Potyvirus virion structure shows conserved protein fold and RNA binding site in ssRNA viruses

Abstract

Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å. The atomic model shows a conserved fold for the CPs of flexible filamentous plant viruses, including a universally conserved RNA binding pocket, which is a potential target for antiviral compounds. This conserved fold of the CP is widely distributed in eukaryotic viruses and is also shared by nucleoproteins of enveloped viruses with segmented (-)ssRNA (negative-sense ssRNA) genomes, including influenza viruses.

Fig. 1. Near-atomic cryoEM structure of WMV virions.

(A and B) Renderings of the 3D cryoEM map [cutaway mode in (B)] calculated for WMV. Segmented densities are depicted as follows: WMV CP core region (light blue), N-terminal arm (dark blue), C-terminal arm (yellow), scattered densities at the inner side of the helix (orange), and density for the ssRNA (red). One of the CP subunits is seen as gray in (A). (C) Two views of the atomic model calculated for WMV CP within the semitransparent density for a subunit segmented from the cryoEM map. (D) Segmented densities for WMV CP subunits are depicted. The subunit N_i is colored on the basis of the calculated electrostatic surface potential.

Fig. 2. Protein-ssRNA interactions in WMV.

(A) Atomic models for WMV CP and ssRNA (ssRNA modeled as a polyU) are seen inside the densities for protein (gray) and RNA (red). (B and C) Closeup views of the RNA binding pocket within WMV CP. The CP is seen in ribbons, and some of the amino acids that interact with the ssRNA are displayed. Density for the RNA is rendered in gray mesh. (D) The ribbon representation for WMV CP is seen as green, but the segments of the protein linking the amino acids that bind to RNA are highlighted in magenta (from Ser¹⁴⁰ to Arg¹⁷²) and yellow (from Arg¹⁷² to Asp²¹⁶).

Fig. 3. Conserved RNA binding pocket in flexible filamentous plant viruses.

(A to C) Close-up views of the RNA binding pockets of the CPs from WMV (A), PepMV [Protein Data Bank (PDB) code: 5FN1 (4)] (B), and papaya mosaic virus (PapMV) [PDB code: 4DOX (5)] (C). For clarity, (A) and (B) show the ssRNA model calculated for WMV virion. The structure for PapMV CP was calculated for isolated and RNA-free protein by crystallography (5). (D) Consensus sequence logos for the CPs from different families of flexuous filamentous plant viruses. The distance was measured by the number of residues (average and SD) between invariant amino acids (Ser, Arg, and Asp), indicated by gray lines and numbers. The number of reference sequences (36) aligned in each family is also indicated. The color scheme of amino acid symbols is as follows: blue for hydrophilic, green for neutral, and black for hydrophobic.

Fig. 4. Conserved fold in eukaryotic ssRNA viruses.

Ribbon representations for the CP and NP regions showing structural homology. The regions of the proteins are depicted in rainbow colors (from N- to C-terminal ends) and are seen together with bound ssRNA (whenever available). TM-scores (15) for the structural alignments between WMV CP and each of the atomic structures are seen inside the orange boxes. Values of TM-scores around 0.5 are indicative of proteins with the same fold. Depicted structures are as follows: WMV CP and ssRNA from the current work, PepMV CP and ssRNA (PDB code: 5FN1) (4), influenza virus A NP (PDB code: 3ZDP) (40), Rift Valley fever virus (RVFV) NP in complex with ssRNA (PDB code: 4H5O) (41), La Crosse virus NP and ssRNA (PDB code: 4BHH) (42), and Tomato spotted wilt virus (TSWV) NP in complex with ssRNA (PDB code: 5IP2) (43).