequency during the pandemic. In this study, we investigated how the proteolytic activity of SARS-CoV-2 3C-like protease (3CLpro) influences the replication of influenza A virus. In silico analysis identified candidate 3CLpro cleavage sites across numerous viral proteins, and biochemical assays confirmed that 3CLpro catalyzes the degradation of influenza virus nucleoprotein (NP) and neuraminidase (NA) in a manner requiring its protease activity. This degradation of NP and PA, which are essential for viral genome packaging and transcription, disrupted the influenza replicative cycle and suppressed viral replication, both upon ectopic 3CLpro expression and during SARS-CoV-2 infection. Our data uncover a direct, enzyme-based mechanism by which SARS-CoV-2 can suppress influenza virus replication during coinfection. We provide a molecular explanation for the sharp, global decline in influenza activity observed during the COVID-19 pandemic and illustrate how enzymatic weapons of one virus can be repurposed to restrain a competing pathogen.
Combined genomic surveillance and structural analyses demonstrated that the investigated Omicron-associated mutations largely preserved the overall architecture of the M protein-Fab interaction interface while modulating residue-level energetic contributions and the conformational dynamics of the E protein.
