Structure of the Mature P3-virus Particle Complex of Cauliflower Mosaic Virus Revealed by Cryo-electron Microscopy Ce ´ lia Plisson 1 †, Marilyne Uzest 2 †, Martin Drucker 2 , Re ´ my Froissart 2 Christian Dumas 3 , James Conway 4 , Daniel Thomas 1 , Ste ´ phane Blanc 2 and Patrick Bron 1 * 1 Universite ´ Rennes I, UMR 6026 CNRS, Campus de Beaulieu, 35042 Rennes, France 2 Station de Recherches de Pathologie Compare ´e, UMR 1231, INRA-CNRS-Universite ´ Montpellier II, 30380 Saint-Christol-les-Ale `s, France 3 Centre de Biochimie Structurale, UMR CNRS 5048 UMR 554 INSERM, Universite ´ Montpellier I, France 4 Laboratoire de Microscopie Electronique, Institut de Biologie Structurale J.-P. Ebel Grenoble 38027, France The cauliflower mosaic virus (CaMV) has an icosahedral capsid composed of the viral protein P4. The viral product P3 is a multifunctional protein closely associated with the virus particle within host cells. The best- characterized function of P3 is its implication in CaMV plant-to-plant transmission by aphid vectors, involving a P3–virion complex. In this transmission process, the viral protein P2 attaches to virion-bound P3, and creates a molecular bridge between the virus and a putative receptor in the aphid’s stylets. Recently, the virion-bound P3 has been suggested to participate in cell-to-cell or long-distance movement of CaMV within the host plant. Thus, as new data accumulate, the importance of the P3–virion complex during the virus life-cycle is becoming more and more evident. To provide a first insight into the knowledge of the transmission process of the virus, we determined the 3D structures of native and P3-decorated virions by cryo-electron microscopy and computer image processing. By difference mapping and biochemical analysis, we show that P3 forms a network around the capsomers and we propose a structural model for the binding of P3 to CaMV capsid in which its C terminus is anchored deeply in the inner shell of the virion, while the N-terminal extremity is facing out of the CaMV capsid, forming dimers by coiled-coil interactions. Our results combined with existing data reinforce the hypothesis that this coiled-coil N-terminal region of P3 could coordinate several functions during the virus life-cycle, such as cell-to-cell movement and aphid-transmission. q 2004 Elsevier Ltd. All rights reserved. Keywords: CaMV/P3; protein structure; cryo-electron microscopy; icosa- hedral reconstruction; aphid-transmission *Corresponding author Introduction Cauliflower mosaic virus (CaMV) is the type member of the family Caulimoviridae (genus Caulimovirus) which, together with hepadnaviruses, constitute the para-phylletic group of pararetro- viruses having a DNA-based genome replicated via reverse transcription of a pre-genomic RNA. 1,2 The approximately 8 kbp circular double-stranded DNA genome of CaMV encodes eight major open reading frames (ORFs). To the products of six ORFs at least one biological function has been assigned. 3 ORF I encodes P1, a protein involved in cell-to-cell movement and systemic spread of the virus within the host. ORFs II and III are described in further detail below as their products (P2 and P3) form a complex with virus particles to promote plant-to- plant vector-transmission, currently the only bio- logical function demonstrated experimentally for the P3–virion complex. ORF IV codes for the precursor of the coat protein (57 kDa), which is post-translationally processed into variable amounts of three major forms of P4 with molecular mass ranging between 35 kDa and 44 kDa. The reverse transcriptase replicating the genome is encoded by ORF V. ORF VI directs the expression of a multifunctional protein, P6, that constitutes the 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. † C.P. & M.U. contributed equally to this work. Abbreviations used: CaMV, cauliflower mosaic virus; ORF, open reading frame; edIB, electron-dense inclusion bodies; elIB, electron-lucent inclusion bodies. E-mail address of the corresponding author: patrick.bron@univ-rennes1.fr doi:10.1016/j.jmb.2004.11.052 J. Mol. Biol. (2005) 346, 267–277