VP4 Protein Appears as a Product of RNA Encapsidation in Triatoma Virus (TRV)

AGUIRRE, J; ALORIA, K; ARIZMENDI, J, M; ILORO, I; ELORTZA, F; G. A. MARTI; NEUMANN, E; REY, F, A; GUERIN, D.M.A.

Encuentro; Biophysical Society annual meeting; 2011

Virus (TrV) is a non-enveloped þssRNA virus belonging to the insectvirus family Dicistroviridae. Its non-enveloped capsid is composed of four proteinsnamed VP1-VP4, plus the minoritary, uncleaved protein precursor VP0,which comprises VP4 and VP3. While the smaller protein VP4 (5.5 kDa versusaround 30 kDa for VP1-3) remained undetected in past studies by standard biochemicalanalyses, the icosahedral (T=1, pseudo-T=3) crystallographic structureof TrV (PDB ID: 3NAP) raised even more suspicions about its existencesince no electron density could be attributed to this peptide. In the presentwork, mass spectrometry (MS) and Tricine-SDS gel electrophoresis wereused to detect the previously elusive capsid protein VP4. Its cleavage siteswere established by sequencing the N-terminus of the protein precursor andMS, and its stoichiometry with respect to the other major capsid proteins(VP1-3) was found to be 1:1. We also characterized the polypeptides comprisingthe naturally occurring non-infectious empty capsids, i.e., RNA-free TrVparticles. The empty particles were composed of VP0-VP3 plus at least sevenadditional polypeptides, which were identified as products of the capsid precursorpolyprotein (P1). We conclude that VP4 protein appears as a product ofRNA encapsidation, and that defective processing of capsid proteins precludesgenome encapsidation. Our results also suggest that the TrV capsid can be builtwithout the scaffolding aid of the nucleic acid.