Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors

J Biol Chem. 2023 Nov;299(11):105258. doi: 10.1016/j.jbc.2023.105258. Epub 2023 Sep 15.

Abstract

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Keywords: RNA virus; polyprotein processing; protease; protein folding; protein-RNA interactions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Circular Dichroism
  • Enzyme Activation
  • Peptide Hydrolases* / chemistry
  • Peptide Hydrolases* / metabolism
  • Poliovirus* / chemistry
  • Poliovirus* / genetics
  • Polyproteins / genetics
  • Polyproteins / metabolism
  • Protein Folding*
  • Protein Stability
  • Protein Structure, Secondary
  • RNA, Viral / genetics
  • RNA, Viral / isolation & purification
  • RNA, Viral / metabolism
  • Viral Proteins* / chemistry
  • Viral Proteins* / genetics
  • Viral Proteins* / metabolism

Substances

  • Peptide Hydrolases
  • Polyproteins
  • RNA, Viral
  • Viral Proteins