A DETAILED VIEW OF POTATO VIRUS X, A FILAMENTOUS FLEXIBLE VIRUS

The cryo-electron microscopy structure of Potato virus X (PVX) has been determined at a resolution of 2.2 Å. The particularly well-defined density of the coat proteins and of the genomic RNA has allowed a detailed analysis of protein-RNA interactions, including those mediated by solvent molecules.

STRUCTURAL BIOLOGY

44 ESRF

PVX is a filamentous plant virus considered to be the prototype of the Alphaflexiviridae family and the archetypal member of the genus potexvirus [1]. It infects several herbaceous plants, mostly Solanaceae, and has been widely studied as a result of its detrimental impact on the global plant economy. It is also important for its potential biotechnological applications, which have not yet been completely explored [2].

Filamentous viruses represent an atypical case for structural studies: they are periodic objects with helical symmetry along one direction, but they do not develop along a straight axis since they are slightly flexible, that is, they bend in the long-medium range. In order to overcome this characteristic, it is necessary to select a small section of the virus particle, choosing a box of appropriate size, and consider this portion as the unit that repeats by translation along the entire length of the virus (and along all the other numerous viral particles present in each field of observation).

Analysis on CM01 made it possible to obtain the structure of PVX at a resolution of 2.2 Å, the highest resolution obtained for a filamentous virus and one of the highest for cryo-EM. In the single particle reconstruction obtained, the density of each cap protein protomer (CP) is clearly defined, as is the density for bound viral ss(+)RNA and density that can be assigned as due to the presence of solvent molecules (Figure 29). The CP comprises three domains: a short N-terminal domain (domain I), a central core

domain (domain II), and a C-terminal domain (domain III). Domain I protrudes from the CP core and, in the assembled virion, embraces domain II of a nearby CP, stabilising the virion structure (Figure 29). The only portion of the virion not visible in the density map is represented by the 28 amino acid residues at the N-terminus of the coat protein, which are disordered.

Fig. 29: a) Top and (b) side views of the density of a portion of the PVX virus. The three domains of the protein are shown in yellow,

green and cyan, the RNA in red.

The viral particle is made up of repeats of 8.8 CP that wrap around to form a left-handed helix. The helical arrangement of the CP monomers results in the alignment of the cavities between domains II and III of each CP subunit, generating a continuous helical crevice, in which the viral RNS is hosted and protected, in the interior capsid surface (Figure 30). Due to the presence of Arg and Lys residues, the internal surface of the crevice is positively charged, facilitating the binding of the negatively charged phosphate groups of the nucleic acid. The density for the genomic ssRNA is an average of segments that contain different sequences and the regions of the nitrogenous bases are mixtures of the four different possible nucleotides. Nonetheless, the density is well defined and has allowed the construction of a 134 poly(U) ssRNA molecule in the repeating unit of the virion.

The stabilisation of the bound RNA is achieved exclusively via direct or water-mediated interactions between its sugar/phosphate backbone and residues of the PVX CP. This allows the capsid to accommodate different sequences of nucleotides. Most of the PVX CP residues involved in RNA packaging are structurally conserved among other family members,

Fig. 30: Two adjacent CP molecules in the virion. One is shown as a surface (yellow) and the second as a

cartoon model (green). The N-terminus of the green CP embraces the nearby CP. The red atoms represent

five nucleotides of RNA.