1 The near-atomic cryoEM structure of a flexible filamentous plant virus shows 2 homology of its coat protein with nucleoproteins of animal viruses 3 Xabier Agirrezabala1, F. Eduardo Méndez-López2, Gorka Lasso3, M. Amelia 4 Sánchez-Pina2, Miguel A. Aranda2, and Mikel Valle1,* 5 1Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC 6 bioGUNE, 48160 Derio, Spain 7 2Centro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de 8 Investigaciones Científicas (CSIC), 30100 Espinardo, Murcia, Spain 9 3Department of Biochemistry and Molecular Biophysics, Columbia University, 10032 10 New York, USA 11 *Correspondence: [email protected] 12 13 14 15 16 17 18 19 20 21 22 1 23 Abstract 24 Flexible filamentous viruses include economically important plant pathogens. 25 Their viral particles contain several hundred copies of a helically arrayed coat 26 protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å 27 resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a 28 representative of the genus Potexvirus (family Alphaflexiviridae). Our results 29 allow modeling of the CP and its interactions with viral RNA. The overall fold of 30 PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus 31 (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main 32 difference between potexvirus CP and phlebovirus NP is in their C-terminal 33 extensions, which appear to determine the characteristics of the distinct 34 multimeric assemblies- a flexuous, helical rod or a loose ribonucleoprotein 35 (RNP). The homology suggests gene transfer between eukaryotic (+) and (- 36 )ssRNA viruses. 37 38 39 40 41 42 43 44 45 2 46 Introduction 47 Flexible filamentous viruses are ubiquitous plant pathogens that have an enormous 48 impact in agriculture (Revers and Garcia, 2015). Their infective particles are non- 49 enveloped and flexible rod-shaped virions (Kendall et al., 2008) and contain several 50 hundreds of copies of a coat protein (CP) arranged in a helical fashion protecting a 51 ssRNA of positive polarity, or (+)ssRNA (Kendall et al., 2008). They are distributed in 52 families Alphaflexiviridae, Betaflexiviridae, Closteroviridae, and Potyviridae, which have 53 different genomic organizations (particularly different between Potyviridae and the rest 54 of the groups) and belong to different superfamilies, but it is thought that their CPs 55 have strong evolutionary relationships (Koonin et al., 2015). Structural studies of 56 virions by X-ray fiber diffraction and cryoEM have indeed revealed a common 57 architecture for flexible plant viruses. The filaments are 120-130 Å in diameter and the 58 CPs are arranged following helical symmetry with slightly less than 9 subunits per turn 59 (Kendall et al., 2008). This overall arrangement is shared by Soybean mosaic virus 60 (SMV) a potyvirus (from the family Potyviridae), and three different potexviruses (family 61 Alphaflexiviridae), Potato virus X (PVX), Papaya mosaic virus (PapMV), and Narcissus 62 mosaic virus (NMV) (Kendall et al., 2013, Yang et al., 2012, Kendall et al., 2008). The 63 flexibility of the virions has limited high resolution structural studies, and most of the 64 previous data were at moderate resolution. Very recently, the cryoEM structure of 65 Bamboo mosaic virus (BaMV), another potexvirus, was determined at 5.6 Å (DiMaio et 66 al., 2015), and the CP was modeled based on the atomic structure of a truncated 67 version of the CP from Papaya mosaic virus (PapMV CP) (Yang et al., 2012). The work 68 revealed that N- and C-terminal extensions of the CP mediate viral polymerization and 69 allow for the flexuous nature of the virions. 70 PepMV is another potexvirus which has emerged recently (Jones et al., 1980), 71 progressing from endemic to epidemic in tomato crops causing severe economic 72 losses worldwide (Hanssen and Thomma, 2010). PepMV is transmitted by mechanical 3 73 contact and virions contain a (+)ssRNA of about 6.4 kb (Aguilar et al., 2002). The 74 PepMV CP is strictly required for cell-to-cell movement of the virus (Sempere et al., 75 2011). 76 We present the cryoEM structure of PepMV virions at 3.9 Å of resolution. The near- 77 atomic 3D map allows for accurate modelling of the CP, the viral RNA and their 78 interaction. In vivo functional studies of several CP mutants confirm the role of several 79 residues in RNA binding and polymerization. Surprisingly, we have also found a clear 80 structural homology between the CP of flexuous viruses and the NP of the genus 81 Phlebovirus, a group of enveloped viruses with a segmented (-)ssRNA genome. The 82 NPs from phleboviruses are associated with the viral genome in loose RNPs 83 (Raymond et al., 2012) protected inside an envelope, in which inserted glycoproteins 84 construct an icosahedral shell (Huiskonen et al., 2009, Freiberg et al., 2008). Despite 85 the divergence of both viral groups, CP from potexviruses and NP from phleboviruses 86 have the same all alpha-helix fold, and their high similarity suggests a horizontal gene 87 transfer event between these evolutionary distant groups of eukaryotic RNA viruses. 88 Results and Discussion 89 CryoEM structure of PepMV 90 We have analyzed by cryoEM PepMV virions isolated from infected Nicotiana 91 benthamiana plants. The 3D map at 3.9 Å of resolution (Figures 1A and 1B and 92 Figure1-figure supplement 3) was calculated by single particle-based helical image 93 processing implemented in Spring software (Desfosses et al., 2014). The cryoEM map 94 reveals a left-handed helix with a diameter of 130 Å and an inner narrow channel of 13 95 Å, and the structure shows a pitch of 34.6 Å and 8.7 CP copies per turn. This overall 96 helical arrangement is in agreement with previous works with other potexviruses 97 (Kendall et al., 2013, Yang et al., 2012), but in the current case and in the recent 4 98 cryoEM structure of BaMV (DiMaio et al., 2015) the attained resolutions allowed a clear 99 assignment of the symmetry. Our cryoEM map reaches near-atomic resolution, to date 100 the highest resolution structural data for a flexible filamentous virion, where bulky 101 protein side chains are discernible (Figure 1C). This allowed us to generate an atomic 102 model for PepMV CP (Figure 1D) by iterative modelling starting with the atomic 103 structure of PapMV CP (see Materials and methods). The PepMV CP structure thus 104 generated has three major regions: the core; an N-terminal flexible arm; and a C- 105 terminal extension. The first 20 amino acids in the N-terminal side are not included in 106 the atomic model since the region projects outwards, and its density vanishes due to 107 high flexibility. The modelled structure for PepMV CP is, as defined for PapMV CP, an 108 all-helix fold, and the low RMSD between the two structures (1.5 Å in the core region) 109 reflects their clear homology (Figure1-figure supplement 5). 110 CP and ssRNA interaction 111 The high level of structural details of our cryoEM map permits the segmentation of the 112 density for the ssRNA and the analysis of the protein-RNA interactions. The ssRNA 113 runs in a helix of 70 Å in diameter (Figure 1B). Each CP binds five ribonucleotides 114 (Figure 1D), so that the entire genome of PepMV would require 1290 copies of PepMV 115 CP spanning 510 nm (3.95 Å of axial rise/subunit), which agrees with the 509 nm 116 length originally reported for the virus (Jones et al., 1980). The signal clearly separates 117 the individual nucleotides, but we could not identify the bases due to the helical 118 averaging of the variable sequence of the ssRNA. In order to explore the protein- 119 ssRNA interactions, we modelled a polyU and included a set of four PepMV CP 120 monomers in a molecular dynamics flexible fitting (MDFF) simulation (Trabuco et al., 121 2008) (Figure 2-figure supplements 1 and 2). The results show that the RNA resides in 122 a continuous groove with a high electropositive potential (Figure 2A) constructed by 123 CPs from consecutive turns of the helix. Local resolution measurements in the cryoEM 5 124 map suggest a high variability in positions 3 and 4 at the segmented density for the 125 ssRNA (Figure 2B). Several amino acids display potential interactions with the 126 phosphate backbone of the ssRNA. S92 and S94 in one face, and Q203 at the 127 opposite side of the ssRNA might interact at positions 1 and 3 (Figure 2C). Backbone 128 torsion angles of 85⁰ and 130⁰ at these positions allow the binding of the base at 129 position 2 into a deep pocket (Figure 2D). Three polar-charged residues, R124, D163 130 and K196 might establish H-bonding contacts with this RNA base. D163 is in the most 131 conserved region within the genus Potexvirus, and the consensus sequence FDFFD 132 (Figure 2-figure supplement 3) constructs the floor of the RNA binding pocket. This 133 pocket is large enough to accommodate pyrimidines or purines, and the presence of 134 three amino acids ensures the interaction with the RNA regardless of the nucleotide.. 135 We tested several PepMV CP mutants in trans-complementation assays (Sempere et 136 al., 2011). Here, a PepMV construct that expresses GFP instead of CP (PepGFPΔCP) 137 acts as reporter of the complementation by the co-expression of selected CP mutants. 138 The fluorescent signal by GFP allowed following the cell-to-cell movement of the virus. 139 The results (Figure 2E) reveal that the three amino acids at the binding pocket for 140 nucleotide at position 2 (R124, D163 and K196) are required to complement the cell-to- 141 cell movement of the CP-defective PepMV mutant, suggesting a disruption of the CP- 142 RNA interaction needed in virus intercellular transport.