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

Despite the wide use of Caenorhabditis elegans as a model organism, the first virus naturally infecting this organism was not discovered until six years ago. The Orsay virus and its related nematode viruses have a positive-sense RNA genome, encoding three proteins: CP, RdRP, and a novel delta protein that shares no homology with any other proteins. d can be expressed either as a free delta or a CP-delta fusion protein by ribosomal frameshift, but the structure and function of both d and CP-delta remain unknown. Using a combination of electron microscopy, X-ray crystallography, computational and biophysical analyses, here we show that the Orsay d protein forms a similar to 420-angstrom long, pentameric fiber with an N-terminal alpha-helical bundle, a beta-stranded filament in the middle, and a C-terminal head domain. The pentameric nature of the d fiber has been independently confirmed by both mass spectrometry and analytical ultracentrifugation. Recombinant Orsay capsid containing CP-delta shows protruding long fibers with globular heads at the distal end. Mutant viruses with disrupted CP-delta fibers were generated by organism-based reverse genetics. These viruses were found to be either non-viable or with poor infectivity according to phenotypic and qRT-PCR analyses. Furthermore, addition of purified delta proteins to worm culture greatly reduced Orsay infectivity in a sequence-specific manner. Based on the structure resemblance between the Orsay CP-delta fiber and the fibers from reovirus and adenovirus, we propose that CP-delta functions as a cell attachment protein to mediate Orsay entry into worm intestine cells.