Resolution of Space-Group Ambiguity and Structure Determination of Nodamura Virus to 3.3 Å resolution from Pseudo-R32 (Monoclinic) Crystals

Abstract

Monoclinic crystals of nodamura virus (NOV) have two virus molecules per asymmetric unit. Packing analysis reveals a pseudo-rhombohedral (pseudo-C2 monoclinic) arrangement of particles in the actual P21 space group (a = 562.1, b = 354.1, c = 612.8 ]k,/3 = 110.9'~). The R32 symmetry is broken rotationally and translationally. The pseudo-symmetry of the unit cell results in three possible monoclinic origins and also restrains the four particles in the unit cell to similar orientations. NOV particles deviate by less than 3 from the ideal orientations, causing overlap of peaks in the rotation function and the generation of peaks that were not interpretable as particle symmetry elements. The space-group ambiguity was resolved by analysing the relationship between the particle orientations determined by high-resolution rotation functions and the attenuation of peak heights in native Patterson maps. Particles were centered less than 1A from the R32 special positions. Three different approaches were required to identify the correct particle center. Following the solutions of the rotation and translation problems, phases were computed using the coordinates of flock house virus (FHV), another member of this virus family. The phases were improved by realspace molecular averaging with a 120-fold non-crystallographic symmetry and by solvent flattening with a spherical mask. The final model for the NOV structure was built using the 3.3 A averaged map. While the overall subunit structure was very similar to that of other nodaviruses, FHV and black beetle virus, NOV showed distinct structural features near particle threefold and quasi-threefold axes and at the protein-RNA interfaces that are consistent with phenotype differences among the related viruses.

Crystal structure of a nematode-infecting virus

“…Meanwhile, the crystal structure of the Orsay VLP reveals several major differences compared with the four known alphanodavirus structures (26)(27)(28)(29). First, the CP of the Orsay virus has two linear domains that are connected by a flexible linker, with the S domain forming a continuous capsid shell.…”

Structure of a pentameric virion-associated fiber with a potential role in Orsay virus entry to host cells

“…g . Flock House virus) [24–27], but has a structural fold closely resembling that of the betanodavirus [28]. …”

Differential segregation of nodaviral coat protein and RNA into progeny virions during mixed infection with FHV and NoV

“…The primary sequence of the FHV and NoV coat proteins is highly divergent and antibodies against one type of coat protein do not cross-react with the other in immunoblot analysis (Kaesberg, 1990). Moreover, the high resolution crystal structures of the two viruses show that subunit-subunit contacts in each particle rely on interactions between distinct sets of amino acid side chains, making the formation of mosaic capsids unlikely (Fisher and Johnson, 1993; Zlotnick et al, 1997). We investigated this experimentally by subjecting F+N particles to immunoprecipitation with FHV antiserum and then tested for the presence of NoV coat protein in the pellet using antibodies against NoV. As shown in figure 6, NoV coat protein was not detectable.…”

“…As shown, mosaic capsids were indeed undetectable and this was consistent with our knowledge of the high resolution structures of the two viruses. While the tertiary fold of the FHV and NoV capsid proteins and the architecture of the virus particles are highly conserved (Fisher and Johnson, 1993; Zlotnick et al, 1997), there are considerable differences in the details. In particular, given the significant divergence of the proteins at the primary sequence level (Kaesberg, 1990), there are notable variations in the amino acids that are used to establish subunit-subunit interactions in the protein shell and based on these differences it was anticipated that FHV and NoV subunits would be highly unlikely to form stable, mosaic particles.…”