Crystal structure of a nematode-infecting virus Yusong R. Guoa, Corey F. Hrycb,c, Joanita Jakanac, Hongbing Jiangd,e, David Wangd,e, Wah Chiub,c, Weiwei Zhonga, and Yizhi J. Taoa,1 aDepartment of BioSciences, Rice University, Houston, TX 77005; bGraduate Program in Structural and Computational Biology and Molecular Biophysics and cVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; and Departments of dMolecular Microbiology and ePathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110 Edited by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved July 25, 2014 (received for review April 22, 2014) Orsay, the first virus discovered to naturally infect Caenorhabditis addition to the viral CP, a CP–δ fusion protein, which is likely elegans or any nematode, has a bipartite, positive-sense RNA ge- generated by ribosomal frameshifting, has been detected in nome. Sequence analyses show that Orsay is related to nodavi- infected cells as well as purified viruses (7). The Orsay CP and ruses, but molecular characterizations of Orsay reveal several RdRP share up to 26–30% amino acid identity with those from δ unique features, such as the expression of a capsid–δ fusion pro- betanodaviruses in the Nodaviridae, but the protein does not tein and the use of an ATG-independent mechanism for translation have any sequence homologs in GenBank. initiation. Here we report the crystal structure of an Orsay virus- The combination of a simple multicellular host organism and a small naturally infecting virus makes C. elegans-Orsay a highly like particle assembled from recombinant capsid protein (CP). – Orsay capsid has a T = 3 icosahedral symmetry with 60 trimeric tractable model for studying host virus interactions (8). To characterize the molecular properties of the newly discovered surface spikes. Each CP can be divided into three regions: an Orsay virus, we report the crystal structure of an Orsay virus-like N-terminal arm that forms an extended protein interaction network = β particle (VLP), which shows a T 3 icosahedral symmetry with at the capsid interior, an S domain with a jelly-roll, -barrel fold a total of 60 trimeric spikes. Each CP molecule can be divided forming the continuous capsid, and a P domain that forms surface into three linear regions, namely the N-terminal arm, the S do- spike projections. The structure of the Orsay S domain is best main forming the continuous capsid shell, and the P domain, = aligned to T 3 plant RNA viruses but exhibits substantial differ- which forms trimeric surface protrusions. The jelly-roll β-barrel ences compared with the insect-infecting alphanodaviruses, which in the S domain closely resembles those from small plant RNA also lack the P domain in their CPs. The Orsay P domain is remotely viruses. The P domain consists of a distorted β-barrel that is only related to the P1 domain in calicivirus and hepatitis E virus, sug- remotely related to the P1 domain from a norovirus CP (9). The gesting a possible evolutionary relationship. Removing the N-ter- N-terminal arm of the Orsay CP forms an extensive network minal arm produced a slightly expanded capsid with fewer nucleic inside the capsid through a β-annulus structure similar to tomato acids packaged, suggesting that the arm is important for capsid bushy stunt virus (10). An N-terminally truncated mutant, CP42–391, stability and genome packaging. Because C. elegans-Orsay serves produces a slightly expanded capsid with less RNA packaged as a highly tractable model for studying viral pathogenesis, our inside, suggesting important roles for the N-terminal arm in results should provide a valuable structural framework for further capsid stabilization and genome packaging. Our results also studies of Orsay replication and infection. demonstrate that the Orsay capsid is structurally distinct from alphanodaviruses. Together with primary sequence-based phy- virology | crystallography | microscopy logeny, these results suggest that the nematode-infecting viruses and betanodaviruses have a common evolutionary history. n the past four decades, the nematode Caenorhabditis elegans Ihas become an important model organism for studying various Significance biological processes such as development, metabolism, aging, the cell cycle, and gene regulation (1). Many key biomedical dis- Since the discovery of Orsay, the first virus that naturally coveries about common diseases, including Alzheimer’s disease, infects nematodes, it has been widely expected that Caeno- diabetes type 2, and even depression, were first made in C. ele- rhabditis elegans-Orsay would serve as a highly tractable gans (2). It has been estimated that around 70% of human genes model for studying viral pathogenesis. Here we report the have homologs in C. elegans, including the majority of human crystal structure of the Orsay virus. The Orsay capsid contains disease genes and pathways (3). 180 copies of the capsid protein, each consisting of a jelly-roll It was not until very recently that Orsay, the first virus that β-barrel and a protrusion domain. Although sequence analyses naturally infects C. elegans in the wild, was discovered (4). In indicate that Orsay is related to nodaviruses, the structure addition, two other nematode viruses, Santeuil and Le Blanc, reveals substantial differences compared with the insect- BIOPHYSICS AND both of which infect C. briggsae, have also been identified (4, 5). infecting alphanodaviruses. Small plant RNA viruses are the Infections by these newly identified viruses cause abnormal in- closest homologs for Orsay when their β-barrel domains are COMPUTATIONAL BIOLOGY testinal morphologies without obvious effect on longevity or compared. Our results have not only shed light on the evolu- brood size. Orsay viral particles have been observed intracellularly tionary lineage of Orsay but have also provided a framework in both intestinal cells and the somatic gonad (4). Sequence for further studies of Orsay–host interaction. analysis indicated that all three nematode viruses are related to viruses in the family Nodaviridae (4, 5). Nodaviruses are non- Author contributions: Y.R.G., D.W., W.C., W.Z., and Y.J.T. designed research; Y.R.G., C.F.H., enveloped RNA viruses that can be further classified as alpha- and J.J. performed research; H.J. contributed new reagents/analytic tools; Y.R.G., C.F.H., nodaviruses and betanodaviruses, which are known to infect and Y.J.T. analyzed data; and Y.R.G., C.F.H., D.W., W.Z., and Y.J.T. wrote the paper. insects and fish, respectively (6). The authors declare no conflict of interest. Orsay virus has a bipartite, positive-sense RNA genome ∼6.3 This article is a PNAS Direct Submission. kb in size (4). The RNA1 segment of the Orsay virus encodes Data deposition: Crystallography, atomic coordinates, and structure factors for the full- a predicted RNA-dependent RNA polymerase (RdRP) of 982 length capsid protein (CP) and CP42–391 reported in this paper have been deposited in the amino acids. RNA2 has two ORFs: one coding for a putative Protein Data Bank, www.pdb.org (PDB ID codes 4NWV and 4NWW, respectively). The EM capsid protein (CP) and the other for a nonstructural protein δ map has been deposited in the EMDataBank (accession no. EMD-5952). of unknown function. The protein translation of the Orsay CP 1To whom correspondence should be addressed. Email: [email protected]. uses an AUG-independent, noncanonical initiation mechanism This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that is likely shared among all three nematode viruses (7). In 1073/pnas.1407122111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1407122111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12781–12786 Downloaded by guest on September 27, 2021 Results in a T = 3 icosahedral symmetry (Fig. 1). A total of 60 trimeric Orsay VLP Expression and Purification. The Orsay CP was overex- spikes are observed on quasi-threefold axes, with each one in pressed in Escherichia coli to produce recombinant VLPs. Be- direct contact with three surrounding spikes. The capsid shell ∼ ∼ cause the translation initiation site of the Orsay CP had not been is 30 Å thick, and the spikes are 35 Å tall. Small plateaus are mapped at the time these studies were initiated, we performed observed on fivefold axes, whereas large depressions are formed a multisequence alignment with existing betanodavirus CPs to at the center of CP hexamers on threefold axes. determine a likely coding region, assuming that CP ORFs should be well-conserved. Our CP construct had a total of 392 amino Structure of the Orsay CP. The structure of each Orsay CP can be divided into three modular regions: an N-terminal arm (residues acid residues, with its N terminus ANKNN- starting from 138 – – residues upstream of the first Met in the originally annotated CP 1 43), a shell (S) domain (residues 44 214), and a protrusion (P) ORF (4). Recombinant CP was purified by chromatography and domain (residues 215 to the end) (Fig. 2 A and B). The S domain exhibits a canonical jelly-roll, β-barrel fold that is frequently seen separated by gel filtration into two size populations (Fig. S1). in nonenveloped icosahedral viruses (13). In the jelly-roll β-barrel, The first, which was found in the void volume, contained VLPs β ∼ eight antiparallel -strands, which are often named sequentially with an 35-nm diameter according to negative-staining electron from B to I, form two twisted β-sheets with four β-strands arranged microscopy (EM). The overall size and morphology of these VLPs in the order of BIDG and CHEF, respectively. The S domain by were indistinguishable from those of authentic virions purified itself forms a continuous capsid shell without any notable holes or from infected worms (7).