Linkage map of protein–protein interactions of Porcine teschovirus

Roland Zell1, Simone Seitz1, Andreas Henke1, Thomas Munder2 and Peter Wutzler1

1 Institute for Virology and Antiviral Therapy, Hans-Knöll-Str. 2, D-07745 Jena, Germany
2 Leibniz Institute for Natural Products Research and Infection Biology, Hans Knöll Institute, Beutenbergstr. 11a, D-07745 Jena, Germany

Correspondence
Roland Zell
i6zero{at}rz.uni-jena.de


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A yeast two-hybrid study was conducted to catalogue the protein–protein interactions of the Porcine teschovirus non-structural proteins. Five homodimer, three reciprocal heterodimer and four unidirectional heterodimer interactions were observed. While several interactions are similar to those described in previous studies using enteroviruses, such as homo- and heterodimeric interactions of the 2B, 3CD and 3D proteins, several were not found previously. Among these is the binding of the leader protein L to the proteinases 3C and 3CD. Unlike the poliovirus 3C, the teschovirus 3C proteinase dimerizes and interacts with 2BC, 3CD and 3D. The strongest interactions were observed for L–3C, L–3CD and 3C–3CD.


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Picornaviruses comprise the genera Aphthovirus, Cardiovirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Parechovirus, Rhinovirus and Teschovirus (Stanway et al., 2005). In addition, porcine enterovirus 8 and some simian enteroviruses have recently been suggested to be closely related species of another new picornavirus genus (Krumbholz et al., 2002; Oberste et al., 2003). Picornaviruses are small non-enveloped, positive-stranded RNA viruses. Genomic RNA (Fig. 1a) is polyadenylated and covalently linked to a virus-encoded peptide, designated 3B (VPg). A single open reading frame (ORF) encodes a polyprotein which is processed post-translationally by one or two viral proteinases. The primary cleavage products are designated by convention as P1, P2 and P3 polypeptides. While P1 is the capsid protein precursor, P2 and P3 are the precursors of the non-structural proteins. Mature P2 and P3 proteins, as well as some stable intermediate precursor products, are thought to be functional during virus replication. The mature non-structural proteins 2CATPase, 3B, 3C protease and 3D polymerase are conserved among all picornaviruses. The ORF is flanked on both sides by non-translated regions (NTR) which are essential for the initiation of viral translation and replication. Beside these similarities among all picornaviruses, there are considerable differences concerning size and structure of the genomes. Genome sizes range from 7·0 to 8·8 kb, and characteristic features comprise the secondary structures of the 5' NTR, the position of the cis-replication element, and the predicted secondary structures of the 3' NTR. In addition, the proteins L, 2A, 2B and 3A are not conserved. Virus replication is a multifactorial process and includes: (i) numerous protein–protein and protein–RNA interactions; (ii) the rearrangement of vesicular and cellular membranes, which modifies membrane permeabilization, signal transduction and protein secretion; (iii) the inhibition of host-cell translation and transcription; and (iv) the destruction of the host-cell cytoskeleton, which finally results in lysis. De novo synthesis of Poliovirus requires cellular extracts, indicating the necessity of yet-undefined host factors and membraneous structures (Molla et al., 1991; Cello et al., 2002). Previous attempts to elucidate picornavirus replication include ultrastructural analyses (Egger et al., 1996; Lyle et al., 2002; Krogerus et al., 2003), in vitro replication assays (Paul et al., 1998), and two-hybrid studies for the detection of protein–protein interactions of Poliovirus and coxsackievirus, respectively (Xiang et al., 1998; Cuconati et al., 1998; de Jong et al., 2002). The latter approach revealed several corresponding homo- and heterodimeric interactions of mature proteins and precursor proteins in both viruses.



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Fig. 1. Results of yeast two-hybrid experiments. (a) Schematic representation of the teschovirus RNA genome. The gene regions are indicated. Gene regions 1A–1D encode capsid proteins while the remaining gene regions encode non-structural proteins (L, P2 and P3). A circle at the 5' end of the genome symbolizes the covalently attached 3B peptide (VPg). A(n) stands for the poly(A) tail. (b) Schematic representation of the yeast two-hybrid system. Sequences of the yeast GAL1 promoter are 5' to the lacZ reporter gene. These consist of three copies of the 17mer upstream activating sequences (UAS) and a polymerase binding site (TATA). (c) {beta}-Galactosidase activities of extracts of yeast transformants. ONPG assays were performed with extracts of yeast double transformants. Interactions were scored positively when ONP release was at least three times greater than in the control experiment [see Fig. 1 (e); i.e. yeast clones co-expressing the respective DBD fusion protein and the unfused AD-GAL4]. {beta}-Galactosidase activity is given in Miller units. In each dataset, the values are presented in the following order (starting from the top): AD–L, –2B, –2BC, –2C, –3A, –3AB, –3C, –3CD, –3D (as indicated in the first dataset). For positively scored co-transformants, the mean values of five independent experiments±standard deviation are presented. These co-transformants are listed in Table 1. (d) {beta}-Galactosidase activity of extracts of yeast transformants expressing truncated AD–3C fusion proteins. (e) Control experiments: {beta}-galactosidase activities of extracts of yeast transformants co-expressing either DBD fusion proteins and unfused AD proteins (upper panel) or unfused DBD proteins and AD fusion proteins (lower panel).

 
In this study, the protein–protein interactions of porcine teschovirus 1 (PTV-1) were analysed with the yeast two-hybrid system (Fields & Song, 1989) and compared to previous results obtained with polio- and coxsackievirus. Teschoviruses are a major cause of porcine polioencephalomyelitis and fertility disorders and are characterized by a number of unique non-structural proteins of unknown function (proteins L, 2B, 3A; see Fig. 1a). Since several attempts to express recombinant teschovirus proteins in bacteria failed (data not shown), the yeast two-hybrid system was employed in order to identify possible protein–protein interactions. Experiments were performed using the commercially available pAS2-1/pGADGH vector system. In pAS2-1 derivatives, the insert is fused to the gene region encoding the DNA-binding domain of the yeast GAL4 protein (DBD-GAL4), while in pGADGH derivatives it is fused to the gene region encoding the GAL4 activator domain (AD-GAL4). Co-transformation of Y190 yeast cells with both plasmids results in constitutive overexpression of both hybrid proteins. If they interact with each other, a functional transactivator is constituted which induces Escherichia coli lacZ reporter gene expression (Fig. 1b). {beta}-Galactosidase activity was assayed by incubating replica filters of co-transformants in X-Gal-containing buffer (filter lift assay) or by performing quantitative o-nitrophenyl {beta}-D-galactoside (ONPG) assays (Breeden & Nasmyth, 1985, 1987). In qualitative assays, a positive protein–protein interaction is indicated by blue spots on the filters, while in quantitative assays the release of o-nitrophenol (ONP) is quantified photometrically.

The gene regions of the non-structural proteins L, 2B, 2BC, 2C, 3A, 3AB, 3C, 3CD and 3D (Fig. 1a) were amplified from cDNA of PTV-1 strain Talfan (Zell et al., 2001) using pfu polymerase and a set of specific primers (sequences not shown). The PCR fragments were purified and cloned into the vectors pAS2-1 (SalI–NcoI restriction sites) and pGADGH (BamHI–XhoI restriction sites), respectively. The gene regions of 2C and 2BC on the one hand, and of 3C and 3CD on the other, were modified in the sequences that encode the nucleotide triphosphate-binding site (GxGxxGKS to GxGxxGQS; Möller & Amons, 1985) and the catalytic site (Cys to Gly), respectively, in order to avoid deleterious effects of the enzymically active proteins in the yeast host cell. To improve the growing properties of the yeast transformants, the 3A gene region was truncated at the 3' end, resulting in a deletion of 24 C-terminal amino acids. In addition, versions of plasmids encoding truncated 2B and 3C proteins were constructed by applying PCR mutagenesis. The structural intactness of all constructs was confirmed by DNA sequencing. The teschovirus 2A protein, which induces an aphthovirus-like release of the capsid protein precursor, was not included in this study, since the pAS-2A construct repeatedly yielded false-positive results due to an artificial transactivation activity (data not shown).

Yeast strain Y190 was transformed (Klebe et al., 1983) with pAS2-1 and pGADGH plasmids and plated on YNB/glucose minimal plates supplemented with histidine and lysine, respectively. As a result, two sets of Y190 clones expressing either GAL4-AD or GAL4-DBD fusions of the non-structural proteins were obtained. After 5 days incubation at 30 °C, the clones were checked for {beta}-galactosidase activity. The assays confirmed that the single transformants did not express the enzyme (data not shown). Then, individual Y190-pAS2-1 clones were transformed by the pGADGH plasmids, yielding a set of co-transformants. It was observed that all yeast clones which were transformed with plasmid constructs expressing wild-type 2C or 2BC were non-viable. This non-viability was attributed to the presumed deleterious ATPase activity of 2C, which has a nucleotide triphosphate-binding motif. In order to achieve viability of the respective yeast transformants, the phosphate-binding site was mutated. The resulting plasmid derivatives produced viable yeast transformants. Likewise, 3A-expressing clones showed a prolonged division cycle, while a truncated version showed normal growth properties. All further experiments were performed with the mutated plasmids. Usually, colonies were scored positive when they turned blue after overnight incubation of the filters at ambient temperature or released ONP in amounts at least three times above background.

In {beta}-galactosidase activity assays, three types of interaction were observed (Table 1, Fig. 1c): (i) homodimeric interactions of 2B, 3A, 3C, 3CD and 3D; (ii) reciprocal heterodimeric interactions of L–3C, L–3CD and 2B–2BC; and (iii) a number of unidirectional (non-reciprocal) heterodimeric interactions in various combinations (DBD–2C/AD–2B, DBD–3C/AD–3CD, DBD–3D/AD–3C, DBD–3D/AD–3CD). Regardless of the type of interaction, the {beta}-galactosidase activities of several co-transformants were low and therefore may either represent weak binding or indicate a disturbed interaction due to the fusion.


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Table 1. Linkage map of protein–protein interactions of teschoviral non-structural proteins

Homodimeric interactions are shown in bold type. r, Reciprocal heterodimeric interactions; u, unidirectional heterodimeric interactions.

 
In a previous study, de Jong et al. (2002) proposed two structural elements of the coxsackievirus 2B protein: an amphipathic helix ranging from amino acids 37 to 54 and a hydrophobic domain ranging from amino acids 63 to 80. The function of 2B is not fully understood, but the enteroviral 2B is located at the cytoplasmic surface of virus-induced membrane vesicles and is thought to alter membrane permeability by the formation of pores (Aguirre et al., 2002). It is difficult to compare teschovirus and enterovirus 2B: the former is considerably larger (146 versus 99 amino acids) and shares no homology with enterovirus 2B. A hydrophobicity plot revealed two prominent hydrophobic stretches ranging from amino acids 50 to 85, but an amphipathic helix was not predictable beyond doubt (data not shown). 2B showed homodimerization and formation of heterodimers with 2C and 2BC. In a first approach to modify teschovirus 2B, three deletion mutants were constructed: (i) 2B{Delta}N had a deletion of the N-terminal 41 amino acids; (ii) 2B{Delta}C had a deletion of the C-terminal 27 amino acids; (iii) 2B{Delta}NC had both deletions. Preliminary two-hybrid experiments revealed changed interactions but did not allow firm conclusions to be drawn (data not shown). The experimental outcome may be interpreted as an indication that homo- and heterodimeric interactions of teschovirus 2B require proper protein folding, which is disturbed in the truncated constructs.

Enteroviral 3A and its precursor 3AB are believed to interact with membranes of the host cell (Paul, 2002). Whether teschoviral 3A has a similar function is unknown, since it has no obvious homology with the enteroviral 3A protein. In the two-hybrid assays, the C-terminally truncated teschovirus 3A protein yielded yeast transformants with improved viability and similar low binding affinities as 3AB (two to three times above background). However, considerable homodimerization was observed for 3A, but not for 3AB. Since the yeast double transformants expressing either AD–3AB or DBD–3AB showed a prolonged division cycle, one may hypothesize that this protein is somehow toxic to yeast cells and that the two-hybrid assay might not correctly indicate all of the naturally occurring 3AB interactions.

Picornavirus 3C is a chymotrypsin-like proteinase consisting of two {beta}-barrel domains connected by an interdomain linker with an RNA-binding motif (Matthews et al., 1994; Bergmann et al., 1997; Mosimann et al., 1997). In a previous two-hybrid study, no interaction of poliovirus 3C with any other P3 protein was observed (Xiang et al., 1998). However, purified recombinant 3C proteinase of coxsackievirus B3 has limited solubility and exhibits a strong tendency to homopolymerization (Ohlenschläger et al., 2004). Teschovirus 3C shows several interactions with non-structural proteins: besides homodimerization, heterodimers of 3C and L, 3CD and 3D were detected. Among these interactions, the 3C–L binding was reciprocal. In order to determine the interacting domain of teschovirus 3C, either the N-terminal or the C-terminal domain was deleted. The results of the two-hybrid experiments revealed that the C-terminal domain of 3C fused to the GAL4 activator domain did not interact with any teschoviral protein, while the N-terminal domain still interacted with 3D, but not with 3CD (Fig. 1d). These results are interpreted as an indication that most of the protein–protein interactions of 3C require the integrity of the 3C proteinase. Like the processed 3C, the 3CD precursor dimerizes and binds to L, 3C and 3D. The 3CD–L interaction is reciprocal.

Within the picornavirus family, aphthoviruses, cardioviruses, erboviruses, kobuviruses, teschoviruses, porcine enterovirus 8 and simian virus 2 have leader proteins. Only the function of the aphtho-/erbovirus L protein is known: it is a proteinase (Guarné et al., 1998). The kobuvirus L protein has been suggested to be involved in RNA replication and encapsidation (Sasaki et al., 2003). The function of the remaining leader proteins which are unrelated to each other remains obscure. Very interesting is the unprecedented binding of the teschovirus L protein to 3C and 3CD. One might hypothesize that the L–3C and L–3CD interactions could either play a role in certain steps of the polyprotein processing or be involved in virus replication. Since 3C and 3CD also bind to each other and to the 3D protein, interaction with the L protein could lead to the formation of higher-order complexes. Ternary and higher-order complexes have previously been suggested for enteroviruses (Paul, 2002); however, such complex interactions cannot be investigated with the yeast two-hybrid system.

Comparison of the protein–protein interactions of teschoviral and enteroviral non-structural proteins (Table 2) shows that both virus groups have in common: (i) the homodimerization of 2B, 3A, 3CD and 3D; (ii) the binding of 2B to 2BC and 2C; and (iii) the interaction of 3D with 3CD. Mature poliovirus 3C does not interact with any of the P3 proteins (Xiang et al., 1998), whereas in teschovirus it dimerized and interacted with 3CD and 3D, but not with P2 proteins (Table 1). None of the teschovirus P2 proteins interacted with any P3 protein. Binding of the enterovirus P3 proteins with P2 proteins has not been investigated previously. In conclusion, the Enterovirus and Teschovirus genera have evolved a number of different protein–protein interactions.


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Table 2. Protein–protein interactions of picornaviral nonstructural proteins

ND, Not done; Unidir., unidirectional heterodimeric interaction; Recipr., reciprocal heterodimeric interaction.

 


   ACKNOWLEDGEMENTS
 
The excellent technical assistance of Sabine Wachsmuth and Ana Vinuela Rodriguez is acknowledged. We thank Matthias Nestler for helpful advice. This work was supported by Deutsche Forschungsgemeinschaft grant ZE 446/2.


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Received 27 April 2005; accepted 22 June 2005.



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