Characterization of P1 leader proteases of the Potyviridae family and identification of the host factors involved in their proteolytic activity during viral infection Hongying Shan Ph.D. Dissertation Madrid 2018 UNIVERSIDAD AUTONOMA DE MADRID Facultad de Ciencias Departamento de Biología Molecular Characterization of P1 leader proteases of the Potyviridae family and identification of the host factors involved in their proteolytic activity during viral infection Hongying Shan This thesis is performed in Departamento de Genética Molecular de Plantas of Centro Nacional de Biotecnología (CNB-CSIC) under the supervision of Dr. Juan Antonio García and Dr. Bernardo Rodamilans Ramos Madrid 2018 Acknowledgements First of all, I want to express my appreciation to thesis supervisors Bernardo Rodamilans and Juan Antonio García, who gave the dedicated guidance to this thesis. I also want to say thanks to Carmen Simón-Mateo, Fabio Pasin, Raquel Piqueras, Beatriz García, Mingmin, Zhengnan, Wenli, Linlin, Ruiqiang, Runhong and Yuwei, who helped me and provided interesting suggestions for the thesis as well as technical support. Thanks to the people in the greenhouse (Tomás Heras, Alejandro Barrasa and Esperanza Parrilla), in vitro plant culture facility (María Luisa Peinado and Beatriz Casal), advanced light microscopy (Sylvia Gutiérrez and Ana Oña), photography service (Inés Poveda) and proteomics facility (Sergio Ciordia and María Carmen Mena). Thanks a lot to all the assistance from lab313 colleagues. Thanks a lot to the whole CNB. Thanks a lot to the Chinese Scholarship Council. Thanks a lot to all my friends. Thanks a lot to my family. Madrid 20/03/2018 Index I CONTENTS Abbreviations………………………………………….……………………….……...VII Viruses cited…………………………………………………………………..……...XIII Summary…………………………………………………………………...….…….XVII Resumen…………………………………………………………......…...…………..XXI I. INTRODUCTION……………………………………………….….….1 I.1 Functions of plant viral proteases…………………………….……….………....7 I.1.1 Replication………………………………………………………………………7 I.1.2 Viral counterdefense…………………………………………….…….…..…….9 I.1.3 Virion maturation………………………………………………….…….……..11 I.1.4 Host range definition……………………………………………….………..…12 I.1.5 Proteolytic activity-unrelated functions………………………………..............13 I.1.6 Functions still uncharacterized…………………………………….….………..13 I.2 Leader proteases in the Potyviridae family……………………….…….………14 I.2.1 Proteolytic diversity of potyvirids………………………………….……….….14 I.2.2 Functions of P1 proteases…………………………………….………….……..16 I.3 Objectives ……………………….…………………….........................................18 II. MATERIALS AND METHODS……………………………………19 II.1 Virus and bacterial strains…………………………………………….…….….…21 II.2 Plant hosts, trees and callus…………………………………………….…….…..21 II.3 Agroinfiltration………………………………………………………….………..21 II.4 Fluorescence imaging and quantification…………………………….………..…22 II.5 DNA plasmids …………………………………………….………….…………..22 II.5.1 Viral constructs………………………………………………………….…….22 II.5.2 Transient expression constructs………………………………………….……27 II.6 DNA constructs to be expressed by in vitro transcription and translation.......…..29 II.7 In vitro transcription and translation assays………………………………….…..31 II.8 Western blot assays………………………………………………….….………...31 II.9 RT-qPCR analysis………………………………………………….……….…….31 II.10 Sequence, phylogenetic and data analysis……………..………………………..32 III II.11 Preparation of BYL extract………………………….…………….…....……….33 II.12 Ion exchange chromatography……………………………………..…….…..….34 II.13 Gel filtration chromatography……………………………..…….………….…..35 II.14 Mass spectrometry……………………………………………………...……….35 II.14.1 In-gel protein digestion and MALDI peptide mass fingerprinting……..……35 II.14.2 Liquid chromatography and mass spectrometry analysis………....................36 II.15 P1 protease activity complementation assays…………………………………...37 II.16 P1 processing activity in N. benthamiana plants in which candidate genes have been down-regulated by virus-induced gene silencing (VIGS)……..….37 III. RESULTS……………………………………………………………39 III.1 P1 protein classification in the Potyviridae family……………………………...41 III.1.1 Self-cleavage activity in WGE and RRL of potyvirid P1 proteins…………..41 III.1.2 RNA silencing suppression activity of potyvirid P1 proteins………..............42 III.1.3 P1 serine proteases do not display in trans activity.…………………………44 III.2 Host range definition of potyviral P1 proteins…………………………………..46 III.2.1 CVYV P1a and PPV P1 in Nicotiana benthamiana…………………….……46 III.2.1.1 Viral accumulation of chimeric PPV bearing P1a is increased in the presence of an extra NIa cleavage site between P1a and HC in N. benthamiana………………………………………………….....46 III.2.1.2 CVYV P1a shows incomplete catalytic activity and the p19 silencing suppressor complements P1a defects in N. benthamiana ……...49 III.2.1.3 cis-supply of CVYV P1a impairs HC silencing suppression activity, which is restored by NIa protease-mediated cleavage in N. benthamiana.51 III.2.2 CVYV P1a and PPV P1 in Cucumis sativus……………………………………53 III.2.2.1 Viral accumulation of chimeric PPV bearing CVYV P1a is highly enhanced in local infections in C. sativus…………………………...……53 III.2.2.2 cis-supply of CVYV P1a effectively sustains PPV HC RSS activity in C.sativus…………………………………………………..………….........54 III.2.3 N-terminal deletion of P1 facilitates PPV replication in a non-permissive host…………………………………………………..……...………………..56 III.2.3.1 Delimitation of the antagonistic domain of PPV P1……………………...56 III.2.3.2 CVYV P1a and TuMV P1 proteolytic domains remain host factor IV dependent in vitro……………………….………………….………..……57 III.2.3.3 A proper N-terminus is essential for plant host factor-independent activity of PPV P1Pro……………………………………………...………59 III.2.3.4 PPV P1Pro activity is not restricted by host specificity in planta and highly facilitates local PPV replication in C. sativus………….………….60 III.2.3.5 Protease domain of CVYV P1a (P1aPro) is host-dependent in planta behaving as full-length CVYV P1a………………………...…….……….64 III.2.3.6 Deletion of the N-terminal antagonistic domain of P1 does not boost systemic infection in Prunus hosts………………………..……..………..67 III.3 Purification and identification of host co-factor(s) of PPV P1 protease….……..69 III.3.1 Preparation of an extract from N. tabacum BY-2 cells that can complement PPV P1 proteolytic activity in RRL translation system……………………...70 III.3.1.1 PPV P1 deficient autocatalytic activity in RRL can be restored by addition of a small percentage of WGE to the translation mixture…….....70 III.3.1.2 A protein extract from N. tabacum BY-2 cells is able to complement PPV P1 processing allowing autocatalytic activity in RRL translation system...71 III.3.2 Purification of the P1 co-factor(s) from BYL extract………………………..73 III.3.2.1 Anion exchange and size exclusion chromatography…………………….73 III.3.2.2 Tandem anion and cation exchange chromatography previous to size exclusion purification…………………………………...…….…………..78 III.3.3 Identification of proteins in purified samples and selection of P1 plant co-factor(s) candidates……………………………………….……………….82 III.3.3.1 Mass spectrometry analyses………………………………………………82 III.3.3.2 Selection of candidates……………………………………………………87 III.3.4 Validation of host factor candidates involved in P1 proteolytic autocleavage.88 III.3.4.1 Use of protein inhibitors to abolish in vitro RRL complementation of P1 self-cleavage………………………………………………....……………88 III.3.4.2 Use of VIGS for candidate validation in N. benthamiana plants…………90 IV. DISSCUSSION……………………………………………………....95 IV.1 Type A vs Type B P1 proteins………………………………….……………….97 IV.2 Type A P1 proteins are involved in host range delimitation……………………99 IV.3 Searching for the proteolytic plant factor(s) of Type A P1 proteins…………...103 V V. CONCLUSIONS…………………………….……………………....107 VI. REFERENCES…………………………………………………..…111 VII. APPENDIX………………………………………………………..131 VI ABBREVIATIONS VII Abbreviations 2, 4-D 2, 4-dinitrophenol 2Apro 2A protease 3Cpro 3C protease 3’UTR 3’ untranslated region 5´UTR 5’ untranslated region Φ Empty vector aa Amino acid ACN Acetonitrile AGO Argonaute protein AlkB Alkylated DNA repair protein bp Base pair BY-2 Nicotiana tabacum Bright Yellow -2 BYL Tobacco BY-2 cell-free lysate BYL-EP BYL prepared from evacuolated protoplasts BYL-EP(DN) Denatured BYL prepared from evacuolated protoplasts BYL-P BYL prepared from protoplasts cDNA Complementary DNA cEF Concentrated eluted fractions cFT Concentrated Flow-through fraction CI Cylindrical inclusion CID Collision-induced dissociation CHCA α-Cyano-4-hydroxycinnamic acid CP Capsid protein dpa Days post agroinfiltration dsRNA Double-strand RNA dsDNA Double-strand DNA DTT Dithiothreitol DUB Deubiquitylating enzyme emPAI Exponentially modified protein abundance index EF Fractions eluted ER Endoplasmic reticulum FI Fluorescence intensity IX FPLC Fast protein liquid chromatography FT Flow-through GFC Gel filtration chromatography GFP Green fluorescence protein GUS ß-Glucuronidase HC Helper component HEL Helicase HSP89.1 Heat shock protein 89.1 HSP70 Heat shock protein 70 IAA Indole-3-acetic acid ICTV International Committee on Taxonomy of Viruses IgG Immunoglobulin G KAc Potassium acetate kDa Kilodalton Nano Liquid Chromatography coupled to Electrospray Tandem Mass LC ESI-MS/MS Spectrometry Lpro Leader protease MALDI-TOF Matrix Assisted Laser Desorption Ionization - Time of Flight MMTS Methyl methanethiosulfonate MS medium Murashige & Skoog basal salts with minimal organics MT/MET Methyltransferase MW Molecular weight marker of protein NIapro Nuclear inclusion A protein NIb Nuclear inclusion B protein NS2 Nonstructural protein 2 NS3