Biochemically investigating the roles of papain-like proteases from +ssRNA viruses in replication and innate immune suppression

Thumbnail Image
Patel, Ankoor
Journal Title
Journal ISSN
Volume Title
Positive-sense single-stranded RNA (+ssRNA) viruses are diverse in the way their genomes are packaged, polyproteins are arranged, and the hosts they infect. They often produce similar proteins despite different hosts, one being the papain-like protease (PLP). PLPs are cysteine proteases that are important in viral replication by cleaving their own polyprotein(s) into functional subdomains. The polyprotein(s) is translated from a single open reading frame within the genome; yet encapsulates multiple individual protein domains that are fused together, maximizing genomic coding capacity. Interestingly, PLPs have been shown to be more than just endopeptidases, which is the basis for this work. +ssRNA viruses have small genomes and utilize programmed ribosomal frameshifting (PRF), allowing for the translation of multiple proteins from one gene. As seen in Porcine reproductive and respiratory syndrome virus, a PLP termed Nonstructural protein 1β, acts as a transactivator of PRF to produce two variant proteins that appear to be essential in immune suppression. Herein, nsp1β is shown to interact with a cellular protein and the viral genome at key residues to facilitate PRF. Biochemical and biophysical investigations illustrate how this complex forms and provides insight into the novelty behind viral and cellular proteins acting as transactivators of PRF. Plant affecting +ssRNA viruses have been studied for decades; however, are far less understood than mammalian viruses. Here we show yet another role for plant-affecting viral PLPs, termed PRO, from genera Marafivirus and Benyvirus acting as deubiquitinases to potentially corrupt the ubiquitin-dependent proteasomal degradation system or tamper with cellular signaling. We have been able to determine that these proteases act as ubiquitin hydrolases and have specificity towards different ubiquitin substrates. We have determined cleavage sites within the marafiviral polyprotein that had not been determined before. Crystal structures of PRO from the marafivirus type member Maize rayado fino virus, show differences within the enzyme in its native and substrate-bound forms. Protein-engineered ubiquitin-like inhibitors were designed against these enzymes to act as competitive inhibitors with hopes of imparting agricultural resilience. A similar strategy was used against a deubiquitinating PLP from Severe acute respiratory syndrome coronavirus 2, termed PLpro.
Virology, protein science, biochemistry, structural biology, biophysics, arteriviruses, nidoviruses, benyviruses, marafiviruses