High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre)

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• Picornaviruses are medically significant RNA viruses.
• Viral genome replication relies on a cis-acting replication element (cre).
• The cre templates the uridylylation of a protein primer (VPg) by the 3D(pol) polymerase.

Purpose of the Study:

• To determine the solution structure of the human rhinovirus 14 cre.
• To understand the structural basis of VPg uridylylation.
• To correlate cre structure with viral RNA replication efficiency.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to determine the 3D structure of the cre.
• In vitro uridylylation assays to assess the functional impact of mutations.
• Bioinformatic analysis of cre sequences from different enteroviruses and rhinoviruses.

Main Results:

• The 33-nt cre adopts a stem-loop structure with a stable 14-nt loop.
• Loop base-stacking interactions stabilize the structure, but internal base-pairing is absent.
• Conserved adenosines in the 5' loop are positioned for VPg-pUpU synthesis during uridylation.
• Loop mutations disrupting base-stacking are detrimental to viral replication.
• 3' loop mutations have minimal impact on the critical 5' loop conformation.

Conclusions:

• The determined cre structure provides a molecular basis for VPg uridylylation.
• The structure explains the functional importance of specific loop nucleotides and the tolerance for 3' loop variation.
• Understanding cre structure-host-pathogen interactions can inform antiviral strategies against picornaviruses.