Institute of Biotechnology, PO Box 56 (Viikinkaari 9), Viikki Biocenter, FIN-00014 University of Helsinki, Finland1
Department of Plant Biology, Genetics Centre, SLU, S-75007 Uppsala, Sweden2
Author for correspondence: Deyin Guo. Fax +358 9 191 59366. e-mail dguo{at}operoni.helsinki.fi
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Abstract |
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YTHS has been used extensively to detect interactions between proteins from many different organisms. The results show a high correlation with affinities that are determined by biochemical methods (Estojak et al., 1995 ). Using YTHS, protein-linkage maps have been established for Escherichia coli bacteriophage T7 (Bartel et al., 1996
), Saccharomyces cerevisiae RNA-splicing machinery (Fromont-Racine et al., 1997
) and poliovirus P2 and P3 proteins (Cuconati et al., 1998
; Xiang et al., 1998
). The technique has also been used to explore the entire proteinprotein interaction network within yeast cells (Ito et al., 2000
; Uetz et al., 2000
).
Potyviruses possess a single-stranded, positive-sense RNA genome of about 10 kb. The genomic RNA is translated into a large polyprotein that is subsequently processed by three virus-encoded proteinases to yield mature functional proteins P1, HC, P3, 6K1, CI, 6K2, VPg, NIaPro, NIb and CP (Riechmann et al., 1992 ). A remarkable feature of potyviruses, compared with other plant RNA viruses, is that most potyviral proteins are multifunctional and several of them participate in both genome replication and virus movement (Revers et al., 1999
; Carrington et al., 1998
), which implies that at least some functions rely on viral proteinprotein interactions.
Some interactions between potyviral proteins have been studied using YTHS (Hong et al., 1995 ; Li et al., 1997
; Merits et al., 1999
; Guo et al., 1999
; Urcuqui-Inchima et al., 1999
). However, interactions between all of the proteins from a single potyvirus have not been studied. The aim of this study was to generate a YTHS-based profile of potyviral protein interaction networks through a comprehensive analysis of interactions among all the major proteins from two potyviruses. Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV), which are not closely related (Berger et al., 1997
) and naturally infect species of different plant families (Solanaceae and Fabaceae, respectively), were chosen for this study; this choice was also favoured by the fact that full-length cDNAs of their genomes were available.
YTHS (Hollenberg et al., 1995 ) and the LexA system (Clontech) were used for the study of PVA and PSbMV proteinprotein interactions, respectively. P3- and NIb-encoding sequences of PVA were subcloned from E. coli expression constructs (Merits et al., 1999
) into the yeast DNA-binding domain (BD) and transcription activation domain (AD) vectors (Table 1
). Other sequences were amplified by PCR from the full-length infectious cDNA of PVA-B11 (accession no. AJ296311) and PSbMV-NY (accession no. X89997; cDNA kindly provided by E. Johansen) using Expand High-Fidelity DNA polymerase (Boehringer Mannheim). PCR fragments were initially cloned into pGEM-T (Promega) and subsequently cloned into yeast fusion vectors (Tables 1
, 2
). All PCR clones and fusion junction sequences in the YTHS vectors were verified by sequencing.
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If one interacting partner in any of the combinations mentioned above was replaced with human lamin C (negative control), the activities of the reporter genes were reduced to background level. The interactions were mediated, therefore, specifically by the viral protein parts in the BD- and AD-fusions. Some interactions also showed directionality. For example, interactions between P3 and NIa from PSbMV were detected only when these proteins were expressed in fusion with BD and AD, respectively (Table 2). Similarly, the interaction between BDNIb and ADNIa was stronger than that between BDNIa and ADNIb (Tables 1
, 2
). Such disparity implies that protein fusions in one direction may have more favourable protein folding or exposure of binding sites than those in the other direction.
The relative strengths of different interactions were compared by quantitative assays of -galactosidase activity. In all experiments,
-galactosidase activity in liquid assays correlated with colour development in filter assays. Colonies harbouring interacting partners, which developed a visible blue colour between 1 and 2 h in filter assays, all produced more than 50 relative units of
-galactosidase activity (Tables 1
, 2
). Colonies that developed a visible colour by 6 h in filter assays gave between 3 and 50 relative units of
-galactosidase activity in liquid assay (Tables 1
, 2
).
-Galactosidase activity could not be detected easily in liquid assays in the colonies that developed a visible colour after overnight incubation; however, after prolonged incubation (12 h), visible colour was at least two times higher than that in negative controls, including the human lamin C fusions (Tables 1
, 2
).
An interaction between CI and HC proteins has not been reported previously for any potyvirus, except, very recently, for Wheat streak mosaic virus (WSMV), which is a member of the family Potyviridae, genus Tritimovirus (Choi et al., 2000 ). The interaction between HC and CI may be relevant to their co-ordinated functions in virus cell-to-cell movement (Cronin et al., 1995
; Kasschau et al., 1997
; Rojas et al., 1997
; Carrington et al., 1998
). Similarly, an interaction between P1 and CI may be relevant to the involvement of these proteins in virus movement and replication mediated by putative protein complexes (Merits et al., 1999
). This is supported by the co-localization of P1 and CI in cells infected by Potato virus Y (Arbatova et al., 1998
).
The VPgVPg interaction reported here is a novel observation for PSbMV and is supported by the more extensive studies carried out on the self-interaction of PVA VPg (Oruetxebarria et al., 2001 ). The potyviral VPg protein mediates aggregation of viral RNA (Luciano et al., 1991
) and the VPgVPg interaction might facilitate recruitment of virus replication and/or translation complexes to the right position on the viral genomic RNA.
The physical interaction between NIb and NIa in yeast cells, shown in this study for both PVA and PSbMV (Tables 1, 2
), was detected previously with two other potyviruses, Tobacco vein mottling virus (TVMV) and Tobacco etch virus (TEV) (Hong et al., 1995
; Li et al., 1997
). However, previous studies had suggested that the domain of NIa which is involved in the interaction with NIb might be different among potyviruses, since the N-terminal domain of VPg interacted with NIb in TVMV (Hong et al., 1995
, Fellers et al., 1998
), whereas the C-terminal proteinase domain (NIaPro) interacted with NIb in TEV (Li et al., 1997
; Daros et al., 1999
). In this study, elevation of reporter gene activity occurred upon co-expression of NIaPro and NIb, in contrast to co-expression of VPg and NIb of PVA (Table 1
), suggesting that the NIaNIb interaction may be more likely to be mediated by the proteinase domain of NIa from PVA, as was shown previously with TEV (Li et al., 1997
). In this and previous studies (Merits et al., 1999
), it has been observed that NIa expressed in yeast undergoes partial autocleavage into VPg and NIaPro and results in fusion proteins containing either VPg or full-length NIa in yeast cells. VPg interacts with itself and, consequently, with NIa, which contains the VPg domain (Tables 1
, 2
). These results might imply that the NIaNIb interaction is mediated by a dimer formed by VPg and full-length NIa in which the NIaPro domain is in contact with NIb. This model is also consistent with a previous report that showed that certain mutations in the VPg domain abolish the interaction between NIa and NIb (Hong et al., 1995
).
Interactions among the putative components of the potyvirus replication complex (NIb, NIa and VPg) shed light on their co-ordinated functions in virus replication. Nevertheless, no detectable interactions were observed between CI and other components of the replication complex, suggesting that CI might be recruited to the replication complex by its RNA-binding properties (Merits et al., 1998 ).
Some of the proteinprotein interactions identified in this study have been observed previously with other potyviruses (Hong et al., 1995 ; Li et al., 1997
; Urcuqui-Inchima et al., 1999
). Very recently, a proteinprotein interaction study was published on WSMV, a tritimovirus with genome organization similar to potyviruses (Choi et al., 2000
). The study was carried out by YTHS expressing random viral cDNAs and this resulted in more interactions between protein domains than those obtained using full-length P1, HC, P3 and CI. It appears that proteins derived from random cDNAs can generate more interactions that are not influenced by global folding. Nevertheless, without structural knowledge of individual proteins, it is difficult to judge which kind of interaction reflects the real situation.
YTHS has emerged as a powerful method for detecting protein interactions (Fields & Song, 1989 ). A large number of proteinprotein interactions have been identified from various organisms by YTHS in the last 10 years, contributing greatly to functional studies of proteins (Vidal & Legrain, 1999
). Nevertheless, YTHS has limitations for studying proteinprotein interactions, as do other methods, such as, for example, in vitro binding assays (Merits et al., 1999
). While the YTHS results are tantalizing, they are not necessarily in agreement with in vitro binding data (Merits et al., 1999
) and confirmation in an in planta system is desirable. In general, the knowledge obtained from YTHS may serve as a basis for further characterization of potyviral proteinprotein interactions and their biological functions.
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Acknowledgments |
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References |
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Received 7 August 2000;
accepted 11 December 2000.