Critical residues for proteolysis activity of maize chlorotic dwarf virus (MCDV) 3C-like protease and comparison of activity of orthologous waikavirus proteases.

Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined.

RIPK1 Is Cleaved by 3C Protease of Rhinovirus A and B Strains and Minor and Major Groups.

Rhinoviruses (RV), like many other viruses, modulate programmed cell death to their own advantage. The viral protease, 3C has an integral role in the modulation, and we have shown that RVA-16 3C protease cleaves Receptor-interacting protein kinase-1 (RIPK1), a key host factor that modulates various cell death and cell survival pathways. In the current study, we have investigated whether this cleavage is conserved across selected RV strains. RIPK1 was cleaved in cells infected with strains representing diversity across phylogenetic groups (A and B) and receptor usage (major and minor groups). The cleavage was abrogated in the presence of the specific 3C protease inhibitor, Rupintrivir. Interestingly, there appears to be involvement of another protease (maybe 2A protease) in RIPK1 cleavage in strains belonging to genotype B. Our data show that 3C protease from diverse RV strains cleaves RIPK1, highlighting the importance of the cleavage to the RV lifecycle.

Natural Phytochemicals, Luteolin and Isoginkgetin, Inhibit 3C Protease and Infection of FMDV, In Silico and In Vitro.

Foot-and-mouth-disease virus (FMDV) is a picornavirus that causes a highly contagious disease of cloven-hoofed animals resulting in economic losses worldwide. The 3C protease (3Cpro) is the main protease essential in the picornavirus life cycle, which is an attractive antiviral target. Here, we used computer-aided virtual screening to filter potential anti-FMDV agents from the natural phytochemical compound libraries. The top 23 filtered compounds were examined for anti-FMDV activities by a cell-based assay, two of which possessed antiviral effects. In the viral and post-viral entry experiments, luteolin and isoginkgetin could significantly block FMDV growth with low 50% effective concentrations (EC50). Moreover, these flavonoids could reduce the viral load as determined by RT-qPCR. However, their prophylactic activities were less effective. Both the cell-based and the fluorescence resonance energy transfer (FRET)-based protease assays confirmed that isoginkgetin was a potent FMDV 3Cpro inhibitor with a 50% inhibition concentration (IC50) of 39.03 ± 0.05 and 65.3 ± 1.7 µM, respectively, whereas luteolin was less effective. Analyses of the protein-ligand interactions revealed that both compounds fit in the substrate-binding pocket and reacted to the key enzymatic residues of the 3Cpro. Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.

Embryotoxic activity of 3C protease of human hepatitis A virus in developing Danio rerio embryos.

The 3C protease is a key factor in picornavirus-induced pathologies with a comprehensive action on cell targets. However, the effects induced by the enzyme have not been described at the organismic level. Here, the model of developing Danio rerio embryos was used to analyze possible toxic effects of the 3C protease of human hepatitis A virus (3Cpro) at the whole-body level. The transient 3Cpro expression had a notable lethal effect and induced a number of specific abnormalities in Danio rerio embryos within 24 h. These effects are due to the proteolytic activity of the enzyme. At the same time, the 3Cpro variant with reduced catalytic activity (3Cmut) increased the incidence of embryonic abnormalities; however, this effect was smaller compared to the native enzyme form. While the expression of 3Cmut increased the overall rate of abnormalities, no predominance of specific ones was observed. The data obtained point to a presence significant impact of picornavirus 3Cprotease at the whole-organism level and make contribution to the study of the infectious process caused by human hepatitis A virus.

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro.

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

A proposed division of the family Picornaviridae into subfamilies based on phylogenetic relationships and functional genomic organization.

The highly diverse virus family Picornaviridae presently comprises 68 approved genera with 158 species plus many unassigned viruses. In order to better match picornavirus taxonomy to the functional and genomic groupings between genera, the establishment of five subfamilies (Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae and Heptrevirinae) is proposed. The subfamilies are defined by phylogenetic analyses of 3CD (precursor of virus-encoded proteinase and polymerase) and P1 (capsid protein precursor) coding sequences and comprise between 7 and 22 currently approved virus genera. Due to the high within-subfamily and between-subfamily divergences of the picornavirus genera, p-distance estimates are unsuited for the demarcation of subfamilies. Members of the proposed subfamilies typically show some commonalities in their genome organisations, including VP1/2A cleavage mechanisms and possession of leader proteins. Other features, such as internal ribosomal entry site types, are more variable within and between members of genera. Some subfamilies are characterised by homology of proteins 1A, 2A, 2B and 3A encoded by members, which do not belong to the canon of orthologous picornavirus proteins. The proposed addition of a subfamily layer to the taxonomy of picornaviruses provides a valuable additional organisational level to the family that acknowledges the existence of higher-level evolutionary groupings of its component genera.

Removal of 3C protease from the 3ABC improves expression, solubility, and purification of the recombinant 3AB of foot-and-mouth disease virus.

During an ongoing outbreak of Foot-and-Mouth Disease Virus (FMDV), it is crucial to distinguish naturally infected from vaccinated seropositive animals. This would support clinical assessment and punctual vigilance. Assays based on 3ABC non-structural protein as an antigen are reliable for this intention. However, the insolubility and degradation of recombinant 3ABC during expression and purification are serious challenges. In this study, alternatively to expressing the recombinant 3ABC (r3ABC), we expressed the 3AB coding sequence (~672 bp) as a recombinant protein (r3AB) with a molecular mass of ~26 KDa. Analytical data from three-dimensional structure, hydrophilicity, and antigenic properties for 3ABC and 3AB exhibited the 3C protein as a hydrophobic, while 3AB as a hydrophilic and highly antigenic protein. The expressed r3AB was recovered as a completely soluble matter after merely native purification, unlike the full expressed r3ABC. Immunoreactivity of r3AB to anti-FMDV antibody in infected sera with different FMDV serotypes was confirmed by the western blot and indirect ELISA. Besides, the authentic antigenicity of purified r3AB was demonstrated through its ability to induce specific seroconversion in mice. Summarily, the removal of 3C: has influenced neither 3D structure nor antigenic properties of the purified r3AB, overcame insolubility and degradation of the r3ABC, and generated a potential superior antigen (r3AB) for herd screening of animals to any FMDV serotype.

Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction.

Stress granules (SGs) are the sites of mRNA storage and related to the regulation of mRNA translation, which are dynamic structures in response to various environmental stresses and viral infections. Seneca Valley virus (SVV), an oncolytic RNA virus belonging to Picornaviridae family, can cause vesicular disease (VD) indistinguished from foot-and-mouth disease (FMD) and other pig VDs. In this study, we found that SVV induced SG formation in the early stage of infection in a PKR-eIF2α dependent manner, as demonstrated by the recruitment of marker proteins of G3BP1 and eIF4GI. Surprisingly, we found that downregulating SG marker proteins TIA1 or G3BP1, or expressing an eIF2α non-phosphorylatable mutant inhibited SG formation, but this inhibition of transient SG formation had no significant effect on SVV propagation. Depletion of G3BP1 significantly attenuated the activation of NF-κB signaling pathway. In addition, we found that SVV inhibited SG formation at the late stage of infection and 3C protease was essential for the inhibition depending on its enzyme activity. Furthermore, we also found that 3C protease blocked the SG formation by disrupting eIF4GI-G3BP1 interaction. Overall, our results demonstrate that SVV induces transient SG formation in an eIF2α phosphorylation and PKR-dependent manner, and that 3C protease inhibits SG formation by interfering eIF4GI-G3BP1 interaction.