S. . i. . 3. ... 1&7. in! memwdws . fl? ‘ .z a. é... , . . . t i finaln . (WI!) 1“? v .13 , u .«.h.m .> 13“.. an" . «dfirzfiwfl. 1 x M .45.? .a v .. as; .3. 5- u , . flamwmfihbs .. . «Mm?» V I! v . Jam“ .. “(Hg . £143.. i. “32.. .. :fiinéin. 5 {7A This is to certify that the dissertation entitled IDENTIFICATION OF HOST PROTEINS INTERACTING WITH POTYVIRAL RNA- DEPENDENT RNA POLYMERASE AND INVESTIGATION OF THE ROLES OF ‘ THE CARBOXY TERMINUS OF CUCUMBER POLY-(A)BINDING PROTEIN 1 INi POTYVIRAL REPLICATION AND CELLULAR TRANSLATION 1 presented by Xiaofeng Wang has been accepted towards fulfillment of the requirements for Ph-D- degree in Geneti cs fit/1 «a J‘ngvfi Major professor Date 5'2 3’ 02’ MS U i: an Affirmative Action/Equal Opportunity Institution 042771 _ LIBRARY Michigan State University PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJCIRCJDateDtnpss-ms IDENTIFICATION OF HOST PROTEINS INTERACTING WITH POTYVIRAL RN A- DEPENDENT RNA POLYMERASE AND INVESTIGATION OF THE ROLES OF THE CARBOXY TERMIN US OF CUCUMBER POLY- (A) BINDING PROTEIN 1 IN POTYVIRAL REPLICATION AND CELLULAR TRANSLATION By Xiaofen g Wang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 2002 ABSTRACT IDENTIFICATION OF HOST PROTEINS INTERACTING WITH POTYVIRAL RNA- DEPENDENT RNA POLYMERASE AND INVESTIGATION OF THE ROLES OF THE CARBOXY TERMINUS OF CUCUMBER POLY- (A) BINDING PROTEIN 1 IN POTYVIRAL REPLICATION AND CELLULAR TRANSLATION By Xiaofeng Wang Viruses rely on host factors to complete their replication and life cycles. The Potyviridae, whose members have a positive sense, single-stranded RNA genome with a viral—encoded, 3’ poly- (A) tail, is the largest and most economically important plant virus family. However, host factors involved in potyviral replication have not been identified. In this work, I sought to identify such host proteins and examine how they are involved in potyviral replication and in host cellular processes. I reported here a cucumber poly- (A) binding protein, CS-PABPl, as a promising candidate that could be involved in potyviral replication. CS-PABP] interacted with RNA-dependent RNA polymerase (RdRp, viral replicase) of zucchini yellow mosaic potyvirus (ZYMV) in the yeast two-hybrid and in vitro binding assays. Since the 3’ poly- (A) of potyviral genome is the site where the minus strand synthesis starts and ZYMV RdRp itself does not bind to poly- (A), we propose that one of the consequences of the interaction is to recruit RdRp or partially assembled replication complexes onto the poly- (A) of viral genome. Deletion analysis indicated that the carboxy terminus of PABP (PABP-CT), which has not been previously well studied, was essential for the interaction. I provided evidence in this work that PABP-CT and its partners were involved in translational regulation. Cucumber PCI6 (PABP-QT interacting), PC1243 and Arabidopsis ERD15 (early responsive to dehydration) interacted with PABP-CT and contain a lZ-amino-acid motif that is present in human PABP-CT interactors. Deletion and point mutation analyses of PC16 indicated that the motif was necessary for the interaction. PC16 inhibited translation in wheat germ and mouse ascites Kresz translation systems. A non- PABP interacting PC16 mutant did not inhibit translation in wheat germ, and caused reduced translation in the Kresz system. PC16 is a wound- and jasmonic acid-inducible protein. The above results suggested a possible translational regulation in gene expression upon stresses or other stimuli. Like other higher eukaryotes, cucumber PABPs exist as a gene family. A second member of the cucumber PABP family, CS-PABP2, which shares 86% identity to CS- PABPl and is expressed constitutively in different tissues, was amplified. While CS- PABP2 interacted with host PABP-CT interactors as well as that of CS-PABPl, it did not interact with ZYMV RdRp. Mutational analysis on a ZYMV RdRp region, which shares similarity to the 12-amino-acid motif, suggested that RdRp might interact with CS- PABPl using a different motif than the host interactors. \l J ACKNOWLEDGEMENTS I would like to thank my committee members: Drs. Richard Allison, Sheng Yang He, Rebecca Grumet, and Michael Thomashow for their consistent support and thoughtful input. I am particularly grateful to my major adviser, Dr. Rebecca Grumet, for giving me this wonderful project, encouraging me at difficult times, and being a honest, well-organized, and insightful model scientist and mentor for me to learn from. A warm thanks to the present and former members from Rebecca’s lab, Sue Hammer, Zakir Ullah, Katerina Papadopoulou, Benli Chai, Mohamed Tawefk. They have made my lab life easier and more productive. My thanks also go to present and former members of Thomashow’s lab: Sarah Gilmore, Eric Stockinger, Ann Gustafson, Kirsten Jjaglo, Dan Zak, Huanying Qing etc. I am particularly in debt to the following individuals who have helped me in several critical experiments and techniques: John Everard, Eric Stockinger, Yaopan Mao, Zakir Ullah. I have worked with four talented students, Emily, Hisako, Karla, and Craig who have contributed a lot to this work. My friends, such as Bin Yu, Hua Zhang, and Lei Li, have provided lots of help and advise. Dr. Nohum Sonenberg at McGill University generously allowed me to work in his lab for the in vitro translation work. Dr. Yuri Svitkin provided mouse ascites Kresz system and gave lots of advise to complete my experiments. All the lab members have made my short stay as a pleasant visit. I want to thank Genetics Program and Department of Horticulture for the administrative help and specially Jeannine from Genetics Program and Lorri and Dr. Loescher from Horticulture Department. I want to thank Graduate School for providing ‘ iv Research Enhancement Fellowship’ to partially support the trip to McGill University and Thoman Fellow Program for the opportunity to learn and discuss hunger related issues. I really appreciate the unlimited support from my parents and my siblings. Their support behind me was the reason to resist the temptation of giving up. I would like to give my special thanks to my wife fro her understanding, encouragement, and her love. Without her, I could not accomplish this much. TABLE OF CONTENTS LIST OF TABLES ............................................................................ VIII LIST OF FIGURES ............................................................................... IX LIST OF ABBREVIATION ....................................................................... X Chapter 1: Literature Review ..................................................................... 1 Introduction ........................................................................................ 1 Host proteins involved in viral replication .................................................. 2 Replication of potyviruses and related viruses ............................................. 5 Objectives of the dissertation ................................................................. 23 References ......................................................................................... 24 Chapter 2: Interaction between zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly-(A)-binding protein .................................... 36 Abstract ............................................................................. . .............. 36 Introduction ...................................................................................... 37 Materials and Methods ........................................................................ 40 Results .............................................................................................. 47 Discussion .......................................................................................... 58 References ......................................................................................... 64 Chapter 3: Identification and characterization of a wound inducible protein that interacts with the carboxy terminus of poly- (A) binding protein and inhibits translation ......................................................................................... 69 Abstract ........................................................................................... 69 Introduction ...................................................................................... 70 Materials and Methods ........................................................................ 71 Results ............................................................................................. 80 Discussion ........................................................................................ 96 References ....................................................................................... 103 Chapter 4: Characterization of the interactions between cucumber poly- (A) binding proteins (PABPS) and PABP-carboxy terminal interactors ............... 108 Abstract .......................................................................................... 108 Introduction ..................................................................................... 109 Materials and Methods ....................................................................... 113 Results and Discussion ........................................................................ 116 References ....................................................................................... 133 Conclusions and future work ................................................................... 137 vi Appendix ........................................................................................... 143 vii LIST OF TABLES Table 2-1. Interactions among ZYMV coat protein (CP), helper component-proteinase (HC-Pro), RNA dependent RNA polymerase (RdRp). and cucumber poly-(A)- binding protein (PABP) in the yeast two-hybrid system ................................... 49 Table 3-1. The cDNAs identified to interact with PABP-CT ................................. 81 Table 3-2. Quatitative B-galactosidase assay for protein-protein interactions in the yeast two-hybrid system ............................................................................... 86 Table 3-3. Quatitative B-galactosidase assay for protein-protein interactions in the yeast two-hybrid system ............................................................................... 90 Table 4-1. Amplification of the paralogs of CS-PABPl ..................................... 117 Table 4-2. Quatitative B-galactosidase assay for protein-protein interactions in the yeast two-hybrid system ............................................................................. 126 viii LIST OF FIGURES Figure 1-1. Organization of the potyvirus genome ............................................... 6 Figure 2-1. PABP is a single or low copy number gene ....................................... 51 Figure 2—2. Predicted amino acid sequence of cucumber poly- (A) binding protein ...... 53 Figure 2-3 Deletion analysis to determine regions in PABP and RdRP responsible for the interaction ............................................................................... 55 Fig 2-4. Cucumber PABP can bind to poly-(A) and RdRp in vitro .......................... 56 Figure 3-1 Amino acid sequence alignment among Arabidopsis ERD15, cucumber PC16 and PC1243 ........................................................................... 82 Figure 3-2. Cucumber PC16 and PC1243 interacted with CS-PABPl in vitro .............. 85 Figure 3-3. Deletion analysis to determine regions in PABP (A) and PC16 (B) responsible for the binding ................................................................. 88 Figure 3-4. PC16 expression is up regulated by wounding and MeJ A ...................... 92 Figure 3-5 Effect of PC16 on poly- (A)-dependent and —independent translation ......... 94 Figure 4-1. Nucleic acid and amino acid sequences of CS—PABP2 ......................... 120 Figure 4-2. CS—PABPI and CS-PABP2 are constitutively expressed in different tissues tested ............................................................................... 121 Figure 4-3. Alignment of the CTC domain of PABPs from different plants ............... 123 Figure 4-4. CS-PABP2 does not interact with RdRp in the yeast two-hybrid system. . ..125 Figure 4-5. Conserved amino acids among all PABP-CT interacting proteins ........... 129 Figure 4—6. In vitro competition between PC16 and ZYMV RdRp for binding to CS-PABPI ...................................................................................... 131 Figure 5-1. A working for potyviral replication ............................................... 140 Figure 6-1. In vitro enzymatic activity assay for ZYMV RdRp ............................. 145 ix BCMNV BMV CI CP CT CTC domain eIF ERD l 5 eRF HC-Pro NCBI NIa NLS NT NTR PABP Paip PAM PAP PCBP PC16 PstV PVA RdRp RRM TEV TuMV TVMV VPg ZYMV LIST OF ABBREVIATION bean common mosaic necrosis potyvirus brome mosaic bromovirus cylindrical inclusion protein coat protein carboxy terminus C-terminal conserved domain eukaryotic initiation factor early responsive to dehydration 15 eukaryotic polypeptide chain release factor helper component-proteinase national center for biotechnology information nuclear inclusion protein a nuclear localization signal amino terminus non-translated region poly— (A) binding protein PABP interacting protein PABP binding motif poly-A polymerase poly- (rC) binding protein PABP-CT interacting 6 pea seed-bome mosaic potyvirus potato virus A RNA-dependent RNA polymerase RNA recognition tobacco etch potyvirus turnip mosaic potyvirus tobacco vein mottling potyvirus viral protein-genome linked zucchini yellow mosaic potyvirus Chapter 1 Literature Review Introduction The Potyviridae, or potyvirus family, is one of the largest and most economically important group of plant viruses. This family of single-stranded RNA viruses includes approximately 200 members that collectively infect most crop species; Many species are infected by more than one potyvirus (Shukla et al., 1994). The potyvirus group has been extensively studied, and much has been learned about many aspects of potyviral replication, including identification of participating viral proteins, viral protein functions, and subcellular localization of the replication machinery (reviewed in Revers et al., 1999; Riechmann et al., 1992; Shukla et al., 1994; Urcuqui-Inchima et al., 2001). Given the limited coding capacity of viral genomes, viruses also rely on host encoded proteins for successful replication. Despite a growing list of host proteins that are involved in the replication of other RNA viruses (Lai, 1998; Strauss and Strauss, 1999), host proteins participating in potyviral replication have not been identified. My first objective was to learn more about the potyviral replication complex by identifying host proteins interacting with the potyviral RNA-dependent RNA polymerase (RdRp, viral replicase). The first two sections of the literature review will review host proteins known to be involved in viral replication and the current status of research on potyviral replication. In the course of this project, host poly- (A) binding protein (PABP) was identified and confirmed to specifically interact with zucchini yellow mosaic potyvirus (ZYMV) RdRp. The carboxy terminus of PABP (PABP-CT) was necessary and sufficient for the SOT VII; gm “as are ; interaction. PABP is an essential cellular protein that plays important roles in mRNA stability, initiation and regulation of translation (Kahvejian and Sonenberg, 2002). While the roles of the amino terminal portion of PABP have been well defined, roles of the carboxy terminus are just now being identified. In the third section of this review, I will review structure and functions of PABP. Host proteins involved in viral replication RNA viruses usually have small genomes, limited coding capacities, and depend on existing or modified host apparatuses, substrates and energy to complete their life cycle. A growing number of host proteins have been proven or implicated to be involved in viral replication, and the majority of these proteins are subverted from RNA- processing or translation machineries of host cells (Lai, 1998; Strauss and Strauss, 1999). These host proteins can be classified into three groups based on how they were identified; some of them were found in different viral replication complexes or directly interact with viral replicase, some were identified as binding protein of the ciS-elements of viral genomes, and some were identified via genetic approaches. The first evidence showing the requirement of host proteins for viral replication was from studies on QB phage (Blumenthal and Carmichael, 1979). The QB viral holoenzyme contains three host proteins; EF-Tu (Elongation Factor), and EF-Ts, which are counterparts of eukaryotic EF-ltx and EF-IB respectively, and 308 ribosomal protein 81. Removal of them will inactivate the enzymatic activity (Blumenthal and Carmichael, 1979). Two decades later, similar results were found in vesicular stomatitis virus (VSV, Das et al., 1998). The purified VSV RNA-dependent RNA polymerase (RdRp) was found inactive unless cellular extracts, specifically EF-IB and EF—lx, were added in the (J 16: frm Vim (Ric. 96110 ends. bilid; GIRL; Pflxe reaction. It was further found that EF-lOt was co-purified with VSV RdRp and EF-lOth subunits were packed in the purified virion, clearly indicating the necessity of host proteins (Das et al., 1998). Besides EF-lOL, different subunits of eIF-3 (eukaryotic Initiation Factor), along with other undefined host proteins, were found in the brome mosaic virus (BMV, Quadt et al., 1993) and tobacco mosaic virus (TMV, Osman and Buck, 1997) replication complexes isolated from BMV and TMV infected plants, respectively. It was further shown that subunit P41 of eIF-3 bound to BMV RdRp (2a protein) in vitro, and addition of wheat germ eIF3 or subunit P41 stimulated BMV viral replication in vitro by three-fold, suggesting eIF3 was directly involved in viral replication (Quadt et al., 1993). Given the difficulty of isolating active replication complexes, other biochemical and genetic methods also have been employed to identify host proteins directly interacting with viral RdRp. Human protein Sam68 (Src-associated in mitosis, 68kDa) was identified to interact with polioviral RdRp protein 3Dpol in the yeast two-hybrid assay (Mcbride et al., 1996). Sam68 was further found to be relocated from the nucleus to poliovirus-induced vesicular compartments in the cytoplasm where viral RNA replication occurs, suggesting that Sam68 could be involved in replication (Mcbride et al., 1996). How Sam68 involved in viral replication is not clear yet. Deletion and point mutation analyses have defined cis-elements of the viral genomes that are important for replication. These are usually located at the 5’ and 3’ ends, but sometimes in the internal region of a viral genome. Viral and host proteins binding to these cis-elements could be involved in viral replication, either by recruitment of RdRp onto these cis-elements, or by the interactions between cis-element-binding proteins. Host proteins such as: poly- (rC) binding protein (PCBP, Gamamik and Andino, 1997; Parsley et al., 1997), La antigen (reviewed in Lai, 1998), polypyrimidine tract- binding protein (PTB, reviewed in Lai, 1998), nucleolin (Waggoner and Samow, 1998), and poly- (A) binding protein (Spagnolo and Hogue, 2000) have been found to bind to different cis-elements of different viruses. Host PCBP binds to the stem-loop B of the Cloverleaf structure at the 5’ end of poliovirus and plays at least two roles, stimulating viral translation (Gamamik and Andino, 1997, 2000; Parsley et al., 1997) and is also required for viral replication (Herold and Andino, 2001; Parsley et al., 1997). Besides host PCBP, polioviral 3CDPro also binds to the Cloverleaf structure to inhibit viral translation and initiate the synthesis of minus strand (Andino et al., 1990). These two proteins could control the transition from translation to replication (Gamamik and Andino, 1998). J anda and Ahlquist (1993) developed a genetic approach to identify host proteins involved in or necessary for viral replication. A yeast-BMV system was developed so that replication of BMV RNA3 could be supported by yeast proteins in combination with BMV la and 2a proteins whose coding sequences were inserted into the yeast genome (J anda and Ahlquist, 1993). At least three recessive mutants that failed to support BMV replication were identified and the corresponding host genes and proteins were investigated. These proteins revealed that host proteins are involved in a broad range of viral replication steps, from translation of the viral replicase gene, to replication complex assembly, to initiation of replication. Yeast Lsmlp (s_mall RNA—like binding protein), which is related to the RNA splicing complex, but found predominately in the cytoplasm, inhibited an early replication step of template selection mediated by viral protein la and a cis-element located within the tRNA-like structure at the 3’ end the RNA3 (Diez, et al., Cm pol red. 2000). Yeast Dedlp is an essential translation-associated protein with ATP-dependent RNA helicase activity. When Dedlp was mutated, BMV replicase 2a translation was specifically inhibited, suggesting Dedlp specifically promotes 2a translation (Noueiry et al., 2000). It has been observed that viral replication complexes are associated with intracellular membranes (Molla et al., 1993; Restrepo-Hartwig and Ahlquist, 1996, 1999; Schaad et al., 1997). Mutation at the OLEl gene, which encodes A9 fatty acid desaturase, caused decreased unsaturated fatty acids synthesis and 90% inhibition of BMV replication, suggesting the composition of ER membrane is critical for viral replication (Lee and Ahlquist, 2001). Replication of potyviruses and related viruses The Potyviridae is the largest plant virus family with about 200 members that cause significant losses in a wide range of crops (Riechmann et al., 1992; Shukla et al., 1994; Revers et al., 1999; Urcuqui-Inchima et al., 2001). Replication complexes are just beginning to be defined for this important group of viruses. Potyviruses have a positive sense, single-stranded RNA genome, which is approximately 10 kb long, with a covalently attached viral protein (VPg, Xiral Protein-genome linked) at the 5’end, and a poly- (A) tail at the 3’ end. The RNA contains a S’NTR, a 3’NTR, and a long open reading frame that is expressed as a polyprotein of approximately 340-370 kD (Fig. l-l). The polyprotein is cleaved into nine individual proteins by three viral encoded proteases (reviewed in Riechmann et al., 1992); P1 (the first protein), HC-Pro (Helper Component- mteinase), and NIa (named Nuclear Inclusion protein a_ because of its tendency to accumulate in the nucleus). P1 protein cleaves the linkage between P1 and HC-Pro and frees Pl from the polyprotein (Verchot et al., 1991); HC-Pro cleaves the junction 5’ NTR 6K1 6K2 3’ NTR r P1 HC-Pro P3 CI VPg-Pro RdRp CP An Fig. 1-1. Organization of the potyvirus genome. Potyviruses have a positive sense, single-stranded RNA genome, approximately 10 kb long, with a poly- (A) tail at the 3’ end, and a viral protein VPg covalently linked to the S’end. At least nine proteins are produced: protein 1 (P1), helper component protease (HC-Pro), protein 3 (P3), 6K1 protein, cylindrical inclusion protein (CI), 6K2 protein, VPg-Pro (Viral protein-genome linked- proteinase, also termed as NIa, nuclear inclusion protein a,), RdRp (RNA- dependent RNA polymerase, also termed as nuclear inclusion b NIb), and coat protein (CP). 1'1 811 14 Car 199 fact III: OH3 O»... a mu. that enh.i between HC-Pro and P3 (the third protein, Carrington et al., 1989); NIa is responsible for the remaining cleavages in the C-terminal two-thirds of the polyprotein (Dougherty and Carrington, 1988). Several intermediate proteins such as CI/6K2, 6K2/NIa, and 6K2/VPg have been observed during TEV infection and may play very important roles (Restrepo- Hartwig and Carrington, 1994; Schaad et al., 1997). Based on their genome structure and gene expression strategy, potyviruses are classified as members of Picomavirus superfamily including the animal-infecting Picomaviridae family, which contains several well-studied viruses, such as: poliovirus, encephalomyocarditis virus and coxsackievirus (Kerekatte et al., 1999). The functions of potyviral proteins have been extensively studied, and we now know that most potyviral proteins play a role in replication. These include P1 protein, HC-pro, P3 protein, CI (Cytoplasmic Inclusion protein), 6 K2 (second 6 kD protein), NIa, and N Ib (Nuclear Inclusion protein b, functions as RdRp or replicase) (Klein et al., 1994; Li and Carrington, 1995; Meritus, 1999; Murphy et al., 1996; Restrepo-Hartwig and Carrington, 1994; Schaad et al., 1996; Schaad et al., 1997; Verchot and Carrington, 1995). Among the above listed proteins, P1, P3 and HC-Pro function as accessory factors, while CI, N13 and NIb (RdRp, see below) are possibly core components of the viral replication complex (Schaad et al., 1997). For example, when the coding sequence of P1 was deleted from the TEV genome, the API mutant accumulated in protoplasts to approximately 2-3% the level of parental genome. In addition, the accumulation of AP] mutant was stimulated in transgenic plants expressing P1 protein. These data suggested that Pl protein is not strictly required for TEV replication, but functions in-trans to enhance replication (Verchot and Carrington, 1995). {It It (1'2 (7. 3 in: CO Ira int. 6K. .\I.: OVC mcr \rpgl HIOD The CI protein of PPV (plum pox virus) has been shown to have a nucleic acid— stimulated ATPase activity and a helicase activity (reviewed in Riechmann et al., 1992). This helicase activity unwound double-stranded RNA molecules and was dependent on hydrolysis of NT P (reviewed in Riechmann et al., 1992). It was also shown in TEV that mutations affecting highly conserved helicase motifs eliminated detectable genome replication, confirming the necessity of the C1 protein in potyviral replication (Carrington et al., 1998). Given the helicase activity, CI protein probably destabilizes secondary structures in potyviral genomes or/and unwinds double-stranded RF (Replication Form) to make minus strand accessible for the replication complex to make progeny RNA genomes. TEV 6K2 protein was first found to be necessary for TEV replication by point mutation and insertional analysis (Restrepo-Hartwig and Carrington, 1994). It was further found that 6K2 was integrated into the ER membrane and to the vesicular compartment derived from the ER membrane in TEV infected plants and in 6K2 transgenic plants (Schaad et al., 1997). Deletion analysis showed that the central hydrophobic region is necessary for the targeting (Schaad et al., 1997). Several intermediate proteins with 6K2 have been observed, including CI/6K2, 6K2/NIa, 6K2/V Pg (Restrepo-Hartwi g and Carrington, 1994; Schaad et al., 1997). When fused to NIa (the cleavage site between 6K2 and Nla was mutated), the 6K2 protein could override the ability of NIa to be targeted to the nucleus and instead targeted N13 to the ER membrane (Restrepo-Hartwi g and Carrington, 1992). It was proposed that Cl, Nla and VPg might be targeted to the ER membrane by 6K2 during TEV infection and assembly along with NIb to make functional replicase. )‘Cu the full~ ind; N la is a multifunctional protein that has RNA binding activity (DarOs and Carrington, 1997), interacts with viral replicase NIb (RdRp, Li et al., 1997; Dares 1999; Hong et al., 1995), and is the major proteinase responsible for cleaving the polyprotein (reviewed in Riechmann et al., 1992). Nla is a modular protein; the N-terminus encodes the VPg and the C-terminus encodes the proteinase. N13 has an internal less-optimal cleavage site for auto-cleavage and can be inefficiently cleaved into N-terminal VPg and C-terminal proteinase. The nature of the inefficient cleavage might be essential for viral replication, since mutations removing the site or accelerating cleavage led to debilitated replication, suggesting the cleavage between two parts at the right time of replication is necessary (Carrington et al., 1993; Schaad et al., 1996). The VPg protein resulting from the cleavage of N la is covalently attached to the 5’ end of RNA genome through the amino acid tyrosine (Tyr) at position 1860 (Murphy et al., 1991). Mutation of the Tyr residue abolished TVMV (_T_obacco Xein Mottling Xirus) replication in protoplasts (Murphy etal., 1996), indicting the importance of the VPg in viral replication. It has been shown in poliovirus that VPg functions as a primer for the initiation of minus strand synthesis (Paul et al., 1998, and see below). Recently, TuMV (flmip Mosaic _\_/irus) VPg was found to interact with Arabidopsis eIF(iso)4E in yeast two-hybrid and in vitro assays (Wittmann et al., 1997). A VPg mutant, which lost the ability to interact with eIF(iso)4E, was identified and replaced the wild type VPg in full-length TuMV cDNA (Le’onard et al., 2000). The mutated TuMV was not infectious, indicating the interaction is necessary for infection (Léonard et al., 2000). Besides Arabidopsis eIF(iso)4E, Arabidopsis eIF4E and wheat eIF(iso)4E (Léonard et al., 2000) interacted with TuMV VPg in a similar degree to that of Arabidopsis eIF(iso)4E. It was I} It also found that tomato eIF4E and tobacco eIF4E interacted VPg of TEV (Schaad et al., 2000), suggesting the interaction between VPg and eIF(iso)4E and/or eIF4E is common and important for host-potyvirus interaction, although the precise role of the interaction remains unknown. NIb was first identified along with Nla since they are located in the TEV infected host nucleus as inclusion body in a 1:1 ratio and were named as nuclear inclusion proteins a and b (Knuhtsen et al., 1974; Dougherty and Hiebert, 1980). It was further found that TEV N Ib has two independent NLS (Nuclear Localization §ignal) responsible for the targeting to nucleus and that the conformation of NIb is important, because any major deletions in the NIb protein abolished the nuclear localization (Li and Carrington, 1993). However, the function of NIb in the nucleus is not clear. Although most of NIb proteins remain in the nucleus, NIb functions as an RNA dependent RNA polymerase in the cytoplasm (Riechmann et al., 1992; Revers et al., 1999; Shukla et al., 1994; Urcuqui- Inchima et al., 2001). All potyviral NIbs have a GDD motif that is the hallmark of an RdRp (Poch et al., 1989; O’Reilly and Kao, 1998), suggesting that it might serve as the catalytic component in the potyviral replication complex. It was later shown in vitro using recombinant NIb protein overexpressed in E.coli that TVMV NIb has RNA- dependent RNA polymerase activity (RdRp, Hong and Hunt, 1996). RdRp apparently functions in-trans, because replication of TEV with the RdRp mutation in the GDD motif can be partially or fully restored in transgenic tobacco protoplasts expressing wild type TEV RdRp (Li and Carrington, 1995). The crystal structure of potyviral RdRps has not been obtained, however, data are available from polioviral RdRp 3Dp01 protein (reviewed in O’Reilly and Kao, 1998). 10 6‘» fl: Polioviral RdRp has a similar structure as other polymerases such as Klenow fragment (E.coli DNA-dependent DNA polymerase), HIV reverse transcriptase, and T7 polymerase (T7 phage DNA-dependent RNA polymerase) (reviewed in O’Reilly and Kao, 1998). The overall shape resembles a ‘right hand’ with finger, palm and thumb subdomains. Comparison of the predicted secondary structure of different RdRps with that of polioviral RdRp has revealed that all RdRps have a similar structure. The palm domain is responsible for the catalysis activity, the finger may determine preference for RNA templates and the thumb domain is likely involved in formation of the clamp on template binding. Besides these three domains, poliovirus and other RdRps have a unique motif at the N -terminus proposed to be involved in oligomeriztion of polioviral RdRp (Hansen et al., 1997). However, there is no evidence that oligomerization is necessary for every RdRp, suggesting that the RdRp unique domain might be involved in other functions. Interactions between potyviral RdRp and other viral proteins, or with itself have been studied in TEV (Darbs et al., 1999; Li et al., 1997), TVMV (Fellers et al., 1998; Hong et al., 1995), PVA (Botato Xirus A) and PstV (Bea seed-home Mosaic Xims) (Guo et al., 2001). In TVMV, RdRp was found to interact with coat protein (CP) in the yeast two-hybrid system (Hon g et al., 1995). The interaction between CP and RdRp may suggest that CP can regulate the relative levels of plus and minus strand TVMV RNA in infected cells (Hong et al., 1995). However, surprisingly, an RdRp mutant ADD, which lost the polymerase activity, was not able to interact with CP. One explanation could be that the region in RdRp responsible for the interaction is close to the GDD motif. Another possibility is that the ADD mutant was not stable, however, it is not likely since the ADD ll ml CO CO) 0ft; mutant interacted with NIa at a comparable level to that between WT RdRp and NIa (Hon g et al., 1995). No interaction between RdRp and CP of PVA or PstV was observed in yeast (Guo et al., 2001). Self-interaction of TVMV RdRp was reported (Hong et al., 1995), which is consistent with poliovirus RdRp where oligomerization of RdRp is critical for replication (Hobson et al., 2001). However, similar RdRp self- interaction was not found in yeast two-hybrid assays for TEV, PVA or PSbMV (Guo et al., 2001; Li et al., 1997). Thus it is not clear whether oligomerization of RdRp is important for potyviral replication. Interaction between N Ia and RdRp was found in TEV (Li et al., 1997; Dares et al., 1999), TVMV (Hong et al., 1995), PVA and PstV (Guo et al., 2001). Disruption of the interaction between Nla and RdRp by mutation in NIa disabled ability of mutant TEV to infect tobacco protoplasts. RdRp mutants recovering the interaction with the NIa mutant partially restored ability to infect, indicating the interaction is necessary for TEV infection (DarOs et al., 1999). Potyviral replication occurs in the cytoplasm in tight association with the host cell membrane (Martin and Garcia, 1991; Schaad et al., 1997). For TEV, the replication complex is localized to the vesicular compartment derived from ER membrane that collapsed into discrete aggregated structures upon TEV infection (Schaad et al., 1997). It was proposed that TEV 6K2 protein initiates assembly of the TEV replication complex by targeting the proteolytic precursors, such as 6K2/N Ia, 6K2/V Pg, and CI/6K2, directly to ER membrane, and by anchoring the replication complex on the ER membrane (Restrepo-Hartwi g and Carrington, 1994; Schaad et al., 1997). Replication of positive sense, single strand RNA viruses starts with the synthesis of the minus strand intermediate using the positive strand as template, followed by 12 It It“ PRU T63: that positive strand synthesis. The synthesis of the minus strand of potyviruses and picomaviruses is initiated at the 3’ end poly- (A) tail of genomic RNA. The poly— (A) tail is necessary for picomavirus replication (Cui et al., 1993; Herold and Andino, 2001) and recent evidence suggested that poly- (A) tail is necessary for potyvirus replication (Tacahashi and Uyeda, 1999). Herold and Andino (2001) investigated the minimal length of poly- (A) necessary for polioviral replication using in vitro transcribed poliovirus replicons with different numbers of A residues ranging from O to 17. When the last nuleotide in the 3’ NTR or poly- (A) tail was ribonucletide, the replicon could eventually replicate even without any A residue, although it did not replicate until several hours later. It was further found that progeny viruses all contained long poly- (A) tails, indicating the restoration of the tail. However, if the last A residue in the poly- (A) was a deoxyadenosine, replicons with 5 A residues could not replicate, confirming that poly- (A) is necessary and more than 5 A residues are required for polioviral replication (Herold and Andino, 2001). Initiation of poliovirus replication was postulated to start from the uridylytion of VPg using poly- (A) tail as template, and uridylated VPg in turn served as primer for synthesis of minus strand (Paul et al., 1998; Aglo et al., 1999). It has been shown in vitro that poliovirus VPg can be uridylated by RdRp (3Dp01 protein) using poly- (A) as template (Paul et al., 1998). RdRps of poliovirus and other picomavirueses do not bind to the poly- (A) tail (Cui et al., 1993; Paul et al., 1994). The poliovirus RdRp is recruited by polioviral 3AB protein onto a secondary structure, which includes the 3’ NTR, and several adenosine residues of the poly- (A) tail (Harris et al., 1994; Agol, 1999). In potyviruses, it appears that important secondary structure exists at the 3’ end of viral RNA genome, which 13 6.8 to Ct 19 includes part of the coat protein coding sequence, and the 3’NTR (Mahajan et al., 1996; Haldeman-Cahill et al., 1998). It was proposed that potyviral NIa recruits RdRp onto viral RNA since Nla has RNA and RdRp binding activity (Schaad et al., 1997). However, how host proteins are involved in recruiting poty and picoma viruses onto the 3’ NTR is not clear. Poly- (A) binding protein (PABP) 1. Functions of Poly- (A) binding protein Most eukaryotic mRNAs have a cap structure, m7GpppN (N is any nucleotide) at the 5’ end, and a poly- (A) tail at the 3’ end (Gingras et al., 1999). Both structures are added post-transcriptionally in the nucleus and are necessary for export of mature mRNAs to the cytoplasm. The poly— (A) is about 50-70 bases long in yeast and 200-250 bases in higher eukaryotes (Amrani et al., 1997; Jacbosen, 1996; Minvielle-Sebrastia et al., 1997). Poly- (A) tail in the cytoplasm is bound by poly- (A) binding protein (PABP, termed Pab in yeast and humans, PABP is used through this dissertation), which is an essential cellular protein for eukaryotes (Sachs et al., 1987; Coller et al., 1998). PABP, together with poly- (A), plays a broad range of roles: pre-mRNA processing (Armani, et al., 1997; Minvielle-Sebrastia et al., 1997), mRN A stability (Caponigro and Parker, 1995; Coller et al., 1998), poly- (A) metabolism (reviewed in J acobsen, 1996; Sachs and Davis, 1989), and initiation and stimulation of translation (Gallie, 1998; Gray et al. 2000; Jacobsen, 1996; Otero et al., 1999; Sachs et al. 1997). PABP has four tandem RRMS (RNA Recognition Motif) located in the N- terrninal two-thirds of the protein, an approximately 75-amino-acid-long C-terminus Conserved (CTC) domain, and a linker rich of methionine and proline in between (Sachs l4 et al., 1986; Kahvejian and Sonenberg, 2002). The RRM, which is the hallmark for a large number of RNA-binding proteins, is composed of about 90 to 100 amino acids with 2 short stretches of conserved amino acids named as RNPl (Riboguclearprotein) and RNP2 (reviewed in Burd and Dreyfuss, 1994). The three-dimensional structure of RRMS has been determined as a four-stranded antiparallel B sheet flanked by two perpendicularly oriented 0t helices. The RNPl and RNP2 are located in the central two B sheets and directly contact RNA (reviewed in Burd and Dreyfuss, 1994). Comparison of the sequences of PABP from different organisms indicated that individual RRMS in a single PABP are more divergent from each other than from the counterpart RRMS in PABPs from different species, suggesting that the individual RRMS are not functionally equivalent (Burd et al., 1991). Deletion analysis indicated that the C-terminus of PABP (PABP-CT, including the linker and CTC domain) is not necessary for poly- (A) binding; however, one individual RRM of yeast PABP was not able to bind to poly- (A). It was also found that RRM1-2 bound to poly- (A) as efficiently and specifically as the RRM1-4 or full-length PABP. RRM3-4 bound to non-poly- (A) RNA and might bind to either a different part of the same mRNA or other RNA (Burd et al., 1991). Point mutation analysis indicated that RRM2 was responsible for the poly- (A) and RRM4 for non-poly- (A) binding (Deardorff and Sachs, 1997). Polyadenylation is one of the posttranscriptional modifications of pre-mRN A and includes the cleavage of the mRNA precursor near the polyadenylation site (AAUAAA) and addition of adenosines to the 3’ end (Wahle and Keller, 1992). The cleavage process requires yeast CF I (_C_leavage Factors 1) and CF 11, and polyadenylation involves CF 1, PF I (_Eolyadenylation Eactor I), and Paplp (poly- (A) polymerase, Amrani et al., 1997; 15 Minvielle-Sebrastia et al., 1997). It was found that PABP was co-purified with CF I (Minvielle-Sebrastia et al., 1997) and specifically interacted with RnalS, a key component of CF I (Amrani et al., 1997). Extracts from temperature sensitive PABP mutants yeast cells grown in non-permissive temperature, were able to cleave the pre- mRNA properly but failed to achieve a poly- (A) tail with proper length (Amrani et al., 1997; Minvielle-Sebrastia et al., 1997). Instead of 50-70 bases, up to hundreds of adenosines were added. When PABP protein was added into the extract, normal length of poly- (A) tail was produced, suggesting that yeast PABP controls the poly- (A) length of newly synthesized mRNAs (Amrani et al., 1997; Minvielle-Sebrastia et al., 1997). In mammalian systems, PABP II, which is a ca.49 kD, nuclear protein with only one RM and distinct from the major member PABPl, was responsible for the 3’ end processing (Whale etal., 1993). Several lines of evidence have indicated that PABP is involved in mRNA stability. Firstly, PABP is negatively involved in decapping (Caponigro and Parker, 1995). One of the major mRNA decay pathways in eukaryotes (yeast) is initiated by poly- (A) shortening, followed by the cleavage of 5’ cap structure (decapping) by Dcplp, and degradation of the rest of mRNA by Xmlp, which is a 5’ to 3’ exonuclease (Decker and Parker, 1993). PABP is an inhibitor of decapping because in yeast cells lacking a functional PABP gene (yeast strain pablA), mRNAs were decapped prior to deadelynation, indicating that PABP protein but not deadelynation is necessary to protect the cap structure (Caponigro and Parker, 1995). Secondly, it was shown that stabilization of mRNA is an intrinsic property of PABP in viva (Coller, et al., 1998). A system was developed to uncouple other functions 16 of PABP from its poly- (A) binding activity. PABP was tethered to the 3’ NTR of reporter mRNAs by fusing PABP to M82 coat protein and placing MS2 binding sites in the 3’ NT R of the reporter gene. Deletion of both RRMl and RRM2 did not destabilize mRNA, suggesting that poly- (A) binding activity is not required for PABP to stabilize mRNA (Coller et al., 1998). Deletions of either RRM3, or RRM4 or 90 amino acids from PABP-CT abolished the stability of reporter mRNA, suggesting that RRM3, 4 and PABP-CT are necessary for the stabilization activity of PABP (Coller et al., 1998; Gray et al., 2000). The poly- (A) tail of reporter mRNAs was shortened similar to other cellular mRNAs, but reporter mRNAs had a longer half-life, suggesting that poly- (A) is not required for stabilization of mRN A. Collectively, Coller et al (1998) concluded that the primary function of poly- (A) is to recruit PABP onto mRN A, and poly- (A) binding and mRNA stabilization activity of PABP are controlled by different portions of the protein. Thirdly, it was found that PABP is positively involved in deadelynation (Caponigro and Parker, 1995; Sachs and Davis, 1989; Sachs and Deardorff, 1992). The poly- (A) tail is gradually shortened after mRN As are transported to the cytoplasm and this shortening is also dependent on the nature of mRNA, especially the sequence of the 3’ NTR (Non-Translated Region, reviewed in Jacobson, 1996). In yeast cells that have been depleted of PABP protein, mRNAs with longer poly- (A) tails were observed (Caponigro and Parker, 1995; Sachs and Davis, 1989). A PABP-dependent poly- (A) ribonuclease (PAN) that shortened the poly- (A) to about 20 bases only in the presence of PABP was identified (Sachs and Deardorff, 1992). Furthermore, longer poly- (A) tails were also found in yeast cells with PAN mutations, confirming that both PAN and PABP are required for poly- (A) tail shortening (Sachs and Deardorff, 1992). 17 pol) lead Stud Eart- elF4 elF-II resul: enhan The H Initlat P01):- factor: and e[ (revim 1997), elFtG Closed. Circuju‘ 5’ 6nd ifi) “ll It has also been documented that translational efficiency of mRNAs with caps and poly- (A) tails was much more than the sum of translational efficiency with either cap or poly- (A) alone. This synergistic stimulation suggests that both ends may communicate to lead to much higher translation (Gallie, 1991; Tarun and Sachs, 1995). Subsequent studies showed that yeast PABP interacted with yeast eIF4G I (eukaryotic Initiation Eactor) and eIF4G II in vitro and in vivo (Tarun and Sachs, 1996, eIF4G is a subunit of eIF4F that contains eIF4E, eIF4G, eIF4A). It was also found in plants that not only wheat eIF4G and eIFiso4G, but also eIF4E, interacted with PABP (Le et al., 1997a). Similar results were also found in mammalian systems (Imataka et al., 1998; Piron et al., 1998). Binding of PABP to poly- (A) and binding of eIF4E to the cap was highly enhanced by the binding between PABP and eIF4G (Le et al., 1997b; Wei et al., 1998). The interaction between eIF4G and PABP provided evidence for the closed-loop model: initiation factor eIF4E, binds to the cap (reviewed in Gingras, 1999); PABP binds to 3’ poly- (A); eIF4G is a platform for the interaction and assembly of multiple initiation factors: eIF4E, PABP, eIF4A, eIF3. The interactions between PABP and eIF4G, eIF4E and eIF4G bring two ends of mRNA close to each other and result in a circular molecule (reviewed in Gallie, 1998; Kahvejian and Sonenberg, 2002; Jacobson, 1996: Sachs et al., 1997). It was observed with the atomic force microscopy that purified yeast PABP, eIF4G and eIF4E circularized capped and polyadenylated RNA in vitro, confirming the closed-loop model (Wells et al., 1998). At least two advantages could result from circularization: l. The 408 ribosome subunit could be easily re-recruited from 3’ end to 5’ end and start another round of translation; and 2, intact mRNA with both cap and poly- (A) will be preferentially translated, avoiding production of truncated proteins (reviewed 18 in inc sufi 199. 2.( abov term: mnon Kahvt 31161111 PIESEr SUperS (Kuhn pOU‘t in Gallie, 1998). It was recently shown that the interaction between PABP and eIF4G indeed was necessary for efficient translation (Tarun et al., 1997; Kessler and Sachs, 1998). For eIF4G mutants that lost ability to bind to PABP, or PABP mutants that lost ability to bind to eIF4G, efficient translation was abolished, suggesting the interaction between these two proteins, or the maintenance of circular molecules were necessary for the efficient translation of mRNA (Tarun et al., 1997; Kessler and Sachs, 1998). PABP deletion analysis has indicated the RRM2 of PABP was necessary and RRMl-2 was sufficient for the interaction with eIF4G and efficient translation (Kessler and Sachs, 1998). 2. C-terminus of poly- (A) binding protein The N -terminus of PABP (PABP-NT) is responsible for most, if not all, of the above functions. The C-terminal third of PABP is not as conserved as that of the N- terminus, however, a CTC domain approximately 75 amino acids long is conserved among all PABPs from different organisms (Deo et al., 2001; Kozlov et al., 2001; Kahvejian and Sonenberg, 2002). The roles of the C-terminus have drawn increasing attention recently. PABP—CT was found to be capable of forming oligomers in the presence of poly- (A). Deletion of Xenopus PABP-CT abolished the gel retardation supershift band, which resulted from auto-polymerization of PABP on the poly- (A) tail (Kfihn and Pieler, 1996). It was further found that deletion of PABP-CT diminished the poly- (A) organizing activity (Ktihn and Pieler, 1996), which renders PABP able to form a higher order complex on poly- (A) with multiple, regularly spaced copies of PABP on a single RNA molecule (Baer and Kornberg, 1983). Coller et al (1998) observed that a yeast PABP mutant with a deletion of 50 amino acids from CT lost the ability to rescue 19 me Fu Stu? \\ h (161: 511:3.- cyc 3.P mult; PABI PABI mum Mmm Basr inmm aI‘ai Iu' CDVA AWA; the inviability of yeast strain pablA, suggesting that CT is necessary for yeast to survive. Furthermore, yeast PABP with a deletion of 90 amino acids from CT lost the ability to stabilize mRNA, suggesting that PABP—CT may be directly involved in mRN A stability which might be necessary for cellular survival (Gray et al., 2000). It was also found that deletion of human PABP-CT caused the accumulation of human PABPl in the nucleus, suggesting PABP-CT was necessary for PABPl to shuttle between the nucleus and cytoplasm (Afonia et al. 1998). 3. Poly- (A) binding proteins in plants Although there is only a single PABP gene in yeast, higher eukaryotes have multiple PABPs. In humans, there are at least three members in the PABP gene family. PABPl is the major member and responsible for the cytoplasmic functions. An inducible PABP (iPABP), which shares 77% amino acid identity with PABPl, is expressed mainly in activated T cells and platelets (Houng et al., 1997; Yang et al., 1995). PABP3 has 92% identity to PABPl and is only expressed in round sperrnatids (Fe’ral et al., 1999). PABP II is a small nuclear protein (49 kD) necessary for polyadenylation but distinct from PABPl in that it has only one RRM (Brais et al., 1998). PABP genes have been isolated from Arabidopsis, wheat, and tobacco, and available data Show that PABPs also occur in plants as a gene family (Hilson et al., 1993; Le et al., 1997b; Le and Gallie, 2000; Belostotsky and Meagher, 1993). In Arabidopsis, cDNAs of at least four members have been isolated: AtPABPl, AtPABP2, AtPABP3, AtPABPS (Hilson et al., 1993; Belostotsky and Meagher, 1993). Two cDNAs from tobacco were also identified (Le and Gallie, 2000). Investigations of these PABPS have 20 IOI dr 1' seq lcar 199 .Arm rem; regit AtP.~‘ Into] et :11 . flour both stud} dEVej that .\ miUI€( Arabi idmnn AtPAI found that they have divergent sequences, differential expression patterns, and potentially different functions. Different members of PABPS in Arabidopsis showed quite different gene sequences and expression patterns. AtPABP2 is expressed in all organs; stems, flowers, leaves, roots, siliques and pollen based on northern and western analysis (Hilson et al., 1993; Palanivelu et al., 200b), suggesting that AtPABP2 is a major member in Arabidopsis. Detailed study of AtPABP2 expression indicated that it is spatially and temporally regulated in different organs; it is expressed strongly in the stele and meristem region of roots, and its expression dramatically decreased in ovules after fertilization. AtPABP2 was also strongly expressed in the transmittal tissues, and possible involvement in pollination-dependent poly- (A) tail shortening was proposed (Palanivelu et al., 200b). AtPABPl is expressed mainly in roots, with a lower expression in immature flowers. AtPABPS and AtPABP3 share only 55% and 65% identity with AtPABP2 and both are only expressed in immature flowers (Belostotsky and Meagher, 1993). Detailed study on AtPABPS found that the expression was restricted to pollen and ovule development and early emryogenesis (Belostotsky and Meagher, 1996). Comparison of amino acid sequences of PABPs from available data suggested that NtPABP3 (tobacco PABP3), NtPABP7 and TsPABP (wheat PABP) are most closely related to AtPABP2; identity among them ranges from about 64% (wheat vs. Arabidopsis) to 72% (tobacco vs. Arabidosis). The two tobacco PABPs share 80% identity (Le and Gallie, 2000). AtPABPS and AtPABP3 are less related not only to AtPABP2, but also to available PABPs from other species, suggesting either that their 21 COL Oiht and p, counterparts have not been identified or they are evolutionally more divergent from others. Functions of Arabidopsis PABPs have been studied in a yeast strain lacking a functional yeast PABP gene. Both AtPABPS and AtPABP2 can partially restore the poly— A shortening, the initiation of translation, and the viability of yeast. However, AtPABPS could not restore the coupling between mRN A deadnylation and decapping, but AtPAB2 could (Belostotsky and Meagher, 1996; Palanivelu et al., 2000a), suggesting a functional difference of these two members. However, the different functions of different members in planta are currently unknown, partially owing to the inability to identify PABP knock- out Arabidopsis. Presumably as in yeast, PABPs are essential in plants (Palanivelu et al., 2000b). 4. Poly- (A) binding protein and viral infections Viruses rely on hosts for energy, substrates, and key proteins. To make full use of the host translational apparatus, viruses often shut down host translation (Pe’ery and Mathews, 2000). Given its important roles in translation and viability of eukaryotes, PABP is one of the major targets for host shut-off during viral infections (Chen et al., 1999; Joachims et al., 1999; Kerekatte et al., 1999; Piron et al., 1998). The N81 protein of influenza virus A binds to human PABP II, which is necessary for polyadenylation, in the nucleus and sequesters PABP from binding to the oligo- (A) tail of host pre-mRNAs. This prevents further adenylation of pre-mRNAs, disrupts the export of mRNAs from nucleus to cytoplasm, and leads to the accumulation of pre-mRNAs and PABP in the nucleus (Chen et al., 1999). Human PABPl was also cleaved at a site in between RRM4 and PABP-CT by the picomaviral 2A protein during enterovirus and coxsackievirus 22 This CHOU 210“ , Tephc eCont CXICn. lfphc idenhf l aSSOCj repllt‘: infections and the cleavage of PABP correlated with the host shut-off (Joachims et al., 1999; Kerekatte et al., 1999). The viral NSP3A protein from Rotavirus was found to have much higher affinity to human eIF4G I than human PABPl has to eIF4G I, and NSP3A could replace PABP proteins that associated with eIF4 complex. This led to two related results: host translation was shut down because PABP no longer participated in host translation, and viral translation was stimulated, because Rotavirus mRNAs are capped but not polyadnylated. NSP3A binds to the 3’ end of the rotaviral genome and bridged the 5’ and 3’ end of viral mRNAs by interacting with the eIF4 complex that binds to viral 5’ cap (Piron et al., 1998). Objectives of the dissertation The interactions between a virus and its hosts are critical for successful infection. This is particularly true for RNA viruses that have a small genome and do not encode enough functions to independently replicate and perform other critical processes. Successful replication requires adaptation of host machinery as is evidenced by the growing number of host factors that have been identified to be involved in viral replication. The potyvirus family, or Potyviridae, is the largest and one of the most economically devastating plant virus families. 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Biol. 15: 6770-6776. 35 Chapter 2 Interaction between zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly-(A)-binding protein This chapter has been published as: Wang, X., Ullah, Z, and Grumet, R. 2000. Interaction between zucchini yellow mosaic potyvirus RN A-dependent RNA polymerase and host poly- (A) binding protein. Virology 275:433-443. Ullah, Z contributed to Fig. 2-3 Abstract Viral replication depends on compatible interactions between a virus and its host. For RNA viruses, the viral replicases (RNA dependent RNA polymerases; RdRps) often associate with components of the host translational apparatus. To date, host factors interacting with potyvirus replicases have not bee identified. The Potyviridae, which form the largest and most economically important plant virus family, have numerous similarities with the animal virus family, the Picomaviridae. Potyviruses have a single stranded, plus sense genome; replication initiates at the viral-encoded, 3' poly (A) terminus. The yeast two-hybrid system was used to identify host plant proteins associating with the RdRp of zucchini yellow mosaic potyvirus (ZYMV). Several cDNA clones representing a single copy of a poly-(A) binding protein (PABP) gene were isolated from a cucumber (Cucumis sativus L.) leaf cDNA library. Deletion analysis indicated that the C-terminus of the PABP is necessary and sufficient for interaction with 36 the RdRp. Full-length cucumber PABP cDNA was obtained using 5' RACE; in vitro- and E. coli-expressed PABP bound to poly-(A)-sepharose and ZYMY RdRp with or without the presence of poly (A). This is the first report of an interaction between a viral replicase and PABP, and may implicate a role for host PABP in the potyviral infection process. Introduction Successful systemic infection by a pathogen depends on compatible interactions between the pathogen and its host. This is particularly true for viral pathogens which have extremely small genomes and limited protein coding capacity. Several host proteins are involved in replication of RNA viruses, either as components of the viral replication complex, or by binding directly to the viral genome (reviewed in: Lai, 1998; Strauss and Strauss, 1999). For RNA viruses, a majority of the factors found in association with the viral replicase, the RNA dependent RNA polymerase (RdRp), are subverted from the host RNA-processing and translational machinery. For example, elongation factors EF-la and different subunits of eIF3 are associated with the replicase complexes of an array of bacterial, plant, and mammalian RNA viruses such as QB phage, brome mosaic virus, tobacco mosaic virus, vesicular stomatis virus, measles virus and poliovirus (Lai, 1998; Strauss and Strauss, 1999). The exact roles of the translational machinery proteins in viral replicase complexes are not fully understood; virus replication and translation might be coupled, or the host proteins may play different roles in virus replication than they do in host translation. The Potyviridae, which resemble the animal virus family, the Picomaviridae, form the largest and one of the economically most important families of plant viruses; 37 approximately 200 members cause serious diseases in a wide range of crop plants (Shukla et al. 1994). The members of this family have a plus sense, single stranded RNA genome of approximately 10 Kb, a VPg (viral protein genome linked) covalently linked to the 5' end, and a poly-(A) tail at the 3' end. The RNA encodes a single polyprotein, which is subsequently cleaved into nine proteins by viral-encoded proteases (Dougherty and Semler, 1993); function of the various potyviral proteins has been a active area of investigation. Among the potyviral proteins, NIb (nuclear inclusion b; originally named for its tendency to accumulate in the nucleus), functions as an RNA-dependent RNA polymerase (RdRp) (Hong and Hunt, 1996; Li and Carrington, 1995). Potyviral RdRps of tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV) interact with other potyviral proteins including P1, P3, and the N13 (nuclear inclusion a) protein, which is composed of the VPg at the amino terminus and the main viral proteinase at the carboxy terminus (Hong et al. 1995; Li et al. 1997; Fellers et al. 1998; Merits et al., 1999; Daros et al. 1999). Replication, which proceeds by copying of the plus strand to the complementary minus strand intermediate, followed by plus strand synthesis, occurs in membrane associated, cytoplasmic fraction (Martin and Garcia, 1991; Schaad et a1. 1997). Cytoplasmic localization of the replication machinery is thought to be achieved by direction of a subset of the potyviral NIa protein proteolytic precursor (including the amino terminal adjacent 6 kDa hydrophobic protein) toward the endoplasmic reticulum rather than the nucleus (Schaad et al. 1997). The RdRp is, in turn, postulated to be recruited to the membrane fraction via interaction with the 6 kDa - VPg - proteinase (Nla) complex. 38 Replication of the minus strand is initiated at the 3' poly (A) tail of the plus sense, genomic RNA. Presence of a poly (A) tail is essential for replication of picomaviruses such as poliovirus, encephalomyocarditis virus and rhinovirus (Cui et al. 1993, Todd and Semler, 1996 Todd et al. 1997; Agol et al. 1999) and recent evidence suggests it is also necessary for potyvirus replication (Tacahashi and Uyeda, 1999). Initiation of poliovirus replication requires uridylylation of the VPg which serves as a primer for minus strand synthesis (Agol et al. 1999). The uridylylation is performed by the poliovirus RdRp (3Dp01 protein) and requires the presence of poly (A) template (Paul et al. 1998). It is not known whether viral poly (A) tails exist as free poly (A)s within the host, or if like eukaryotic mRNAs, they are found in association with host poly (A) binding protein (PABP). Given the reported high abundance of PABPS, studies have indicated an approximately 75-95% fold excess of free PABP over binding sites on cytoplasmic poly (A) (Drawbridge et al., 1990; Gorlach et al. 1994), association of PABP with viral RNAs seems likely. PABP-dependent translation of viral genomes, anal gous to the PABP- dependent translation of eukaryotic mRNAs, has been proposed (Gallie, 1998). Poliovirus and other picomavirus RdRps do not bind to poly (A) directly (Cui et al. 1993; Paul et al. 1994). The poliovirus 3Dpol is recruited to a complex secondary structure in the 3' non-translated region (NTR) upstream of the poly (A) tail via interaction with the RNA-binding 3AB proteins (analagous to the potyviral 6 kDa-VPg; 3A is involved membrane association; 3B is the VPg) (reviews: Xiang et al. 1997; Agol et al. 1999). These interactions are thought to provide template specificity. Potyviruses appear to have important secondary structure near the 3' end of the genome which may include sequences within both the 3' NTR and the adjacent coat protein coding region 39 (Mahajan et al. 1996; Haldeman-Cahill et al. 1998). Although mutations in these regions interfere with replication, specific binding of viral proteins to these sequences has not been demonstrated for potyviruses. Specific host factors interacting with potyviral replicases have not been identified yet. In this study we examined interactions between zucchini yellow mosaic potyvirus (ZYMV) and its cucumber (Cucumis sativus L.) host, by identifying cucumber proteins that interact with ZYMV RdRp. Yeast two-hybrid analysis demonstrated reproducible and specific interactions between ZYMV RdRp and cucumber poly (A)-binding protein. Materials and Methods Strains and plasmids Plasmid pBluescript KS and Esherichia coli strain XLl-Blue (Stratagene) were used for general DNA manipulation. Yeast (Saccharomyces cerevisiae) strain YRG2 and the GAL-4 based two-hybrid plasmids pBD-GAL4, p53, pLAMINC, pSV40, and pAD- GAL4 were purchased from Stratagene. pBD-GAL4 and pAD-GAL4 (binding domain and activation domain, respectively) were used to express coat protein (CP), helper component-proteinase (HC-Pro), RNA-dependent RNA polymerase (RdRp), and poly- (A) binding protein (PABP) in yeast as GAL4 binding domain- or activation domain- fusion proteins. pGEM®-T EASY vector (Promega) was used for cloning 5’ RACE (rapid amplification of cDNA ends) products. pGEX-Sx-l (Phamacia) was used to produce fusion proteins GST-PABP or GST-RdRp in E. coli strain Xa 90, and pET-28a- 1 (Novagen) was used to produce His-PABP in E. coli strain BL21(DE3). The ZYMV RdRp gene was amplified by PCR with Vent DNA polymerase (New England Biolabs) using ZYMV cDNA [Connecticut (CT) isolate; Grumet and Fang, 40 1990] as template. The amplified product was inserted as an EcoRI - Sall fragment into pBD-GAL4, pAD-GAL4 and pGEX-Sx—l vectors in frame to form pBDRdRp, pADRdRp, and pGEXRdRp respectively, and sequenced. The primers were designed as follows (restriction sites are italicized): 5' end primer, 5' CCGGAAITCAGCAAGCGA GAAAGATG 3', and 3' end primer 5' AGAGTCGAC'ITGGAGCATCACAGTGT 3'. The helper component-protease (HC-Pro) gene was amplified as above using the ZYMV NAA isolate cDNA (Gal-On et al., 1992) as template, inserted as an EcoRI - Sal] fragment into pBD—GAL4 and pAD-GAIA vectors in frame to form pBDHC and pADHC, and sequenced. The primers were: 5' end primer 5' CCGGAATTCAGCG AAGTTGACCAC 3' and 3' end primer 5' AGAGTCGACACCAACTCTGTAATG 3'. The ZYMV—CT isolate coat protein (CP) gene in the pTL37-CP construct (Fang and Grumet, 1993), was digested with NcoI, filled in with Klenow fragment (Gibco BRL), and then digested with PstI. The blunt-Pst] CP fragment was then ligated to pUC119, which had been digested with EcoRI, filled in with Klenow, then digested with PstI. The EcoRI - PsII CP fragment was then inserted into pBD-GAL4 and pAD-GAL4 vectors to form pBDCP and pADCP, and sequenced. The yeast two-hybrid full-length cucumber PABP clones were generated as follows. PCR (Vent polymerase) was used to amplify the 5' end of the full length PABP clone (obtained by 5' RACE as described below) and to add an EcoRI site to the start codon. The 5' end primer was 5' TAGAAITCATGGCTCAGG'ITCCACC 3'. The 3' end primer was the GSP2 primer used for 5' RACE (see below). The PCR product was digested with EcoRI and Clal, ligated to the EcoRI - Clal digested plasmid pADNIS, which had the partial PABP clone, to form pADPABP. The full length PABP was also 41 inserted as an EcoRI - Sal] fragment into pBD-GAL4, pGEX-Sx-l, and pET—28a-1 to form pBDPABP, pGEXPABP, and pETPABP. Construction of the cucumber leaf two-hybrid cDNA library in A HybriZAP-2.1 vector Total RNA was isolated from young leaves of cucumber cultivar ‘Straight 8' as described by Chomczynski and Sacchi (1987); mRNA was further purified using the Promega PolyATract® mRNA isolation system 11. Approximately five ug mRNA was used for cDNA synthesis following the protocol of the HybriZAP® 2.1 two-hybrid cDNA synthesis kit, and cDNAs (ranging from 0.5 Kb to 2.5 Kb) were inserted into the A HybriZAP-2.l vector as EcoRI - XhoI fragments. The primary library contained ~ 5X106 PFU (plaque forming units). The primary A. cDNA library was amplified once and then converted to a plasmid (pAD-GAL4) library by in vivo mass excision according to the Stratagene protocol. Screening the library with RdRp and testing for interactions between viral proteins Yeast transformation, growth media, and X-gal (5-bromo-4-chloro-3-indolyl B—D- galactopyranoside) filter assays were performed following the supplier’s procedures (Stratagene). Yeast YRGZ cells were first transformed with pBDRdRp, and then transformed with CsCl—purified cDNA library plasmid DNA. The transformants were plated onto SD (synthetic dropout) medium without leucine, tryptophane, or histidine (SD-L-T-H). Colonies that grew on selection medium were restreaked onto SD-L-T-H, transferred to nitrocellulose membrane (Schleicher & Schuell), and assayed for 42 fill the RC lll'br 385‘ expression of B-galactosidase activity (Lac Z) by X-Gal filter assay. Colonies that survived in medium without histidine and turned blue in the X-Gal assay were considered as putative positives. Plasmids were recovered from putative positive yeast colonies using Zymoprep yeast plasmid miniprep (Zymo Research, CA), transformed to E. coli, and amplified. The recovered plasmids were then transformed back to YRG2 yeast either alone, or in combination with pBD-GAL4, p53, pLamin C, or pBD-RdRp. The transformants were plated onto SD-H or SD-L—T-H, and assayed for Lac Z activity. Those that can turn on two reporter genes only in the presence of pBD-RdRp, but can not turn on reporter genes, either alone, or in combination with pBD-GAIA, p53 or pLamin C were considered to be postitives. 5’ RACE (rapid amplification of cDNA ends) A 5' primer, GSPl, 5’-TCATTCTTCCATT CATCTCAGCAA-3’) and 3' primer, GSP2 (5’-TTGTCATCATCGATGCTATCAT) complementing the 5’ end of the longest PABP cDNA (NI 8) near the SacI and Clal sites respectively, were synthesized as gene specific primers for 5’RACE. The fragments were then amplified according to the 5' RACE protocol (Gibco BRL), and cloned to pGEM®-T EASY vector using AT cloning (Promega protocol). The two longest cDNA fragments, 1.2 and 1.4 kb, were sequenced. The overlapping sequences of the two cDNAs were the same. One had a 397 bp 5' NTR, the other had a 216 bp 5' NTR. RdRp and PABP deletions All the PABP deletions were made from the longest cDNA obtained from the two hybrid screen, N18 (Fig. 2-3). N18A300 was amplified by PCR, using RG110 and RG157 as 5’ and 3’ terminal primers. RG110 was located at the 5' end of N18, and included an 43 EcoRI site, 5'-AAAGAAITCGGCTI‘TGTAAATITI‘GAG-T. RG 105 complemented the region from position 1801 to 1819 (position denoted according to full-length Cs- PABP cDNA) of N18 and included a XhoI site, 5’-TCTCTCGAGCAAATGTAGAACC TCAGT-3’. The PCR product was inserted into pADGAL4 as an EcoRI-Xhol fragment to form pADN18A300. NIAmlu was amplified with RG110 and RG115, which complemented the region from position 1476 to 1495, with XhoI, Sphl, and Sac] sites at the 5’end, 5'- GTACTCGAGCATGCGAGCTCAAAGGTACAGGCTGCTGG-3’. The PCR product was inserted as an EcoRI-Xhol fragment into pADGAL4 to form pADNI8Amlu, and into pBluescript to form pBSN18Amlu. pBSNI8Amlu was then digested with Sphl, and reli gated to form pBSNI8Asph. The EcoRI-Xhol N18Asph fragment was then subcloned to pADGAL4 to form pADNISAsph. All the RdRp deletions were made from full-length RdRp (Fig. 2-3). RdRpAl, A4, A5, A6, A7, and A8 were amplified by PCR using primers as indicated in Fig. 2-3; all the PCR products were inserted into pBDGAL4 as EcoRI-Sall fragments. The sequences of primers RG100, 101, 121, 125, 126, and 127 were: 5’-AGAGTCGACCCTGACT'ITCT CAAGC-3 ’, 5’ -CCGGAATTCTGC GCTGCGATGATT-3 ’; 5 ’ -ACTGAATTCCTCGAG AAAGAGAGAAT-3’; 5 ’ -GTGGAA ITCCCAA'ITCTTGCTCCTGA-3’; 5’-TTAGAA ITCGAGCTCAGGCCGCTT-3 ’, and 5 ’ -TTCGTCGACI‘CTCGAGTTTTGGAGTG-3’, respectively. RdRpA2 was generated as follows: pBDRdRp was digested with NcoI, filled in with Klenow fragment, and digested with EcoRI. This RdRp fragment was ligated to pBDGAL4, which had been digested with Sal], blunted, then cut by EcoRI to form pBDRdRpA2. pBSRdRp (RdRp in pBluescript) was cut with EcoRI and X1201, the EcoRI—XhoI RdRpA3 fragment was then subcloned to pBDGAL4 as pBDRdRpA3. 44 i‘ | _ 1. In vitro expression and poly-(A)-sepharose binding of PABP and RdRp Full-length PABP and RdRp were cloned to pET28a-l to produce histidine- tagged PABP or RdRp. Each pET plasmid, and the Promega TNT® system control construct expressing luciferase without a histidine tag, was expressed in a 5011] volume using 2ug plasmid DNA in the Promega TNT® quick coupled transcription/translation system with the addition of 0.4 mM magnesium acetate, 30 mM KC], and 35$- methionine. 5 ul of 50 ul reaction product was incubated with poly-(A)-sepharose (Sigma) for 1 hour on ice in 100 pl binding buffer [100 mM sodium acetate, 5 mM MgC12, 5 mM KC], 40 mM HEPES, pH 7.6, 10% glycerol, protease inhibitor cocktail (Sigma), 0.2% Triton X-100, 5 mM B-mercaptoethanol]. The beads were washed six times with 500 u] binding buffer plus 200 mM NaCl. The beads were then boiled in 24 ul loading buffer and analyzed by SDS-PAGE followed by autoradiography overnight. Expression of PABP and RdRp in E. coli The E. coli stain Xa 9O harboring pGEXPABP was grown at 37 C in LB with 50 mg/l ampicillin to O.D.600 = 1.0. IPTG (B-D-thiogalactoside) was added to 0.1 mM and the cells grown for another 3 hours at 37 C, then harvested. The E. coli stain Xa 90 harboring pGEXRdRp was grown at 37 C in LB with 50 mg/l ampicillin to O.D.6oo = 0.3- 0.4. IPTG was added to the medium and the cells were then grown at 16 C overnight prior to harvesting. GST-tagged PABP and RdRp were purified as recommended by the supplier (Phamacia); yield of protein was estimated by Biorad assay. E. coli strain BL21 (DE3) harboring pET-PABP was grown at 37 C in LB with kanamycin to O.D.(,oo = 0.6. After addition of IPT G to 1.0 mM, the cells were grown for 3 h at 37 C. His-tagged PABP was purified as recommended by the supplier (Novagen). 45 In vitro analysis of protein-protein interaction Five pg GST, or GST-PABP purified from E. coli was immobilized on 30 pl glutathion-sepharose (Pharmacia), and incubated with 5 pl of in vitro 355 labeled RdRp or luciferase (prepared using Promega TNT quick coupled transcription/translation system) in 90 pl binding buffer plus 10 pl bovine serum at room temperature for 1 hour. The beads were then washed 6 times with 500 p1 binding buffer plus 200 mM NaCl, boiled in 24 p1 loading buffer, and analyzed by SDS-PAGE followed by autoradiography overnight. For binding to poly (A), 5 pg His-PABP was immobilized on poly-(A)- sepharose, 5 pl in vitro 3 5 S labeled luciferase or RdRp were added. Binding, washing, SDS-PAGE and autoradiography were as above. DNA and RNA hybridization Genomic DNA was extracted from young cucumber leaves according to the protocol of Dellaporta et al. (1985). Ten pg restriction enzyme-digested DNA was subjected to electrophoresis on a 1% agarose gel and transferred to nylon membrane (Micron Separations) by capillary blotting (Sambrook et a1, 1989). Total RNA was isolated from cucumber leaf, root, immature and mature flowers as described by Chomczynski and Sacchi (1987); 10 pg of each was used for northern blot (Sambrook et al., 1989). Full-length CS-PABPI was labeled using the DIG DNA labeling and detection kit (Boeringer Mannheim). High stringency hybridization and wash conditions were done according to Sambrook et al. (1989). Low stringency hybridization and wash conditions were done following Hilson et al. (1993). 46 Results Screening of the cucumber yeast two-hybrid library with ZYMV RdRp The yeast two-hybrid cDNA library was constructed from mRNA from leaf tissue of ZYMV- susceptible cucumber (cv. Straight 8). The library contained 5 X 106 pfu; greater than 90% of the excised plasmids encoded cDNA inserts ranging in size from 0.5- 2.5 kb. Quality of the library was assessed by successful PCR amplification of the low c0py cucumber gene, CS -ACSI (ACC synthase; Trebitsch et al. 1996). Following two separate screenings of the library using ZYMV RdRp expressed in the GAL4 binding domain vector as bait, eleven cucumber clones were found to have specific, reproducible interaction with the RdRp. Each clone induced the two reporter genes allowing for growth in the absence of histidine and expression of B-galactosidase activity. In each case, plasmids encoding putative interactors were isolated and retransformed back to yeast either alone, or in combination with ZYMV RdRp, human p53, Lamin C, or binding domain vector alone. Each interacting clone failed to induce the reporter genes alone, or in any combination except with RdRp. Of the eleven verified clones showing interaction with RdRp, three enocoded distinct, unknown proteins, one had significant sequence homology to DNA J, one to protein phosphatase, and six encoded a protein having high homology (ca. 70% nucleotide identity) to poly-(A) binding protein (PABP) of Arabidopsis thaliana and wheat. The PABP-homologous cDNAs ranged in size from 0.5 kb to 1.5 kb, the longer the cDNA, the stronger the observed interaction with RdRp. The six RdRp—interacting cDNAs were reverse transcribed from at least three different mRNAs: cDNA NI 8, 60, and N1 351 have the same polyadenylation sitesl49 nucleotides downstream of the stop 47 codon; NI 7 has a poly-(A) addition site 201 nucleotides from the stop codon; and N1 439 and 447 have poly-(A) addition sites 250 nucleotides from the stop codon. Although PABPs are encoded by a multi gene family in Arabidopsis, and multiple isoforms have been observed in wheat (Belostotsky and Meagher, 1993; Hilson et a1. 1993; Le et al., 1997), the overlapping sequence of all six cDNAs showed complete sequence identity, indicating that all of the clones were transcribed from the same cucumber PAPB gene. The putative PABP clones were tested for interaction with two other ZYMV proteins, coat protein (CP) and helper component protease (HC-Pro) (Table l-l). Although both the CP and HC clones were capable of self-interaction, as would be predicted based on ability of CP to polymerize to form capsids, HC-Pro to dimerize, and previous yeast two-hybrid studies (Hong et al. 1995; Thombury et a1. 1985; Ureuqui- Inchima et al. 1999; Wang and Pirone, 1999), neither the ZYMV CP nor HC-Pro interacted with cucumber PABP. These results further suggest a specific interaction between cucumber PABP and the potyviral RdRp. The interaction between PABP and ZYMV RdRp did not occur when tested with RdRp fused to the GAL4 activation domain and PABP to the DNA binding domain. One way interactions have been observed in numerous other two-hybrid combinations and may be due to conformational changes arising from the fusion protein (Fields and Stemglanz, 1994). PABP also did not interact with itself; this is consistent with previous studies indicating that multimerization of PABP requires the presence of poly (A) (Kuhn and Pieler, 1996). The ZYMV RdRp did not interact with itself, CP, or HC-Pro. 48 Table 2-1. Interactions among ZYMV coat protein (CP), helper component- proteinase (HC-Pro), RNA dependent RNA polymerase (RdRP), and cucumber poly-(A)-binding protein (PABP) in the yeast two-hybrid system. BD-CP BD-HC-Pro BD-PABP BD-RdRp AD-CP +++a - ND - AD-HC-Pro - ++ ND _ AD-PABP - - - +++ AD-RdRp - - - _ ND: Not determined. a The strength of the interactions were based on the X-Gal filter assay. +++, colonies turned dark blue within 3 hours; ++, colonies turned dark blue within 6 hours to overnight; +, colonies turned light blue overnight; -, did not turn blue. All controls, including the clones by themselves or in combination with human p53 or Lamin C, remained white. 49 Characterization of the cucumber PABP gene and interacting domains of the PABP and RdRp proteins To further examine the interaction between the ZYMV RdRp and the putative cucumber PABP, the full length cDNA was obtained by 5’ RACE (5’ rapid amplification of cDNA ends). In total, a 2597bp cDNA sequence was obtained for the cucumber PABP gene (CS-PAPB] ; submitted to GenBank, accession no. AF240679). In the 397bp 5' NTR of the cDNA, there are 9 oligo-(A) clusters, ranging from 4 A to 9 A. These A-rich sequences were also found in PABPS from Arabidopsis, wheat and other organisms (Belostotsky and Meagher, 1993; Hilson et al. 1993; Le et al., 1997). The 3’ NT R is 250bp and three polyadenylation signal sequences are located at positions 2099, 2151, and 2201 bp. The coding sequence is 1950 bp and predicts a 649 amino acid protein with a molecular weight of 70.5 kD which is similar to other plant and animal PABPS (Belostostsky and Meagher, 1993; Le et al. 1997). This gene (CS-PABPI) is most similar to the shoot-expressed Arabidopsis PABP2 gene (Belostotsky and Meagher, 1993; Hilson et al. 1993). Comparison of percent amino acid identity shows greater homology between the putative cucumber Cs-PABPI gene and Arabidopsis PABP2 (69.7%), than between Arabidopsis PABP2 and Arabidopsis PABPS (49.4%). Consistent with apparent transcription from a single gene, Southern blot analysis of cucumber genomic DNA indicated that the cucumber PABP clone hybridized with a single distinct genomic band with most restriction enzymes (Fig. 2-1); additional bands were not observed in low stringency washes (data not shown). Although there is no EcoRI site within the coding region of the CS-PABPI gene, the two bands observed in the EcoRI digest are likely due 50 1...-..) L" "a" _ 9.4 kb - - ' ——- 4.3 kb and __ 2.3 kb , — 2.0 kb na- 1 2 3 4 5 6 Fig. 2-1. PABP is a single or low copy number gene. Cucumber DNA (~10pg) was digested with X120] (1), XbaI (2), Pstl (3), HindIII (4), EcoRI (5), and BamHI (6), transferred to nylon membrane, and hybridized with Di g-labeled PABP coding sequence. PABP cDN A sequence does not have internal recognition site for these restriction endonucleases. 51 an EcoRI site within an intron. Northern blot analysis of leaves, roots, immature and mature flowers from susceptible ‘Straight 8' plants, and leaves from the resistant lines ‘TMG-l' and ‘Dina-l', showed expression of CS-PABPI in all tissues tested; in each case hybridization was with a single band (data not shown). Higher expression was observed in leaves and immature flowers; there was no difference in expression between the susceptible and resistant genotypes. The amino terminal two-thirds of the predicted PABP contains 4 RRMS (RNA recognition motifs), which are found in PABPS from all sources, including yeast and animals (Le et al., 1997) (Fig. 2-2). Each RRM in turn has two conserved sequences, RNP-l and RNP-2, which come in direct contact with RNA (Kuhn and Pieler, 1996). RRM-1 is responsible for the interaction with eIF4G in yeast (Kessler and Sachs, 1998). The two smaller cucumber cDNAs (NI439 and NI359) lacked these RRMS, suggesting that the RRMS are not involved in binding with ZYMV RdRp. The carboxy-terminal third of the PABP protein is not as highly conserved as the amino terminus, and functions of the C-terminus are less well defined. At the C-terminus (amino acids 553-624), there is a 71 amino acid region that is conserved among Arabidopsis, wheat, and cucumber. This CTC (C-terminal conserved) domain was also found within other characterized PABPS (e. g. yeast, vertebrate) and has been implicated in homodimerization and efficient poly (A) binding (Kuhn and Pieler, 1996). The smallest PABP cDNA obtained from the two-hybrid screen (NI 351) encodes the last 130 amino acids and interacts with the ZYMV RdRp. This suggests that the CTC domain might be involved in the interaction with RdRp. Consistent with these results, deletion of the C—terminal 50 amino acids of PABP abolished the interaction in yeast, indicating that 52 A. MAQVPPQPQVPNSGADPAANGGANQHVTTSLYVGDLDVNVTDSQLYDLFNQ VGQVVSVRVCRDLTSRRSLGYGYVNYSNPVDASRALDVLNFTPLNGNPIRV MYSHRDPSVRKSGSGNIFIKNLDKAIDHKALHDTFSAFGSILSCKVATDSSGQ SKGFGFVQFDTEEAALKAIEKLNGMLLNDKQVFVGPFLRKQERESVSEKTKF NNVFVKNLAETTSEEDLKNMFGEFGPITSVVVMRDGEGKSKCFGFVNFENA DDAARSVEALNGKKVDGKEWYVGKAQKKSEREVELKSRFEQSVKEAADKYQ GANLYVKNLDDSIDDDKLKELFTGFGTITSCKVMRDPNGISRGSGFVAFSSPE EAARALAEMNGRMIVSKPLYVALAQRKEDRIARLQAQFSQMQPMAMASSVA PRGMPMYPPGGPGIGQQIFYGQAPPTIISSQPGFGYQQQLMPGMRPGGGPMPNFF VPMVQQGQQGQRSGGRRAGAIQQTQQPVPLMQQQMLPRGRVYRYPPGRGLPD LPMPGVAGGMFSVPYEMGGMPPRDAVHPQPVPVGALASALANATPDQQRTM LGENLYPLVEQLEPDNAAKVTGMLLEMDQTEVLHLLESPEALKAKVAEAM EVLRSVAQQSGNAADQLASLSLTDNLDS Interaction with RdRp‘I B. RRM1 RRM2 RRM3 RRM4 CTC -m:.l1:_.ll.-x -- I1 -: +4.... 1 29 107117 139203 235 311 333 556 629649 N18 245 ' I l +++ Clal Sac] l 1 +++ N17 273 l 1 +4- Nl439 395 . l l .., N135] 519 Fig. 2-2. Predicted amino acid sequence of cucumber poly- (A) binding protein. A. Amino acid sequence predicted from the full-length cDNA. The first 4 regions in bold are RNA recognition motifs (RRM), the last region in bold is the C-terminal conserved domain (CTC domain). B. Schematic presentation of full-length PABP protein and products for two-hybrid interacting PABP cDNAs (numbering of the clones is as in Table 1 ). 53 the C-terminus of PABP is essential for the binding with RdRp (Fig. 2-3A); we cannot rule out, however, the possibility that the failure to detect interaction with the C-terminal deleted protein is due to lack of stability of the deleted protein. Deletion analysis was used to examine regions of the RdRp responsible for interaction with PABP (Fig. 2-3B). Deletions from both the amino and carboxy termini of the RdRp abolished the interaction in yeast. Although we cannot eliminate the possibility that certain deletion products were unstable, it may be that a large portion of the RdRp is necessary for the interaction with PABP. In vitro verification of the RdRp - PABP interaction The in vitro expression product of the full-length PABP cDNA migrated in SDS- PAGE gels as predicted for an ca. 70 KDa protein. The labeled cucumber PABP was able to bind to poly-(A)-sepharose in vitro (Fig. 2-4A, lane 7), confirming that the cloned cDNA encodes a true PABP. Like other picomaviral RdRps (Cui et a1. 1993; Paul et al. 1994), ZYMV RdRp alone did not bind directly to poly (A) (Fig. 2-4A, lane 6). To demonstrate that the cucumber PABP can bind to ZYMV RdRp in vitro, full length PABP was expressed in E. coli as either a His-PABP fusion protein or GST-PABP fusion protein and was immobilized on poly-(A)-sepharose or glutathione-sepharose. Approximately 10-20% of the input RdRp bound to these fusion proteins (Fig. 2-4B, 4C), but none bound to poly-(A)-sepharose (Fig. 2-4B) or GST-sepharose alone (Fig. 2-4C). Luciferase did not bind to the PABP bound to polyA- or glutathione-sepharose. These results suggested that the RdRp and PABP bind to one another specifically in vitro. In analogous experiments where RdRp was expressed in E. coli as a GST-RdRp fusion protein and immobilized on glutathione-sepharose, approximately 10-20% of the in vitro 54 A NI 8" N1 8 A300 NI 8 Amlu NI 8 Asph B RdRp RdRpAl RdRpA2 RdRpA3 RdRpA4 RdRpAS RdRpA6 RdRpA7 RdRpA8 Sphl Mlu I 6 F r f r 730 1251 1521 1802 2099 RG1 I? iGlfl R0110" *Rous I g Sac I Nco 1 Xho I Sac I 11 ll .fi . 4|! I I I I I 525 645 12281340 1551 I '7 1 RG89Ei i RG100 E 3’ r _._l L —l RGl0l J iRGSS RG121ERGS8 l RG126i 1R688 R612; ' RG127 RG125 i I RG88 Interaction with RdRpb +++ Interaction with N18 +++ Fig. 2-3 Deletion analysis to determine regions in PABP and RdRP responsible for the interaction. A. Different sizes of cDNA NI 8 were amplified by PCR or generated by restriction enzyme digestion. All the deletions were tested against full-length RdRp in the yeast two-hybrid system. B. Different sizes of RdRp were amplified by PCR or generated by restriction enzyme digestion. All the deletions were tested against NI 8 in the yeast two-hybrid system. Numbering in each case corresponds to the full length cDNA sequence. 55 25% Input poly- (A) bound A. Luc RdRp PABP Luc RdRp PABP 1 2 3 4 5 6 B. poly-A+ poly-A+ poly-A+ PABP+ PABP+ 10% Input RdRp Luc RdRp RdRp Luc C. G-PABP G—PABP GST+ 10% Input +RdRp +Luc RdRp RdRp Luc “1...... .3; v' — W‘L—w Fig 2-4. Cucumber PABP can bind to poly-(A) and RdRp in vitro. A. PABP can bind to poly-(A)-sepharose in vitro. Lane l-Lane 3: 25% input of in vitro labeled luciferase (Luc), RdRp, and PABP. Lane 5- Lane 7: proteins on poly-(A)-sepharose after six washes. B. PABP can bind to RdRp in vitro in the presence of poly-(A). Lanes 1 and 2: His-PABP purified from E. coli was immobilized on poly-(A)-sepharose, and incubated 56 with 35S-RdRp (Lane 1) or 35S-Luc (Lane 2), followed by six washes. Lane 3: RdRp was directly incubated with poly-(A)—sepharose without His-PABP. Lanes 4 and 5:10% input of 35S-RdRp and 35S-Luc. C. PABP can bind to RdRp in vitro without the presence of poly-(A). GST-PABP (Lane 1, 2 ) or GST (Lane 3) was immobilized on glutathione- sepha-rose, and then incubated with 35S-RdRp (Lane 1, 3) or 35S-Luc (Lane 2) followed by six washes. Lanes 4 and 5 are 10% input of 35S-RdRp and 3SS-Luc. 57 labeled PABP was retained (data not shown). Since a comparable percentage of binding was observed whether the RdRp or PABP was immobilized and whether RdRp was expressed in vitro or in E. coli suggests a modest affinity between the two molecules. Whether this modest affinity reflects affinity in planta where other interacting factors may be involved, is not known. Consistent with the ability of the C-terminal portion of the PABP to interact with the RdRp in yeast, the binding in vitro did not require the presence of poly-(A) (Fig. 2- 4C). The NI 8 and N1 439 clones also weakly bound RdRp in vitro, but the shortest clone, N1 351, did not Show detectable binding (data not shown). The difference between these results with the short clones in vitro, and the yeast two hybrid assay may reflect differences in sensitivity of the two methods. The shorter clones also showed reduced interactions in the yeast two-hybrid system as assayed by speed and intensity of blue color development. Discussion The above-described results indicate that the ZYMV potyviral RdRp is capable of specifically interacting with host PABP in the yeast two-hybrid system and in in vitro binding assays. To our knowledge, PABP has not been implicated as a component of any viral replicase complex, nor identified to play a role in virus replication (Lai, 1998; Strauss and Strauss, 1999). The repeated and high frequency isolation of PABP (6 of 11 clones) indicated that the interaction is reproducible. The interaction of the RdRp with PABP is particularly intriguing Since potyviruses are polyadenylated at the 3' end of the positive strand which is the site of initiation of minus strand synthesis. 58 Interaction was abolished by either amino or carboxy terminal deletions of the RdRp. This may reflect complex secondary or tertiary structure of the RdRp. RdRps, like other types of DNA and RNA polymerases, consist of finger-palm-thumb domains resulting in intramolecular interactions between the amino- and carboxy-terminal portions of the molecule (O’Reilly and Cao, 1998; Lesburg et al. 1999); such interactions also may be important for association with the PABP. Similar problems in assigning functions to specific RdRp domains were observed with the tobacco etch potyvirus (TEV) RdRp. Loss of interaction between the TEV RdRp and the Nla protein were observed with both amino and carboxy terminal deletions of the TEV RdRp, and nuclear localization capacity was eliminated by deletions from either terminus and by small insertions at several positions in the protein (Li and Carrington, 1993; Li et al. 1997). In this study ZYMV RdRp did not interact with itself. Previous two-hybrid analysis with TEV and tobacco vein mottling virus (TVMV) potyviral RdRps have provided different results (Hong et al., 1995; Li et al. 1997). Self-interaction was observed for TVMV RdRp but not TEV RdRp. The reasons for these differences among the potyviral RdRps is not known, it may reflect real differences or may be an artifact due to an interfering effect of the GAL4 domains in one or both fusion proteins. We also did not observe the interaction between RdRp and CP as was reported for TVMV (Hong et al., 1995). In that study interaction was observed only when the RdRp was fused to the binding domain. Despite known interactions between HC-Pro and CP in vivo (e. g., for aphid transmission), and demonstrations of interaction using in-vitro binding assays (Blane et al. 1997; Peng et al. 1998), we did not observe interaction between HC-Pro and CP in our yeast two-hybrid assay. The failure to bind in the yeast two-hybrid assay may 59 reflect burying of the amino terminus of the CP in the fusion protein construct. The CP amino terminus is normally externally located on the virion and is critical for the CP/HC- Pro interaction. PABP belongs to a large family of RN A-binding proteins that contain highly conserved RNA recognition motifs (RRMS). Although PABPS generally exist as multigene families, complete sequence identity in overlapping regions of the interacting cucumber clones indicated that a single cucumber PABP was interacting with the ZYMV RdRp. Southern blot analysis showed only a single hybridizing band, which may reflect the high sequence variability among different members of the PABP family (Belsotosky and Meagher, 1993; Hilson et al. 1993). These results suggest specificity in the RdRp- PABP interaction, although the possibility of developmental or tissue specific expression of the different PABPS such that only a single gene family member was expressed in leaves at the time they were harvested for cDNA synthesis, cannot be eliminated; within Arabidopsis different members of the PABP gene family are expressed in different tissues (Belostosky and Meagher, 1993: Hilson et al. 1993). Northern analysis indicated that the CS-PABPI gene was expressed in all tissues tested including leaves, roots, immature and mature flowers. PABP has been the subject of a good deal of recent research indicating that it plays a critical role in eukaryotic translation (Jacobson, 1996; Gallie, 1998; Sachs et al. 1997). PABP is an essential component of eukaryotic cells; deletion of PABP in yeast can cause lethality (Sachs et al. 1987) and reduction in PABP levels, either by cleavage or sequestration by viral proteins, can result in shut down of host translation (Chen et al. 1999; Joachims et al. 1999; Piron et al. 1998). Recent evidence has shown that PABP 60 also facilitates initiation and maintenance of efficient translation by promoting interactions between the 5' and 3' termini of messenger RNAs (reviews: Gallie, 1998; Sachs et al. 1997). A typical mRNA molecule has a 5’ cap and 3’ poly-(A) tail, the two termini function synergistically to promote translation through protein-protein interactions: at the 5’ end, eIF4E, which is a subunit of eIF4F (containing subunits eIF4E, eIF4G, and eIF4A), binds to the cap structure; at the 3’end, PABP binds to the poly-(A) tail, PABP then binds to eIF4G. These interactions result in a circular mRNA molecule. By bringing the 5’ end and 3’ ends close to each other, translation of full-length message is promoted and re-initiation of translation is facilitated. Although not capped, the potyviral 5' NTR of TEV conferred synergistic enhancement of translation when in combination with a poly (A) tail (Gallie et al., 1995). Viral RdRps frequently have been shown to interact with components of the host translational apparatus (Lai, 1998; Strauss and Struass, 1999). The results presented here showing interaction of PABP with the ZYMV RdRp suggests that an additional component of the host translational machinery associates with a viral replicase and raises some intriguing questions. Perhaps, similar to its role in eukaryotic translation, PABP facilitates intramolecular interactions relevant to potyviral replication. Cellular proteins binding viral RNA may serve to bring spatially separate regions, including 3' and 5' termini, of viral RNA template together to form replication complexes. The joining of plus and minus strand leader sequences is a critical step in mouse hepatitis virus RNA synthesis (Lai, 1998). Perhaps the interaction between RdRp and host PABP serves to promote interaction between RdRp and the viral poly (A) tail, either by helping to recruit RdRp to the poly (A) tail, or by facilitating removal of PABP from the poly (A) tail and 61 allowing access of the RdRp for initiation of replication. Deletion analyses and partial clones obtained in the yeast two-hybrid screen indicated that interaction with the RdRp occurred via the carboxy terminus and not the RNA binding motifs in the amino terminal half of the protein. Studies with Xenopus PABP showed that the conserved CT C domain was important for polymerization of PABP leading to enhanced PABP binding (Kuhn and Pieler, 1996). Perhaps association of the RdRp with PABP interferes with the PABP polymerization and facilitates removal from the poly (A) tail. Recent studies have shown that viral-induced shut down of host protein synthesis, which is thought to facilitate viral infection by increasing accessibility of host factors for viral purposes, can be mediated, at least in part, by sequestration or cleavage of PABPS. PABPS were sequestered by NSl protein during influenza A virus infection (Chen et al., 1999), were removed from interaction with eIF4F during rotavirus infection (Piron et al., 1998), and were cleaved by viral proteases during Picomavirus infection (J oachims et al. 1999). In each case there was an associated reduction in host protein synthesis that could be related to an effect on PABP. Interestingly, the interaction between influenza A N81 and human PABPII, which takes place in the nucleus and results in hnRNAs with poly (A) tails that are too short to allow for export, occurs via the carboxy terminus of PAPBII. Shut down of host translation is less well studied for plant viruses. In the one system that has been examined, infection by pea seed borne mosaic potyvirus (PSbMV), inhibition of host gene expression and virus-mediated mRNA degradation occurred in a reversible manner during the course of infection (Wang and Maule, 1995; Aranda et al. 1996; Aranda and Maule, 1998). There appears to be a widespread loss of many host 62 mRNAs, indicating degradation. If RdRp serves to remove PABP from the poly (A) tail, this could result in decreased mRNA stability. Such an observation would not be inconsistent with sequestration of PABP. During potyvirus infection, RdRp is expressed in large quantities and can accumulate in the nucleus as an inclusion body (NIb). Its function in the nucleus is unknown since the viral life cycle is completed within the cytoplasm. It is possible that the NIb/RdRp sequesters PABP and keeps it from binding to hnRNA in the nucleus, inhibiting RNA processing. The possible involvement of the RdRp-PABP interaction in viral replication by recruitment to the poly (A) tail or participation in translational inhibition are not necessarily mutually exclusive. RdRp may interact with PABP to facilitate viral replication, while at later stages, increasing quantities of RdRp might inhibit host translation. Our future research will examine possible functions of the interaction between potyviral RdRp and cucumber PABP. Acknowledgements: We thank Dr. Eric Stockinger for help with yeast manipulation and protein purifications and Mr. Yaopan Mao for assistance with protein-protein binding assays. We appreciate Drs. Richard Allison and Suzanne Thiem for critical reviews of the manuscript. 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Virol. 8,256-273. 68 Chapter 3 Identification and characterization of a wound inducible protein that interacts with the carboxy terminus of poly- (A) binding protein and inhibits translation Abstract A cucumber protein, PC16 (RABP-QT Interacting), was identified based on its ability to interact with the carboxy terminus (CT) of poly- (A) binding protein (PABP) both in yeast two-hybrid and in vitro binding assays. PC16 shares a conserved amino acid domain (SmLanApvap) in common with a second cucumber PABP-CT interactor (PC1243), human PABP-CT interactors, and with Arabidopsis ERD15 (Early Responsive to thydration). Deletion analysis and point mutations indicate that the presence of this domain is necessary for the interaction. PC16 expression is inducible by both wounding and jasmonic acid; mRN A levels increased sharply 4-8 hours post-treatment in both wounded and non-wounded upper leaves, indicating systemic induction. Recent studies with human PABP interacting proteins have indicated a newly identified mechanism of translational regulation. PC16, like the human PABP-CT interactors, influenced translation in mouse ascites Krebs2 and wheat germ translation systems. Wild type PC16 inhibited translation in wheat germ, whereas the non-interacting mutant, PC16-23A, did not. These results demonstrate an interaction between PABP and a wound responsive protein, with possible implications for translational regulation. 69 Introduction Poly- (A) binding protein (PABP), which binds to the poly- (A) tail of mRNA, is an essential component for a broad range of eukaryotic cellular functions (Sachs et al, 1987). Together with poly- (A), PABP participates in pre-mRNA processing (Amrani et al, 1997; Minvielle-Sebasia et al. 1997), regulation of mRNA stability (Caponigro and Parker, 1996; Coller et al, 1998), and initiation and stimulation of translation (Gallie, 1998; Gray et al, 2000; Jacobsen, 1996; Otero et al, 1999; Sachs et al. 1997). Deletion of the PABP coding sequence in yeast leads to a lethal phenotype (Sachs et al. 1987) and reduction of PABP levels, either by cleavage or sequestration during viral infection can inhibit host translation (Chen et al. 1999; Kerekatte et al. 1999; Joachims et al. 1999; Piron et al. 1998). PABP facilitates initiation and maintenance of efficient translation by promoting interactions between the 5’ and 3’ ends of mRNAs (Jacbson, 1996; Gallie, 1998; Sachs et al, 1997; Tarun and Sachs, 1996; Le et al, 1997b). A typical mRNA has a 5’ cap and 3’ poly— (A) tail; the two termini function synergistically to promote translation through protein-protein interactions. At the 5’ end, eIF4E (eukaryotic Initiation Eactor E), which is a subunit of eIF4F complex (containing subunits of eIF4E, eIF4A eIF4G), binds to the cap structure (reviewed in Gingras et al, 1999); at the 3’ end, PABP binds to the poly- (A) and then to eIF4G. These interactions result in a circular mRNA molecule (Wells et al, 1998). By bringing the 5’ and 3’ ends close together, translation of full-length mRNA is promoted and re-initiation of translation is facilitated (Jacbson, 1996; Gallie, 1998; Kahavejan et al, 2002; Sachs et al, 1997). A key distinguishing feature of PABP is the presence of four highly conserved RNA recognition motifs (RRMS) found in PABPS from all sources, including plants, 70 animals, and yeast (Belostotsky and Meagher, 1993; Hilson et al, 1993; Le and Gallie, 2000). The RRMs, which are located in the amino terminal two-thirds of the protein, include two conserved sequences that come in direct contact with RNA (Burd and Dreyfuss, 1994). The second RRM is responsible for interaction with eIF4G (Kessler and Sachs, 1998). The carboxy terminal (CT) third of the PABP protein is not as highly conserved as the amino terminus, and functions of the CT have been less well defined. Coller et al. (1998) observed that deletion of the C-terrninal 50 amino acids of yeast Pablp (yeast PABP) abolished the ability to rescue yeast cells lacking a functional PABI gene. Cleavage of PABP between the RRMS and CT by coxsackievirus or poliovirus 2A proteinases diminished host translation, and the cleaved fragments were unable to stimulate translation of capped, polyadenylated mRN As in vitro (Kerekatte et al. 1999). These experiments indicate the importance of PABP-CT in cellular functions. PABP-CT also has been implicated in homo-dimerization and proper nuclear shuttling (Kiihn and Pieler, 1996; Afonia et al. 1998). PABP-CT has drawn increasing attention recently and roles played by PABP-CT are emerging due to the identification of its interactors. Several proteins recently have been identified to interact with PABP-CT; three human proteins, eukaryotic polypeptide chain releasing factor 3 (eRF3, Hoshino et al, 1999), Paip 1 (PABP-interacting protein 1, Craig et al, 1998), and Paip 2 (Khaleghpour et al, 2001a, 2001b); a yeast protein Pbplp (PABP binding protein, Mangus et al, 1998); and a viral protein, zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase (RdRp) (Wang et al, 2000). A conserved 12- amino-acid motif was found among Paipl, Paip2, and eRF3 and the 15 amino-acid region containing this 12 amino acids was termed PAM2 in Paipl and 2 (PABP interacting 71 Motif) (Roy et al, 2002). Deletion analyses and binding studies with a short peptide containing the PAM2 domain showed that this region is sufficient for the binding to PABP-CT (Khaleghpour et al, 2001b; Kozlov et al, 2001; Roy et al, 2002). Searching the NCBI (National Qenter of _B_iotechnology Information) gene bank with the motif showed several Arabidopsis proteins with this motif, however, functions of these proteins are not known and the interaction with PABP-CT was not verified (Khaleghpour et al, 2001; Kozlov et al, 2001). Gene expression can be regulated not only transcriptionally, but also post- transcriptionally, for example, at translation steps (Mathews et a1, 2000). Translational regulation is rapid and direct and plays important roles in the control of cell growth, development, and stress responses (Reviewed in Conlon and Raff, 1999; Gingrus et al, 1999; Schneider, 2000). Modulation of translation is usually exerted at the initiation step (Mathews et al, 2000); a body of evidence indicated that ribosome recruitment step is the major target (Reviewed in Gingras et al, 1999). Recent studies with human PABPI interactors Paipl and Paip2 provide the first demonstration of a proposed mechanism of translational regulation involving the 3’ end of mRNA, in which a co-activator(Paip1) and a repressor (Paip2) modulate translation (Kahvejian and Sonenberg, 2002). The two molecules compete with each other for binding to PABP, and either promote or inhibit translation (Craig et al, 1998; Khaleghpour, 2001a). It is not known if this mechanism occurs more broadly. We previously identified an interaction between a potyviral RNA-dependent RNA polymerase (RdRp, viral replicase) and cucumber PABP-CT (Wang et al, 2000) and proposed that the interaction could be involved in potyviral replication. To address the 72 possible roles of PABP-CT in host functions, we sought to identify and examine host proteins interacting with PABP-CT. Plant proteins interacting with PABP-CT had not yet been identified. I report here the identification of two novel proteins, PC16 (RABP-QT Interacting) and PC1243, that interact with PABP-CT and full-length PABP. Both proteins share the 12-amino-acid motif common to the mammalian interactors Paipl, Paip2 and eRF3 (Khaleghpour et al, 2001b; Roy et al, 2002). I further demonstrated that two amino acids, leucine at 16th and phenylalanine at 23rd positions, are critical for interaction with PABP-CT. Expression of PC16 at the transcriptional level is up-regulated by wounding and jasmonic acid (J A). Addition of PC16, but not a non-PABP interacting PC16 mutant, inhibited translation in the in vitro wheat germ translation system. Materials and Methods Screening the cucumber leaf cDNA library using the C-terminus of PABP (PABP-C254) The cucumber leaf two-hybrid cDNA library, yeast transformation, growth media, and X-gal (5-bromo-4-chloro—3-indolyl B-D-galactopyranoside) filter assays have been described previously (Wang et al, 2000). Yeast (Saccharomyces cerevisiae) strain YRG2 cells were first transformed with pBDPABP-C254 (referred to as NI439 in Wang et al, 2000), which produces a fusion protein with the GAL4 binding domain and the C- terrninal 254 amino acids of cucumber (Cucumis sativus L.) PABPl, and then transformed with CsCl-purified plasmid DNA from the cucumber leaf cDNA library. The transformants were plated onto SD (synthetic dropout) medium without leucine, 73 tryptophan, and histidine (SD-L—T-H). Colonies that grew on selection medium were restreaked onto SD-L-T-H, transferred to nitrocellulose membrane (Schleicher & Schuell), and assayed for expression of B-galactosidase activity (Lac Z) by X-Gal filter assay. Colonies that survived medium without histidine and turned blue in the X-Gal assay were considered as putative positives. Plasmids were recovered from putatively positive yeast colonies using the Zymoprep yeast plasmid miniprep protocol (Zymo Research, CA), transformed to E. coli, and amplified. The recovered plasmids were then transformed back to YRGZ yeast either alone, or in combination with pBDPABP-C254, vector pBDGAL4, or two negative controls: p53 and pLamin C. The transformants were plated onto SD-H or SD-L-T-H, and further assayed for Lac Z activity. Those that expressed both reporter genes only in combination with pBDPABP-C254, but did not turn on reporter genes, either alone, or in combination with pBDGAL4, p53 or pLamin C were considered postitives. Deletions and mutants of PCIS, ERD15 and CS-PABPl: The coding sequences and 3’NTRS of PC16 and PC1243 were amplified by PCR with Vent DNA polymerase (New England Biolabs) using the original clones isolated from the yeast two-hybrid cDNA library as template and inserted as BamHI —XhoI fragments into pAD-GAL4 vectors in frame to form pADPCI6, and pADPC1243. The primers RG168 (ATGGATCCMQGATGTTG’ITACTCAAA, PC16 5’ primer) and RG93 (CAGTATCTACGATTCATA, complementary to the sequence 935-952 on pAD vector), RG188 (AAGGATCCAIQGCTCTAGCATCTGTI‘, PC1243 5’ primer) and RG93 were used to amplify PC16 and PC1243, respectively. The PCR fragments were digested with BamHI and XhoI and inserted into the pAD vector. To make pBDPCI6, the plasmid DNA 74 recovered from yeast was digested with Xho] thoroughly and EcoRI partially and ligated to the pBDGAL4 vector. This full-length cDNA insert contains 5’ and 3’ NTRs and coding sequence. Primers RG189 (AAGGATCCCCCATGGCCTATAGAACG, which has the PC16 nucleic acid sequence from 76 to 94 as 5’ primer) and RG93 were used to amplify PCI6dl. The BamHI-XhoI fragment was inserted into the pAD vector to form pADPCI6d1. Plasmids pADPCI6d2 and pADPCI6d3 (encoding first 83 and 101 amino acids, respectively) were constructed as follows: pADPCI6 was digested thoroughly with BamHI and partially with EcoRI, two BamHI-EcoRI PC16 fragments were recovered and subcloned to the pADGAL4 vector. Fragments PCI6d4 and PCI6d5, which encoded the first 56 and 45 amino acids, respectively, were amplified using RG168 and RGl90 (AACTCGAGGCGTTCTTGAAGCCAT), or RG168 and RG191 (AACTCGAGG GAGGACTGGATGAGC). Point mutations were introduced into PC16 with PCR based site-directed mutagenesis (Ho et al, 1989). A typical process is as follows: RG168 and an antisense primer with the intended mutation (RG237, RG244, RG246, or RG248, Table 3-1) were used to amplify the 5’ end of PC16 with the introduced mutation; RG93 and a sense primer (RG236, RG 243, RG245, or RG247, Table 3-1) were employed to amplify the 3’ end of PC16 with the same mutation. The PCR products were then gel-purified and 100 ng of each of two ends were used as templates to amplify full-length PC16 with the specific mutation. RG236 (CGCTCC’ITTGGCCGTTCCCAT) and RG237 (CATGG 75 GAACGGCCAAAGGAGCG), RG243 (GTITCCATGTCGAATCCCAAC) and RG244 (GTTGGGATTCGACATGGAAAC), RG245 (GTTGAATCCCGCCGCT CC'I'IT G) and RG246 (CAAAGGAGCGGCGGGA'I‘TCAAC), RG247 (GAATCCCAACTCT CCTIT G TT) and RG248 (AACAAAGGAGA GTTGGGATTC) were used to mutate Phe-23 to ala, Leu-16 to Ser, Ala-l9 to Ser, and Glu-20 to Ala, respectively. The coding sequence of Arabidopsis ERD15 was amplified with Vent using plasmid 157B7T7 from the Ohio Stock Center as template and inserted as an EcoRI-Sall fragment into pBD-GAL4 and pAD-GAL4. The primers RG 205 (AAGAA'ITCflGC GATGGTATCAGG) and RG 206 (AAGTCGACTCAGCGAGGCTGGTGG) were used. Plasmids pBDPABP, pBDPABP-C406 (encoding the C-terminal 406 amino acids of CS-PABPl, termed N18 previously), pBDPABP-C254, pBDPABP-Cl31 (encoding the C-terminal 131 amino acids of CS-PABPl, termed NI351 previously) and pBDPABPACSO (deletion of 50 amino acids from the CT, termed PABPA320 previously) were constructed by digesting the corresponding activation vector pADGAL4 plasmids and inserting the cDNA fragments into the pBDGAL4 vector as EcoRI-XhoI fragment (Wang et al, 2000). In vitro analysis of protein-protein interaction: Full-length PC16, PCI-23A mutant, and PC1243 were cloned to pET28a-l (Novagen) to produce histidine-tagged PC16, PC16-23A, or PC1243. All genes were expressed using the Promega TNT‘ quick coupled transcription/translation system with 2 ug DNA, 0.4 mM magnesium acetate and 30 mM KCl in the presence of 35S-methionine. His-PABP was over-expressed and purified from E. coli strain BL21 (DE3) as described before (Wang et al, 2000). Five pg of His-PABP was immobilized on 30 ul of poly-(A)- 76 sepharose (Sigma), 5 pL of in vitro 35 S labeled PC16, PC16-23A, PC1243, ZYMV RdRp or luciferase was added and incubated in 90 pl binding buffer (100 mM NaAc, 5 mM MgC12, 5 mM KCl, 40 mM HEPES, pH 7.6, 10% glycerol, protease inhibitor cocktail (Sigma), 0.2% Triton X-100, 5 mM B-mercaptoethanol) plus 10 pl bovine serum at room temperature for 1 hour. The beads were then washed six times with 500 pl binding buffer plus 200 mM NaCl, boiled in 24 pl loading buffer, and analyzed by SDS-PAGE followed by auto-radiography overnight. B-Galactosidase activity assay: B-Galactosidase activity of yeast cells was measured following the protocol from Clontech ‘Yeast Protocols Handbook’. The yeast colonies were picked and cultured overnight in 2.5 ml SD-L-T. Either 0.2 ml or 1.0 ml yeast cells (depending on the affinity of two proteins according to the X-gal filter lifting assay) were harvested at OD6OO of 0.6-1.0. Cell pellets were then resuspended in 100 pl Z-buffer (60mM NazHPO4, 40mM NaHzPO4, 10mM KCl, lmM MgSO4) without B-mercaptol ethanol (B-ME), frozen in liquid nitrogen and thawed in a 30°C water bath three times. Z-buffer (0.7 ml) with B- ME, 0.16 ml 4 mg/ml o-nitrophenol-B-galactopyranoside (ONPG) was added and incubated in a 30°C water bath for 15 minutes or 45 minutes. The reaction was then stopped by adding 0.4 ml 1M N azCO3 and absorbance at 420 was measured. The B- galactosidase activity was calculated as described by Miller (1972). Plant materials and treatment: Cucumber (cv. Straight 8) seeds were germinated and grown in containers (10x10x7.5cm) containing commercial soil mix (sphagnum peat 70-80%, pH5.5-6.5) 77 (Baccto, Michigan Peat Company, Houston, TX) in a growth chamber with 14-h (24°C)/10-h (20°C) light/dark cycles. For wounding treatment, plants were grown to the 4-leaf stage and the tips of the second leaves were wounded with forceps. Tissues were harvested from the wounded second leaves and non-wounded third leaves at 0, 1, 2, 4, 8, and 24 hours after wounding. For drought treatment, two types of treatments were used. For the half-leaf drought treatment, the second leaves were detached from the plant and cut into two pieces along the main vein. One piece was put in between wet Whatman filter papers and sealed in petri dish as control. The other half was put between two dry Whatman filter papers. Control and treated half-leaves were frozen in liquid nitrogen at 0, 1, 2, 4, 24 hours after treatment. For whole-leaf drought treatment, whole leaves were detached and put either in wet or dry filter papers. For methal jasmonate (MeJ A) treatment, plants were put in a lucite box (31 cm x 27 cm x 14 cm). One pl of MeJ A was diluted into 60 pl ethanol and 10 pl solution was dropped on each of 5 cotton swabs that were distributed evenly in the box. The box was then sealed, one pair of plants was removed from the box at the time defined and the third and fourth leaves were harvested. Control plants were placed in a lucite box and either left untreated or treated with treated with 50 pl of ethanol. Northern Analysis: Total RNA was isolated using ‘Concert Plant RNA reagent’ from Invitrogen. Five pg or 10 pg of RNA were used for northern blots with PC16 as probe and 15 pg was used with PC1243 as probe. Both PC16 and PC1243 RNA probes were made using the ‘MAXIscript’ kit from Ambion and labeled with Digoxigenin-l l-UTP (Dig, Roche). Pre- hybridization and hybridization were conducted at 50 °C in hybridization buffer 78 containing 50% formamid (Roche protocol). The membrane was washed twice (15 minutes each) at room temperature using 2xSSC/0.2% SDS, and 4 times at 68 °C (15 minutes each) using O.1>