When a virus enters a host cell it can be sensed by pattern recognition receptors (PRRs) that sensed conserved features of a virus called a pathogen associated molecular pattern (PAMP). For RNA viruses, this is often double stranded RNA, in the form of a replication intermediate or a structured region of the viral genome. Activation of PRRs lead to downstream signaling that induces type I interferons (IFNs) (Fig. 1 A and C) that can then activate the IFN-response pathway (Fig. 1 B) to induces hundreds of IFN-stimulated genes, which can have direct antiviral properties. As innate immunity is the first line of defense against viruses, they have evolved several strategies to evade sensing by the host cell. One mechanism that is commonly used is cleavage of host innate immune proteins by viral proteases. The most well studied is the HCV protease complex, NS3/4A, cleavage of MAVS, an innate immune adaptor protein. There has been limited study of Coronavirus cleavage of host innate immune proteins by the viral protease 3Cysteine-like protease (3CLpro, nsp5). It is known that two porcine coronaviruses use the 3CLpro to cleave NEMO, an NF-kB inhibitor protein, but other host targets have not been studied. Our research focuses on testing whether the coronavirus 3CLpro is involved in innate immune evasion, cleaves predicted host targets within the innate immune pathways, and whether there are host species differences in the ability of 3CLpro to cleave the target protein.
Figure 1. Innate immune signaling pathways. (A,C)Viruses are sensed by Pattern Recognition receptors and activate signaling pathways to induce Type I IFN and other cytokines. IFN can then bind to receptors on neighboring cells to activate the IFN response pathway which induces hundreds of ISGs that help in clearing the virus. It is known that nsp5 cleaves NEMO to disrupt NF-κB signaling.
Coronavirus genomes contains ~10 open reading frames (ORFs). The first 2 overlapping ORFs (ORF1a and ORF1an) encode the nonstructural proteins (nsps) and the remaining ORFs encode structural and accessory proteins. There are 16 nsps that are translated into 2 large polyproteins. The individual proteins are then cleaved by viral proteases (nsp3 and nsp5) to generate mature proteins. Nsp5 is responsible for 11 cleavages (dark grey arrow heads). Projects in the Beachboard lab will focus on the role of nsp5 in evading host innate immunity.
Figure 2. Coronavirus Genome organization. The Mouse hepatitis virus (MHV) genome is shown. Nsp5 is responsible for 11 cleavages (dark grey arrow heads). The stucture of SARS-CoV-2 nsp5 is shown (PDB: 6M2N)