Structure and RNA Recognition in Recombinant STNV Capsids

Abstract

We have expressed a recombinant form of the coat protein of satellite tobacco necrosis virus (STNV) in E. Coli using a codon-optimized gene, and shown it assembles spontaneously into capsids closely resembling the wild-type virus. The T=1 virus-like particles (VLPs) package the recombinant RNA transcript, and conditions have been established for disassembly and reassembly in vitro. We have solved the X-ray crystal structure of the VLP refined it to R/Rfree 17.4/20.7% at 1.4Å resolution. We also collected low resolution X-ray data in the range 140-6Å, and the 60-fold averaged electron density map clearly shows well ordered RNA fragments lodged near the inside surface of the capsid, close to basic clusters of N-terminal triple helices that extend into the interior of the particle. The RNA consists of a 3 bp helical stem, with a single unpaired base at the 3’ end and probably consists of a number of short stem-loops, where the loop region is disordered. Using immobilised coat protein monomers placed under reassembly conditions with ‘free’ coat protein subunits, we have prepared a range of partially assembled coat protein species for RNA aptamer selection. SELEX directed against the RNA-binding faces of the STNV coat proteins resulted in the isolation of a series of clones, with motifs that match the STNV1 genome at a number of positions. The motifs are predicted to fold into stem-loops allowing us to propose a model for packaging of the RNA genome as a series of stem-loops joined by single-stranded linkers.