Atomic Structure of Cucumber Necrosis Virus and the Role of the Capsid in Vector Transmission

Abstract

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome packaged in a T‫3؍‬ icosahedral particle. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. CNV undergoes a conformational change upon binding to the zoospore that is required for transmission, and specific polysaccharides on the zoospore surface have been implicated in binding. To better understand this transmission process, we have determined the atomic structure of CNV. As expected, being a member of the Tombusvirus genus, the core structure of CNV is highly similar to that of Tomato bushy stunt virus (TBSV), with major differences lying on the exposed loops. Also, as was seen with TBSV, CNV appears to have a calcium binding site between the subunits around the quasi-3-fold axes. However, unlike TBSV, there appears to be a novel zinc binding site within the ␤ annulus formed by the N termini of the three C subunits at the icosahedral 3-fold axes. Two of the mutations causing defective transmission map immediately around this zinc binding site. The other mutations causing defective transmission and particle formation are mapped onto the CNV structure, and it is likely that a number of the mutations affect zoospore transmission by affecting conformational transitions rather than directly affecting receptor binding. C ucumber necrosis virus (CNV) is a member of the genusTombusvirus and has a monopartite positive-sense RNA genome (1, 2). CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus (2-4). The type species of the Tombusvirus genus is Tomato bushy stunt virus (TBSV), and the structure of TBSV was the first virus structure determined by X-ray crystallography (5).These viruses have a Tϭ3 icosahedral protein shell with a diameter of ϳ300 Å formed from 180 identical coat protein (CP) subunits. The three conformationally distinct copies of the CPs, called A, B, and C, are shown in Fig. 1. The C subunits lie next to the icosahedral 2-fold axes, while the A and B subunits surround the 5-fold axes. Each capsid protein is comprised of three domains: the first ϳ60 residues at the N-terminal region (R), the shell (S) domain, and the protruding (P) domain (Fig. 2A). The first ϳ90 residues are disordered in the A and B subunits, while the first ϳ60 residues are disordered in the C subunit. Residues ϳ60 to 78 in the C subunit form a structure called the ␤ annulus that is thought to stabilize the capsid. The first ϳ60 residues at the N-terminal region interact with the RNA interior. Between this region (R) and the S domain are ϳ30 residues that act as a flexible connecting arm. The S domain is ϳ170 residues and forms a tight protein shell around the RNA genome. The final ϳ115 residues form the P domains, which combine with the adjacent subunit's P domains to form dimeric protrusions from the shell. The A subunits lie immediately adjacent to the icosahedral 5-fold axes, and their P domains interact with the P domains of adjacent B subunits to form 60 of the 90...

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“…The common feature of these viruses is that all of the subunits possess the canonical jellyroll fold, which is composed with eight antiparalleled β-sheets defined as a β-barrel structure (Stirk et al, 1992). For the viruses in the first group, the subunit is composed of two βÀbarrel structures: one contributes to the surface domain and the other to the protruding domain (Olson et al, 1983;Hogle et al, 1986;Morgunova et al, 1994;Wada et al, 2008;Li et al, 2013). For the viruses in the second group, the subunit is composed of only one β-barrel structure that contributes to the surface domain (Oda et al, 2000;Makino et al, 2013).…”

“…Based on structures registered in the PDB (protein data bank), viruses in the family Tombusviridae can be divided into two groups: one group is characterized by obvious protrusions on the surface while the other has a rather smooth surface. The first group includes Tomato bushy stunt virus (TBSV) (Olson et al, 1983), Carnation mottle virus (CarMV) (Morgunova et al, 1994), Turnip crinkle virus (TCV) (Hogle et al, 1986), Melon necrotic spot virus (MNSV) (Wada et al, 2008) and Cucumber necrosis virus (CNV) (Li et al, 2013). The second group is represented by Panicum mosaic virus (PMV) (Makino et al, 2013) and Tobacco necrosis virus A (TNV-A) (Oda et al, 2000).…”

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

“…The virus concentration was determined spectrophotometrically (the absorbance at 260 nm of a 1-mg/ml suspension of CNV is 4.5). CsCl purification of CNV was performed as previously described (29).…”

“…To meet the requirements of quasiequivalence, the CP adopts three different conformations (A, B, and C). The arms are disordered in the A and B subunits, while C subunits are characterized by ordered arms that extend to form the ␤-annulus at the particle 3-fold axis (29,30). In analogy to Tomato bushy stunt virus (TBSV), Ca 2ϩ ions stabilize the interactions between adjacent A, B, and C subunits in the shell domain by bringing together aspartate residues at the particle quasi-3-fold axis (Q3) (31,32).…”

“…Each of the CP subunits folds into three major domains: an RNA binding domain (R), the shell domain (S), and the protruding domain (P). The P and the S domains are connected by a short hinge (h) region while the arm (a) region flexibly tethers the R and S domains, both allowing for Tϭ3 icosahedral symmetry and quasiequivalent subunit interactions (29,30). To meet the requirements of quasiequivalence, the CP adopts three different conformations (A, B, and C).…”