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Poly(rC) binding proteins and the 5' cloverleaf of uncapped poliovirus mRNA function during de novo assembly of

Brian J Kempf1, David J Barton

  • 1Department of Microbiology, University of Colorado Denver, School of Medicine, 12800 E. 19th Ave., Aurora, CO 80045, USA.

Journal of Virology
|April 11, 2008
PubMed

Insights

Poliovirus mRNA uses a unique structure to avoid degradation by protecting itself from exonucleases. This interaction is crucial for forming stable polysomes during viral replication.

Area of Science:

  • Molecular Biology
  • Virology
  • RNA Biology

Background:

  • Poliovirus (PV) mRNA lacks a 5' cap, making it susceptible to degradation by the 5' exonuclease XRN1.
  • A 5'-terminal cloverleaf structure on PV mRNA interacts with poly(rC) binding proteins (PCBPs) to confer protection against XRN1.
  • Understanding the mechanisms protecting uncapped viral mRNA is vital for comprehending viral replication strategies.

Purpose of the Study:

  • To investigate the de novo polysome formation of poliovirus mRNA in a cell-free system.
  • To elucidate the role of PCBP-RNA interactions in protecting PV mRNA during translation.
  • To characterize the dynamics of translation factors and viral proteins during polysome assembly.

Main Methods:

  • Utilized HeLa cell-free translation-replication reactions to study polysome formation.
  • Analyzed polysome assembly kinetics and cofractionation of viral mRNA, proteins, and translation factors.
  • Introduced mutations (e.g., C24A) in PCBP-binding elements to assess their impact on polysome formation and mRNA stability.

Main Results:

  • PV mRNA efficiently formed polysomes, with ribosome density correlating to the open reading frame (ORF) length.
  • Nascent viral polypeptides cofractionated with polysomes, while mature proteins were released.
  • Cleavage of eukaryotic initiation factor 4GI (eIF4GI) occurred during polysome assembly, with its C-terminal portion associating with polysomes.
  • PCBP 1 and 2, along with Poly(A) binding protein, were found to associate with viral polysomes.
  • A mutation disrupting the PCBP-cloverleaf interaction significantly impaired polysome formation and stability.

Conclusions:

  • The PCBP-5' cloverleaf RNA interaction is essential for protecting uncapped PV mRNA from 5' exonuclease during early translation.
  • This interaction is critical for the formation and stability of nascent poliovirus polysomes.
  • Viral proteins and translation factors, including eIF4GI, play dynamic roles in the assembly and maturation of viral polysomes.

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