Abstract
Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.
Highlights
To solve the crystal structure of a Turnip yellow mosaic virus (TYMV) PRO/double up as ubiquitin hydrolases (DUB)·ubiquitin complex and because the affinity of a single module of Ub for the enzyme is low [24, 34], we used a modified form of Ub (Ub-VME) in which the C-terminal Gly76 is substituted with a vinyl methylester function that spontaneously and irreversibly forms a covalent linkage with the catalytic cysteine of DUBs in a Michael addition [36, 37]
TYMV PRO/DUB and Ub-VME were incubated at 25 °C, leading to the formation of a covalent complex as evidenced by SDS-PAGE (Fig. S1A), which was purified by size-exclusion chromatography (Fig. S1B)
We will limit our analysis to the single well-ordered complex composed of chains A (TYMV PRO/DUB, ordered residues 732–876 by polyprotein numbering) and B (Ub-VME, residues 1–76 including the terminal glycyl-vinylmethylester covalently linked to the catalytic Cys783)
Summary
Deubiquitination of cellular proteins by viral DUBs can down-regulate the production of diverse antiviral molecules such as interferons or cytokines and allow viruses to evade host immune responses [12, 13]. Another example is the deubiquitination of viral proteins by viral DUBs that avoids their targeting to the proteasome, a process that can be viewed as a rescue of these viral proteins.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
