Date Published: February 27, 2017
Publisher: Public Library of Science
Author(s): Yanlin Fan, Yusong R. Guo, Wang Yuan, Ying Zhou, Matthew V. Holt, Tao Wang, Borries Demeler, Nicolas L. Young, Weiwei Zhong, Yizhi J. Tao, Félix A. Rey.
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 δ protein that shares no homology with any other proteins. δ can be expressed either as a free δ or a CP-δ fusion protein by ribosomal frameshift, but the structure and function of both δ and CP-δ remain unknown. Using a combination of electron microscopy, X-ray crystallography, computational and biophysical analyses, here we show that the Orsay δ protein forms a ~420-Å long, pentameric fiber with an N-terminal α-helical bundle, a β-stranded filament in the middle, and a C-terminal head domain. The pentameric nature of the δ fiber has been independently confirmed by both mass spectrometry and analytical ultracentrifugation. Recombinant Orsay capsid containing CP-δ shows protruding long fibers with globular heads at the distal end. Mutant viruses with disrupted CP-δ 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 δ proteins to worm culture greatly reduced Orsay infectivity in a sequence-specific manner. Based on the structure resemblance between the Orsay CP-δ fiber and the fibers from reovirus and adenovirus, we propose that CP-δ functions as a cell attachment protein to mediate Orsay entry into worm intestine cells.
For the past four decades, the nematode Caenorhabditis elegans (C. elegans) has been used as an important model organism for studying biological processes such as development, metabolism, aging, cell cycle and gene regulation . However, it was not until the year 2011 that Orsay, the first virus that naturally infects C. elegans in the wild, was discovered . Two other nematode viruses, Santeuil and Le Blanc, which both infect C. briggsae, have also been identified [2, 3]. Infections by these newly identified viruses cause abnormal intestinal morphologies without obvious effects on longevity or brood size [2, 4].
Results from our study indicated that the CP-δ fusion protein plays a specific function in host cell entry during Orsay infection based on the following evidence: (1) Orsay δ forms pentameric fibers; (2) CP-δ is incorporated into viral capsid as a minor structural protein; (3) the δ portion of CP-δ forms a long projecting fibers with a globular head domain at the distal end; (4) disrupting the structural integrity of CP-δ results in non-viable virus mutants; and (5) the addition of recombinant δ to worm medium reduced Orsay infectivity. The use of recombinant VLPs enabled us to directly visualize the CP-δ fibers due to the enhanced amount of CP-δ in the VLP sample. In contrast, the native virion sample contains only ~5% CP-δ , which corresponds to only one to two CP-δ fibers in average in each particle. It would be difficult to identify these long fibers by EM unless they lie flat on sample grids and interact evenly with heavy atom stains, thus explaining the difficulties we had trying to visualize such fibers using native virion samples.