LS-6-P-6031 Novel lateral contact is essential for structural integrity of helical Barely stripe mosaic virions.

Barley stripe mosaic virus is a part of Hordeivirus genus, which includes plant viruses with tripartite +RNA genome. Three genomic RNAs are incapsidated separately. Virus spreads via contacts between plants, via seed and pollen and course diseases from light mosaic to lethal necrosis. BSMV virion has a shape of a rigid rod with helical distribution of RNA and coat protein.
Structure of members of Hordeivirus genus is poorly studied, whereas structure of close Tobamovirus genus is well-discribed. The aim of this work is to determine the structure of BSMV using methods of electron microscopy and image processing (Clare and Orlova,2010).
It was estimated that the helix has 5 turns per period and 111 subunits per period. The angle between subunits was ~16.2°, and helical rise between subunits was ~1.18Å.
Multivariate Statistical Analysis (MSA) of obtained electron microscopical data showed that there was a structural heterogeneity in the dataset: diameter of viral particles varied. The images of wide and narrow particles (with diameter of 224Å and 216 Å correspondingly) were extracted and used for 3d reconstruction. It turned out that narrow particles have slightly different helical parameters: 106 subunits per period, the angle between subunits was ~17°, and helical rise between subunits was ~1.24Å. The resolution of the final electron density map was 5.7 Å for a wide particle and 5.1Å – for a narrow. On the vertical slice of both structures there is visible a groove between protein subunits, where RNA is located (Fig. 1). The reached resolution makes it possible to trace the α-helices in the coat protein of BSMV and the sites of RNA-protein interactions are determined (Fig. 1). The flexible fitting of homology model of BSMV coat protein (CP) showed that there is a loop at a high radius. The loop protrudes from hydrophobic core and makes a contact with the next subunit (Fig. 2). To analyze the functional importance of interacting arm formed by the BSMV CP internal loop, we constructed two CP mutants, BSMV-del10 with a ten-residue deletion in the loop and BSMV-IY/GG, in which two residues Ile86 and Tyr91 involved in the contact with neighboring CP subunit were replaced with Gly residues (Fig. 3a). Recombinant viruses carrying the mutant CP gene were able to systemically infect N. benthamiana plants and accumulated to levels similar to recBSMV (Fig.3a,b). However, virions were not found in tissues infected with recBSMV-del10 and recBSMV-IY/GG, while readily detected in recBSMV-infected plants (Fig. 3c,d), showing that both mutants were unable to form stable virions. These data demonstrate the crucial importance of the inter-subunit interacting arm in formation and/or maintaining the structure of BSMV virions.