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Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
Correspondence
Aniko V. Paul
apaul{at}notes.cc.sunysb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Biochemistry, University of Alberta, Canada. 
A supplementary table showing primers used to make yeast two-hybrid constructs is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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7500 nt) of PV encodes most of the necessary factors (proteins and RNA signals) required for efficient viral translation and replication. To avoid competition for resources, the virus has evolved to inhibit host cell transcription and translation and to affect other cellular processes, e.g. perturbation of protein and vesicular membrane transport, as well as the timing of apoptosis.
The PV RNA genome encodes one large polyprotein that is proteolytically processed into precursor and individual mature proteins (Fig. 1a
). The products, according to their processing order within the polyprotein, are divided into three regions. The P1 region encodes the structural proteins that form the capsid of progeny virions. The P2 region encodes 2Apro, 2B, 2CATPase and the processing intermediate 2BC. These proteins are primarily involved in the induction of the biochemical and morphological changes that occur in the infected cell. 2Apro is a viral proteinase (Toyoda et al., 1986) that liberates P1 from the rest of the viral polyprotein. It also cleaves several host factors to achieve host translation and transcription shut off (Dasgupta et al., 2002; Zamora et al., 2002) and induces apoptotic cell death (Goldstaub et al., 2000). Protein 2B is responsible for the blockade of cellular secretory transport (Doedens & Kirkegaard, 1995), disintegration of Golgi membranes (Sandoval & Carrasco, 1997) and membrane permeabilization (Aldabe et al., 1996). The function of 2BC is primarily associated with the induction of vesicles during viral infection. The expression of 2BC or 2CATPase causes a membrane rearrangement in the cytoplasm similar to that during polioviral infection (Cho et al., 1994). 2CATPase is a viral ATPase (Mirzayan & Wimmer, 1994; Rodriguez & Carrasco, 1993) that binds zinc (Pfister et al., 2000) and viral RNA (Rodriguez & Carrasco, 1995). All these activities involve multiple motifs that are essential for viral RNA synthesis (Paul et al., 1994b; Pfister & Wimmer, 1999; Rodriguez & Carrasco, 1993) (Fig. 1b). In infected cells, 2CATPase is found in abundance in the membranous replication complex where viral RNA synthesis takes place, an association probably mediated through its N-terminal sequence (Bienz et al., 1983; Echeverri et al., 1998; Teterina et al., 1997). The proteins of the P3 region are the ones most directly involved in RNA synthesis. Initial processing of the P3 domain yields two important and relatively stable precursors, 3AB and 3CDpro. 3AB is a small basic protein with multiple functions in viral RNA replication. In vitro, 3AB was found to stimulate the polymerase activity of 3Dpol (Paul et al., 1994a; Richards & Ehrenfeld, 1998), the auto-proteolysis of 3CDpro (Molla et al., 1994) and to form a ternary complex with 3CDpro and the cloverleaf RNA structure at the 5' terminus of PV RNA (Harris et al., 1994; Xiang et al., 1995). In vivo, 3AB is believed to recruit 3Dpol to the membranous replication complex where RNA synthesis takes place, via direct protein–protein interaction and 3AB's affinity to RNA and membranes (Giachetti et al., 1992; Lyle et al., 2002; Paul et al., 1994a; Xiang et al., 1998). Furthermore, the observations that recombinant 3AB multimerizes and that mutations that destroy 3AB/3AB interaction severely impact viral viability, suggest that a high-order structural role is provided by 3AB in the replication complex (Lyle et al., 2002; Towner et al., 1996; Xiang et al., 1998). Proteins 3Cpro and 3CDpro assume most of the processing of the PV polyprotein (Lawson & Semler, 1992) but they also inhibit host mRNA translation and transcription via their proteolytic property (Dasgupta et al., 2002; Zamora et al., 2002). 3CDpro is an RNA-binding protein that has specific affinity for the 5' cloverleaf of the PV RNA genome, a property essential for viral replication (Andino et al., 1993; Harris et al., 1994; Parsley et al., 1997). In addition, 3CDpro binds to an RNA hairpin in the 2CATPase-coding sequence [cre(2C)], which serves as the template for the uridylylation of VPg by 3Dpol in vitro (Paul et al., 2000a; Yin et al., 2003). The final cleavage products of 3AB are a small membrane-bound protein 3A and the 22 aa long-terminal peptide, VPg. The latter is the primer for the RNA polymerase 3Dpol during RNA synthesis (Paul et al., 1998), while the 3A protein itself also exhibits functions that are distinct from those in the context of 3AB. It inhibits endoplasmic reticulum (ER) to Golgi membrane and secretory traffic (Doedens et al., 1997), induces specific translocation of different members of the ADP-ribosylation factor family to membranes (Belov et al., 2005) and inhibits tumour necrosis factor-induced apoptosis (Neznanov et al., 2001). 3Dpol is a template- and primer-dependent RNA polymerase (Flanegan & Baltimore, 1977; Paul et al., 1998), which also possesses RNA binding (Pata et al., 1995) and polymerization-dependent RNA-duplex unwinding activities (Cho et al., 1993).
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; Hope et al., 1997; Xiang et al., 1998). Another report on the P2 linkage map in coxsackie B3 virus, using a mammalian cell-based two-hybrid system, showed results that parallel those obtained with the PV P2 proteins in the yeast two-hybrid system (De Jong et al., 2002). Two recent publications give partial protein linkage maps for the non-structural proteins of PV (Teterina et al., 2006) and porcine teschovirus (Zell et al., 2005), respectively. Here, we extended the analysis of the PV protein linkage map to consist of all possible pair-wise combinations of the non-structural proteins, which included testing the partnership between the final cleavage products as well as some of their processing intermediates. We also carried out a detailed biochemical analysis of the 2CATPase/3AB interaction. Our results indicate the presence of an extensive network of protein–protein interactions between the P2 and P3 cleavage products. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Construction of plasmids.
Two PV cDNA plasmids were generated via site-directed mutagenesis. pT7PVM G4438A contains a silent mutation in the 2CATPase-coding region to remove an XhoI site. pT7PVM G5877C has a C147S change in the catalytic triad of 3Cpro that reduces proteinase activity of 3Cpro without affecting the folding of the polyprotein or protein–protein interaction involving 3CDpro in the yeast (Hammerle et al., 1991; Xiang et al., 1998).
For the plasmids used in the P2-P3 linkage map construction, the coding regions of non-structural viral proteins were amplified via PCR from pT7PVM G4438A or pT7PVM G5877C (for all 3Cpro containing constructs) with primers described in Supplementary Table S1 (available in JGV Online). The PCR products were digested with EcoRI/XhoI and inserted into the yeast two-hybrid vectors EG202 and JG4-5, restricted with the same enzymes.
Yeast transformation, 
-galactosidase assay and total yeast protein extraction.
Yeast strain EGY48 was transformed as described previously (Paul et al., 2000b). Three plasmids are used in this inducible yeast two-hybrid system: pSH12-34 contains a reporter LacZ gene downstream of eight LexA-binding sites in tandem and the URA3 gene (Zervos et al., 1993). EG202 encodes the fusion protein with ‘bait’ protein and the 202 aa DNA-binding domain of LexA protein plus the HIS3 gene that allows the auxotrophic yeast strain EGY48 to grow in the absence of histidine. The expression of LexA fusion proteins is constitutive in yeast. JG4-5 encodes the TRP1 gene, which confers EGY48 the ability to grow in the absence of trytophan, and a fusion of ‘target’ protein and the B42 acidic transactivation domain. Transformed yeast cells were selected on ura–, his–, trp–, D-raffinose+medium. The expression of the B42-target fusion protein is under the control of a Gal1 promoter inducible upon the addition of D-galactose into the medium. The inducibility of this system minimizes the toxicity of foreign proteins to yeast cells.
The expression levels of fusion proteins were examined by Western blotting of total yeast protein extracts using antibodies against HA-tag (AD fusion) and LexA-tag (DB fusion) (Santa Cruz Biotechnology). To prepare the extract for Western analyses, 2.0 OD600 of yeast culture (OD600 of 0.7–1.2 ml–1) was harvested and spun down, the pellet was subjected to two cycles of freeze–thawing at –80 °C and three cycles of freeze–thawing in liquid nitrogen. The yeast lysate was boiled, centrifuged to remove debris and the supernatant was loaded onto a 12.5 % SDS-polyacrylamide gel for electrophoresis before Western blot analysis.
To assay -galactosidase activity, 10 colonies from a yeast transformation plate were picked and cultured in yeast synthetic medium containing S-raffinose as the carbon source and lacking histidine, uracil and tryptophan until the OD600 reached 0.6. Galactose (2 %) was added to induce the expression of AD fusion protein for 4 h. Yeast cells were pelleted and broken with glass beads. -Galactosidase activity was measured by incubating lysate and substrate (chlorophenol red -D-galactopyranoside) at 37 °C and calculated in Miller units as 1000 times OD574/(OD600xthe product of T and V) with T being the time of the colorimetric reaction in minutes and V the volume (ml) of the culture (Bartel et al., 1993).
Expression and purification of GST and GST-2C.
GST (Glutathione S-transferase), GST-2CATPase and three GST-2CATPase mutants were expressed using a protocol similar to the one described previously (Pfister & Wimmer, 1999).
GST pull-down assay.
To assay for the binding of the GST or GST-2CATPase proteins with 3AB, we used a GST pull-down assay as described previously (Cuconati et al., 1998). The standard condition was 2.4 µg GST-2CATPase (or 1 µg GST) with 0.4 µg 3AB in 140 mM NaCl, 20 mM HEPES pH 7.4 at 30 °C (or 4 °C). The proteins associated with the glutathione–Sepharose (GSH) beads were analysed on 14.5 % SDS-PAGE followed by Western blot analysis using antibodies against 2CATPase (monoclonal) and 3B (polyclonal), respectively. The 3B antibody is against the C-terminal 7 aa of VPg.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), on the one hand, and to the P3 domain (Xiang et al., 1998), on the other. Here, the same strategy was followed to link these two maps.
Protein linkage map between the P2 and P3 proteins of PV
The interactions were first tested with P2 polypeptides expressed as LexA-tagged DB fusions and P3 polypeptides as transcription AD fusions (HA-tagged) (orientation I) (Tables 1 and 2). As a positive control we also included 3B and 3Dpol, whose interaction was previously shown to be very strong (Xiang et al., 1998). Western blot analyses were carried out and verified comparable expression levels of fusion proteins in all combinations (data not shown). The strongest binding of the P2 and P3 partners was observed between 2Apro and 3Cpro (Tables 1 and 2). The interaction was moderately strong between 2Apro and 3A, and between 2CATPase and 3AB or 3A (Tables 1 and 2). We detected only weak interaction between 2B and 3A, between 2B and 3AB and between 2Apro and 3CDpro or 3Dpol or 3B (Tables 1 and 2).
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and 2). For example, 2Apro binding to 3A was weak, and the binding of 2B or 2CATPase to 3A was insignificant (Tables 1 and 2). Western blot analyses were used to ascertain that the expression level of fusion proteins in all combinations was comparable (data not shown). Control experiments in which one of the two fusion expression constructs was replaced with the parental vector containing no insert sequence displayed only background –galactosidase activity (data not shown), indicating specificity of the observed interactions. The level of -galactosidase activity for each combination is presented in Table 1. A dependence of the interaction scored in the yeast two-hybrid system upon the polarity of some binding partners (3Dpol-3AB; 3B-3Dpol; 3B-3CDpro; 3CDpro-3AB) has been observed before (Xiang et al., 1998).
The formation of the binding pairs 2CATPase/3A and 2CATPase/3AB, but not of a 2CATPase/3B pair, suggests that the 3A moiety of 3AB is the one involved in the 2CATPase/3AB interaction. Indeed, 3B was found not to interact with any of the P2 proteins except weakly with 2Apro. 3AB was found to bind both 2B and 2CATPase regardless of whether these proteins were provided in the bait or the prey construct. In contrast, 3AB did not bind with their processing precursor 2BC, an observation suggesting that 2BC does not express the appropriate binding determinant under the conditions of the experiments. Interestingly, the strongest interactions observed between the P2 and P3 proteins are those between 2Apro (a soluble polypeptide) and 3A (a membrane-bound polypeptide) and between 2Apro and 3Cpro (both soluble polypeptides). A summary of the interacting pairs between the P2 and P3 proteins is shown in Table 2.
Biochemical analysis of the 2CATPase/3AB interaction in vitro using GST pull-down assay
The possible functions of PV 2CATPase, an ATPase (Mirzayan & Wimmer, 1994; Pfister & Wimmer, 1999; Rodriguez & Carrasco, 1993) and of 3AB, an RNA- and membrane-binding protein (Paul et al., 1994a; Towner et al., 1996; Xiang et al., 1995), have been studied in great detail and both polypeptides are essential for viral proliferation. Yet their precise role in PV replication is still obscure. Relative to other binding pairs, the interaction between 2CATPase and 3AB is moderately strong (Tables 1 and 2). In view of the importance of these proteins in PV replication, we have further examined the 2CATPase/3AB binding in vitro with purified recombinant proteins. Due to their lipophilic properties, these proteins are water insoluble. However, we were able to employ GST-pull-down assays since the GST-2CATPase fusion protein, which retains ATPase activity, could be readily expressed in Escherichia coli and purified (Pfister & Wimmer, 1999). 3AB, on the other hand, can be purified in the presence of mild detergent without losing some of its activities in vitro (Harris et al., 1994; Molla et al., 1994; Paul et al., 1994a). As shown in Fig. 2(a), GST-2CATPase was able to bind purified 3AB from solution onto GSH beads, whereas GST alone did not (compare lanes 1–7 with lane 8). The amount of 3AB bound increases with increasing protein concentration until a molar ratio of 1 : 1 is reached (Fig. 2a, lane 4), which may mimic the stoichiometry of these viral proteins in the infected cell. However, when 3AB was present in the assay at up to a 10-fold molar excess over the GST-2CATPase protein, the apparent amount of 3AB in the pull-down fraction was reduced (Fig. 2a, compare lane 4 with lanes 5–7; Fig. 2c, compare lanes 1 and 2). This result may reflect our previous observation that 3AB has a strong tendency to form multimers at higher concentration (Xiang et al., 1995) and is supported by the dimeric nature of the N-terminal 3A domain as shown by nuclear magnetic resonance studies (Strauss et al., 2003). We speculate that the 3AB multimers bind either inefficiently or not at all to GST-2CATPase and, thus, multimerization of 3AB would effectively reduce the concentration of monomeric 3AB for its binding to 2CATPase.
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) (Fig. 1b). A mutant PV carrying the A3 mutation is quasi-infectious, yielding revertants to the wild-type sequence, whereas the B4 mutations lead to a lethal phenotype (Mirzayan & Wimmer, 1992). As shown in Fig. 2(c), mutants C2 and A3 interact with 3AB at an affinity comparable to that between wild-type 2CATPase and 3AB in vitro (Fig. 2c, compare lanes 3 and 4 with lane 2). Interestingly, the binding to 3AB by mutant B4 was weakened (Fig. 2c, compare lanes 2 and 5), indicating the possible involvement of this region of 2CATPase in its interaction with 3AB. Furthermore, in agreement with our yeast two-hybrid findings, the interaction between 2CATPase and 3AB is likely a result of the affinity between 2CATPase and 3A, as the GST-2CATPase does not seem to pull down detectable amounts of VPg under the conditions of these assays (Fig. 2c, lanes 8–11).
Effect of salt and detergent on 2CATPase/3AB interaction in vitro
Since the PV 2CATPase protein exhibited ATPase activity in vitro in the form of a GST-2CATPase fusion (Pfister & Wimmer, 1999), we tested the effect of ATP on the GST-2CATPase/3AB interaction. As shown in Fig. 2(c), in the presence of 1 mM ATP (concentration used for in vitro ATPase assays of GST-2CATPase) the interaction between 2CATPase and 3AB was almost unchanged (compare lanes 2 and 6).
At 4 °C, the GST-2CATPase/3AB interaction is much weaker than that at 30 °C, (Fig. 2c, compare lanes 2 and 13), an observation suggesting a preference of the 2CATPase/3AB complex formation for physiological temperature. We then altered the buffer condition used in this pull-down assay to determine whether ionic or hydrophobic interactions account for the association between these two proteins. It should be noted that with samples containing VPg (Fig. 2c, lanes 8–11) we did not obtain any signal at the position expected of VPg (data not shown). In additional experiments, we observed that both salt concentration and detergent content affect this interaction, albeit in different ways (Fig. 3). Low salt concentration is favoured over high salt concentration (Fig. 3b, lanes 1–6). The observation that increasing the concentration of non-ionic detergent (up to 0.04 %) favours the binding of 3AB to GST-2CATPase (Fig. 3a, lanes 1–3) suggests that only monomeric 3AB interacts with 2CATPase and that the amount of monomeric 3AB reaches an optimum at a Triton concentration of about 0.04 %. The results of these experiments are consistent with a requirement for both ionic and hydrophobic contacts for the interaction between the two proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). They overcome the problem of low coding capacity by assigning multiple functions to individual proteins. This complexity of protein function has been clearly established for the non-structural proteins encoded by PV (Fig. 1a). The assignment of specific functions to some viral polypeptides is difficult, since the processing intermediates play a role in replication distinct from that of the cleavage products (e.g. 3AB, 3A, and 3B, 3CDpro, 3Cpro and 3Dpol). Moreover, some viral proteins change properties once in association with other viral polypeptides (Paul et al., 1994a; Molla et al., 1994).
Polypeptides 2Apro, 2B, 2CATPase, 2BC, 3AB, 3A, 3Cpro, 3CDpro, 3Dpol and the uridylylated form of VPg have all been found in membranous replication complexes in vivo that are the sites of PV RNA synthesis (Bienz et al., 1983, 1990; Takegami et al., 1983). It was predictable, therefore, that these proteins have binding partners amongst themselves and, possibly, with cellular proteins. The PV polyprotein is most rapidly cleaved between the P1 and P2 domains (catalysed by 2Apro) and between P2 and P3, catalysed by 3Cpro/3CDpro (Fig. 1a). These large precursors are then further processed to smaller precursors and mature proteins. We have previously provided evidence for interactions both between members of the P2 proteins (Cuconati et al., 1998) and between members of the P3 domain (Xiang et al., 1998). Interestingly, some functions of the P2 and P3 proteins overlap. For example, both 2B and 3A alter the permeability of membranes (Aldabe et al., 1996; Lama & Carrasco, 1992) and block secretory transport at the ER/Golgi step (Doedens & Kirkegaard, 1995; Doedens et al., 1997). Genetic experiments have suggested an interaction between 2B and 3A (Towner et al., 2003; Fujita et al., 2007). In other studies, with chimeric PV viruses containing the amphipathic helix of HRV14 in 2CATPase, revertants were observed that indicated interactions between 2CATPase and 2B or 3A (Teterina et al., 2006). To confirm these interactions and to test the possibility that other members of the P2 and P3 proteins interact with each other during RNA replication, we established a complete P2-P3 linkage map.
The linkage map of P2-encoded polypeptides has revealed that proteinase 2Apro does not engage in homo- or heteromultimer complex formation within that group of viral proteins (Cuconati et al., 1998). In contrast, 2Apro interacts with several proteins of the P3 domain, most notable with 3Cpro, confirming the results of a mammalian two-hybrid study by Teterina et al. (2006). Both proteins are involved in proteolytic processing not only of the viral polyprotein but also of cellular translation and transcription factors. In addition, both proteins have functions in RNA replication (Li et al., 2001; Lu et al., 1995; Molla et al., 1993) distinct from peptide bond hydrolysis. PV 3Cpro is an RNA-binding protein although its affinity to RNA is greatly augmented in the form of its precursor, 3CDpro (Gamarnik & Andino, 2000; Harris et al., 1994; Parsley et al., 1997; Xiang et al., 1995; Yin et al., 2003). It is possible that a 2Apro/3Cpro complex confers a specific RNA-binding function to 2Apro when 2Apro is involved in the regulation of translation or RNA replication.
In the yeast two-hybrid screen, 2Apro formed a complex with 3Dpol in both orientations although the signal is weak (Tables 1 and 2). During the processing of the viral polyprotein from PV type 1, 2Apro cleaves the polyprotein within the 3Dpol-coding region to generate 3C' and 3D' (Fig. 1a). Whether the 2Apro/3Dpol interaction reflects anything more than an enzyme/substrate relationship is not known. In spite of the affinity of 2Apro to 3Cpro and to 3Dpol, its interaction with 3CDpro is so weak (and scored only in one orientation) that it is impossible at present to assign any significance to it. This very weak binding either results from the masking of binding determinants in 3CDpro, or it is an experimental artefact. Similarly, we do not yet know whether binding between 2Apro and 3A is of biological significance since the binding is also found to be quite weak and only in one orientation.
Interaction of 2B and 2CATPase with 3A and 3AB
In the yeast two-hybrid assay, the P3-encoded proteins 3A and 3AB interacted with the P2 proteins 2B and 2CATPase but, surprisingly, not 2BC. All of these proteins are membrane-associated, generating profound morphological changes in the cytoplasm of PV-infected cells. In contrast, the 2CATPase/3AB interaction was not detected in the mammalian two-hybrid system but the interaction of 2CATPase with 3A and of 2BC with 3A/3AB was observed (Teterina et al., 2006).
2B and 2CATPase are membrane-associated via their amphipathic helices (Echeverri et al., 1998; van Kuppeveld et al., 1997), whereas 3A and 3AB are anchored through a hydrophobic domain near the C terminus of 3A (Giachetti et al., 1992; Towner et al., 1996). We have recently shown that in synthetic membranes 3AB adopts only a non-transmembrane configuration, while 3A exists as a mixture of transmembrane and non-transmembrane forms (Fujita et al., 2007). 2B and 3A have been associated with numerous changes in cytoplasmic metabolism, most notably membrane permeabilization and inhibition of protein secretion. The association of these proteins may augment their effects on the cytoplasmic membranes.
Interestingly, a genetic screen of a chimera in which the hydrophobic domain of PV 3A was exchanged to that of HRV14 3A yielded, amongst mutations in the 3A-coding region, a PV variant with a second-site mutation in 2B (Towner et al., 2003). To explain their results, the investigators proposed that 2B and 3A(B) interact in the replication complex. We came to the same conclusion when examining revertants derived from mutants in the hydrophobic anchor of 3A (Fujita et al., 2007). The data presented here provide direct evidence for such interaction, in agreement with studies of Teterina et al. (2006). In fact, the 2B-3AB association is likely to occur between 2B and 3A since 3B did not show any affinity for 2B in our yeast two-hybrid system in either orientation.
3AB is a membrane bound RNA-binding protein that has affinity to 3Dpol and 3CDpro (Giachetti et al., 1992; Hope et al., 1997; Lyle et al., 2002; Paul et al., 1994a; Xiang et al., 1998). 2CATPase is an ATPase (Pfister & Wimmer, 1999) with RNA-binding activity (Rodriguez & Carrasco, 1995) that is membrane-associated in PV-infected cells (Echeverri et al., 1998) (Fig. 1b). Guanidine HCl, a potent inhibitor of PV RNA replication (Barton & Flanegan, 1997; Wimmer et al., 1993), inhibits the ATPase activity of purified recombinant GST-2CATPase protein in vitro (Pfister & Wimmer, 1999). The amino acid sequence of 2CATPase reveals not only characteristic NTP-binding motifs (A and B) but, in addition, a C motif (helicase superfamily III) that suggests a helicase function. All attempts, however, to provide biochemical evidence for a helicase activity of 2CATPase have failed so far (Pfister & Wimmer, 1999). One might speculate that an interaction between the two membrane-associated polypeptides 3AB and 2CATPase may modify 2CATPase function and induce helicase activity that may be essential for the initiation of plus-strand RNA synthesis. In confirmation of the 3AB/2CATPase interaction, we have also observed that immunofluorescent staining of PV-infected cells with anti-3AB and anti-2CATPase antibodies showed that these viral proteins colocalize to perinuclear regions around the time of peak hours for viral RNA replication (data not shown). It should be noted that this interaction was not observed in the mammalian two-hybrid study (Teterina et al., 2006).
The key polypeptide in the rearrangement of cytoplasmic membranes to vesicle structures supporting PV genome replication in vivo is 2BC (Egger et al., 2002). It has been suggested that 3A is a co-factor of 2BC in the formation of replication-competent vesicles (Suhy et al., 2000). Although we have not found an interaction between 2BC and 3A or 3AB, such complexes were detected in the mammalian two-hybrid system (Teterina et al., 2006). Since we have observed complex formation between 2CATPase and 3A or 3AB, our failure to detect such interaction is most likely due to a lack of expression of the appropriate binding determinants in 2BC under the conditions of our experiments.
Our biochemical studies of the 2CATPase/3AB interaction suggested that such complex formation is optimal only when 3AB is in a monomeric form. In addition, we have observed that both hydrophobic and ionic groups mediate the interaction between these two proteins. Studies aimed at determining the site of interaction between 2CATPase and 3AB suggested that motif B in the 2CATPase protein is involved in this process.
Our results indicate the presence of an extensive network of protein–protein interactions between the P2 and P3 cleavage products. The full understanding of these interactions should be useful for the elucidation of how viral translation and replication occur at the protein level. Although our current results enhance our understanding of the interactions between the PV non-structural proteins, more studies will be needed to fully appreciate the complexity of the PV replication complex.
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ACKNOWLEDGEMENTS |
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Received 14 December 2006;
accepted 2 April 2007.
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