Structural basis of nanobody-recognition of grapevine fanleaf virus and of virus resistance loss

Orlov, I.; Hemmer, C.; Ackerer, L.; Lorber, B.; Ghannam, A.; Poignavent, V.; Hleibieh, K.; Sauter, C.; Schmitt-Keichinger, C.; Belval, L.; Hily, J.-M.; Marmonier, A.; Komar, V.; Gersch, S.; Schellenberger, P.; Bron, P.; Vigne, E.; Muyldermans, S.; Lemaire, O.; Demangeat, G.; Ritzenthaler, C.; Klaholz, B.

doi:10.1101/728907

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bioRxiv · 10.1101/728907

Structural basis of nanobody-recognition of grapevine fanleaf virus and of virus resistance loss

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Abstract

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently and shown to be highly effective in plants including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb23 complex which provides the basis for the molecular recognition by the nanobody. The structure reveals a composite binding site bridging over 3 domains of the capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.\n\nSignificanceGrapevine fanleaf virus (GFLV) is a picorna-like plant virus that severely impacts vineyards worldwide. While Nanobodies (Nb) confer resistance to GFLV in plants the underlying molecular mechanism of action is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb complex. It uncovers the conformational epitope on the capsid surface which is a composite binding site into which the antigen loop is accommodated through an induced fit mechanism. Furthermore, we describe several resistance-breaking isolates of GFLV with reduced Nb binding capacity. Those that carry a C-terminal extension also fail to be transmitted by nematodes. Together, these data provide structure-function insights into the Nb-GFLV recognition and the molecular mechanism leading to loss of resistance.

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Orlov, I., Hemmer, C., Ackerer, L., Lorber, B., Ghannam, A., Poignavent, V., Hleibieh, K., Sauter, C., Schmitt-Keichinger, C., Belval, L., Hily, J.-M., Marmonier, A., Komar, V., Gersch, S., Schellenberger, P., Bron, P., Vigne, E., Muyldermans, S., Lemaire, O., Demangeat, G., Ritzenthaler, C., Klaholz, B.. 2019-08-08. Structural basis of nanobody-recognition of grapevine fanleaf virus and of virus resistance loss. https://doi.org/10.1101/728907

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