Virulence determinant of Senecavirus A: 3C protein functions as a protease to cleave viral polyprotein and host factors

Figure 1.. Schematic representations of SVA virion and genome. Front (A) and section (B) views of SVA virion. The virion is an icosahedral particle, composed of 60 protomers, each of which contains four viral proteins, namely, VP1, VP2, VP3, and VP4. Schematic representation of SVA genome (C). The genome consists of approximately 7300 nucleotides. Arrows indicate start and end sites of genetic elements at the genome. Genetic elements are proportionally scaled to their actual lengths in SVA genome.

Figure 2.. Schematic representation of SVA infection, propagation and packaging. (i) SVA virion binging to cellular receptor; (ii) entrance of virion into cell via endocytosis; (iii) virus uncoating to release genome; (iv) IRES-mediated binding of ribosome to viral genome; (v) translation of polyprotein; (vi) cleavage of polyprotein by different mechanisms; (vii) RdRp-mediated replication of antigenome; (viii) RdRp-mediated replication of genome; (ix) release of nascent genome from template; (x) genome packaging to assemble virion; (xi) cell lysis for releasing virion.

Figure 3.. Putative RNA secondary structure of 3Cpro sequence. The secondary structure is computationally predicted by the RNAfold web server based on fold algorithms of minimum free energy.

Figure 4.. Phylogenetic analysis of 50 SVA isolates. The phylogenetic tree is constructed using the neighbor-joining method. The evolutionary distances are computed using the maximum composite likelihood method, and are in the units of the number of base substitutions per site. SVA isolates from China, the USA and other countries were shown with blue, red and green names, respectively.

Figure 5.. Characteristics of protein sequence of SVA 3Cpro. The number of each amino acid at SVA (CH-LX-01–2016) 3Cpro (A). Hydrophilic, hydrophobic, acidic and basic amino acids are represented with red, green, brown and blue columns, respectively. Putative secondary structure of SVA 3Cpro (B), computationally predicted by the CFSSP web server. R1, R8 and R9: α-helix regions; R2, R3, R4 and R6: β-sheet regions; R5: β-turn region; R7: coil region. SAAP sites at 3Cpro based on the multiple sequence alignment of 50 isolates (C). The heights of symbols are scaled according to the frequencies of amino acids at each position. Topology diagram of SVA 3Cpro (D). SVA 3Cpro is split into two domains, independently indicated with light green and red shadows. Red cylinders: α helices; green arrows: β sheets.

Figure 6.. Properties of 3Cpro-mediated cleavage of polyprotein. 3Cpro-targeted sites of SVA polyprotein for cleavage (A). Blue arrowheads: 3Cpro-targeted sites; red arrowhead: ribosome skipping (RS) site; brown arrowhead: site with unknown proteolytic mechanism. Schematic representation of 3Cpro-recognized 8-aa motif of polyprotein substrate (B). Multiple sequence alignment of eight 3Cpro-identified motifs in SVA polyprotein (C). The alignment analysis is carried out based on 58 representative isolate sequences in GenBank. The heights of symbols are scaled according to the frequencies of amino acids at each position. Electrostatic surface of SVA 3Cpro reacting with key motif in substrate (D), adapted from the reference [70] with minor modifications. P4 to P4’ residues are represented as stick models. S4 to S3’ subsites are indicated with dotted lines. Thirteen 3Cpro residues, indicated with solid lines, potentially interact with the substrate.

Figure 7.. Roles of SVA 3Cpro in interfering with innate immunity of host. An SVA virion enters a cell by endocytosis and then releases its RNA genome, which can subsequently trigger two IFN response pathways, RIG-I/MDA5–MAVS ① and TLR3 TRIF ② signaling pathways. Both pathways regulate the release of type I IFN and pro-inflammatory cytokines by signal transduction. SVA 3Cpro, however, is able to interact with different components concerning both pathways, including RIG-I, MAVS, TRIF, TRAF3, TANK, TBK1, IRF3/7 and NF-κB, for suppressing IFN secretion or function. Red and black T-shaped symbols represent the inhibiting effects mediated by 3Cpro and others, respectively.