Orsay Virus CP-δ Adopts a Novel β-Bracelet Structural Fold and Incorporates into Virions as a Head Fiber

Abstract

Fiber proteins are commonly found in eukaryotic and prokaryotic viruses where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via non-covalent interactions. The small RNA virus Orsay, which specifically infects the laboratory model Caenorhabditis elegans, encodes a fibrous protein δ that can be expressed as a free protein and as a capsid protein-δ (CP-δ) fusion protein. Free δ has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP-δ remained relatively unknown. Here, we demonstrate that Orsay CP-δ is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of δ (1-101) (i.e. a deletion mutant containing the first 101 amino acid (aa) residues of δ) reveals a pentameric, 145-Å long fiber with an N-terminal coiled coil followed by multiple β-bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP-δ fibers with dimensions similar to free δ. δ proteins from two other nematode viruses Le Blanc and Santeuil, which both specifically infect Caenorhabditis briggsae, were also found to form fibrous molecules. Recombinant Le Blanc δ was able to block Orsay virus infection in worm culture and vice versa, suggesting these two viruses likely compete for the same cell receptor(s). Thus, we propose that while CP-δ likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus. IMPORTANCE Viruses often have extended fibers to mediate host cell recognition and entry, serving as promising targets for antiviral drug development. Unlike other known viral fibers, the δ proteins from the three recently discovered nematode viruses are incorporated into infectious particles as protruding fibers covalently linked to the capsid. Crystal structures of δ revealed novel pentameric folding repeats, which we term β-bracelets, in the intermediate shaft region. Based on sequence analysis, the β-bracelet motif of δ is conserved in all three nematode viruses and could account for ∼60% of the total length of the fiber. Our study indicated that δ plays important roles in cell attachment for this group of nematode viruses. In addition, the tightly knitted β-bracelet fold, which presumably allows δ to survive harsh environments in the worm gut, could serve applicable to bioengineering applications given its potentially high stability.

Story of an infection: viral dynamics and host responses in theCaenorhabditis elegans-Orsay virus pathosystem

“…OrV has a positive-sense, bi- segmented RNA genome of ∼6.3 kb which encodes four proteins: an RNA-dependent RNA polymerase (RdRP), a viral capsid protein (CP), protein (8), and a protein fusion of CP-8 (Félix et al, 2011; Jiang et al, 2014). Free 8 mediates nonlytic viral release (Yuan et al, 2018) and the CP-8 fusion is incorporated into the virions as pentameric head fibers (Fan et al, 2017; Guo et al, 2020).…”

Experimental evolution of an RNA virus inCaenorhabditis elegans

“…In RNA2, we found a hotspot in the first 20 nucleotides, and two others between nucleotides 400-500, within the S domain of the CP (Guo et al, 2014), and 1500-1600, from the end of the second alpha sheet to beta sheet 6 of the 8 protein (Fig. 4A, B) (Guo et al, 2020).…”

“…The other common SNV (RNA 1: 302G>A) was not above the threshold in sample JUv1580_vlc RL2. (B) High variability hotspots (magenta) represented on the predicted structure of RdRp and PDB structures 4NWV and 5W82 of the CP trimer and 8 pentamer (amino acids 1-110), respectively (Guo et al, 2014, Guo et al, 2020).…”

“…Data in all panels are presented as median ±IQR. represented on the predicted structure of RdRp and PDB structures 4NWV and 5W82 of the CP trimer and d pentamer (amino acids 1-110), respectively (Guo et al, 2014, Guo et al, 2020.…”

Collagen and actin network mediate antiviral immunity against Orsay in C. elegans intestinal cells

“…It has been shown that the free δ and the CP-δ fusion protein play different roles during viral exit and viral entry, respectively. While free δ was found to colocalize with terminal web and mediate nonlytic viral release (6), CP-δ is incorporated into infectious viral particles as a pentameric head fiber (7). The CP-δ head fiber adopts a novel bracelet structure with a terminal globular domain that is likely involved in host receptor engagement.…”