SARS-CoV-2 Mpro oligomerization as a potential target for therapy.

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Autoinhibition of suicidal capsid protease from O'nyong'nyong virus.

Alphaviruses pose a significant threat to public health. Capsid protein encoded in the alphaviral genomes constitutes an interesting therapy target, as it also serves as a protease (CP). Remarkably, it undergoes autoproteolysis, leading to the generation of the C-terminal tryptophan that localizes to the active pocket, deactivating the enzyme. Lack of activity hampers the viral replication cycle, as the virus is not capable of producing the infectious progeny. We investigated the structure and function of the CP encoded in the genome of O'nyong'nyong virus (ONNV), which has instigated outbreaks in Africa. Our research provides a high-resolution crystal structure of the ONNV CP in its active state and evaluates the enzyme's activity. Furthermore, we demonstrated a dose-dependent reduction in ONNV CP proteolytic activity when exposed to indole, suggesting that tryptophan analogs may be a promising basis for developing small molecule inhibitors. It's noteworthy that the capsid protease plays an essential role in virus assembly, binding viral glycoproteins through its glycoprotein-binding hydrophobic pocket. We showed that non-aromatic cyclic compounds like dioxane disrupt this vital interaction. Our findings provide deeper insights into ONNV's biology, and we believe they will prove instrumental in guiding the development of antiviral strategies against arthritogenic alphaviruses.

Pseudanabaena galeata CCNP1313-Biological Activity and Peptides Production.

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549ACE2/TMPRSS2) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts.

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses.

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

Zika virus: mapping and reprogramming the entry.

BACKGROUND: The flaviviridae family comprises single-stranded RNA viruses that enter cells via clathrin-mediated pH-dependent endocytosis. Although the initial events of the virus entry have been already identified, data regarding intracellular virus trafficking and delivery to the replication site are limited. The purpose of this study was to map the transport route of Zika virus and to identify the fusion site within the endosomal compartment. METHODS: Tracking of viral particles in the cell was carried out with confocal microscopy. Immunostaining of two structural proteins of Zika virus enabled precise mapping of the route of the ribonucleocapsid and the envelope and, consequently, mapping the fusion site in the endosomal compartment. The results were verified using RNAi silencing and chemical inhibitors. RESULTS: After endocytic internalization, Zika virus is trafficked through the endosomal compartment to fuse in late endosomes. Inhibition of endosome acidification using bafilomycin A1 hampers the infection, as the fusion is inhibited; instead, the virus is transported to late compartments where it undergoes proteolytic degradation. The degradation products are ejected from the cell via slow recycling vesicles. Surprisingly, NH4Cl, which is also believed to block endosome acidification, shows a very different mode of action. In the presence of this basic compound, the endocytic hub is reprogrammed. Zika virus-containing vesicles never reach the late stage, but are rapidly trafficked to the plasma membrane via a fast recycling pathway after the clathrin-mediated endocytosis. Further, we also noted that, similarly as other members of the flaviviridae family, Zika virus undergoes furin- or furin-like-dependent activation during late steps of infection, while serine or cysteine proteases are not required for Zika virus maturation or entry. CONCLUSIONS: Zika virus fusion occurs in late endosomes and is pH-dependent. These results broaden our understanding of Zika virus intracellular trafficking and may in future allow for development of novel treatment strategies. Further, we identified a novel mode of action for agents commonly used in studies of virus entry. Schematic representation of differences in ZIKV trafficking in the presence of Baf A1 and NH4Cl.