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1 Department of Molecular Biosciences, Section of Veterinary Immunology and Virology, Swedish University of Agricultural Sciences, Box 588, S-751 23 Uppsala, Sweden
2 Department of Biomedical Sciences and Veterinary Public Health, Section of Parasitology and Virology, Swedish University of Agricultural Sciences, Box 588, S-751 23 Uppsala, Sweden
Correspondence
Sirje Timmusk
Sirje.Timmusk{at}vmm.slu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Two genotypes of PCV have been identified: PCV1, which is non-pathogenic, and PCV2, which has been identified as the aetiological agent of an emerging swine disease, the post-weaning multisystemic wasting syndrome (PMWS) (Clark, 1997; Harding & Clark, 1997). The sequence identity between PCV1 and PCV2 isolates varies between 68 and 76 % (Cheung, 2003a), and the two types of viruses have similar genomic organization. Serological surveys indicate that both PCV1 and PCV2 are widespread in swine populations all over the world (Allan & Ellis, 2000).
PCV are small, icosahedral, non-enveloped viruses with a circular single-stranded (ss) DNA genome of 1.7 kb (Todd et al., 1991; Meehan et al., 1998). PCV are among the smallest known mammalian viruses (Mankertz et al., 1997). The PCV virion has a diameter of around 17 nm (Tischer et al., 1982) and is composed of coat protein subunits assembled in 12 pentameric units (Crowther et al., 2003). Partial sequence similarity suggests that animal circoviruses may be derived from plant nanoviruses that may have infected a vertebrate host via an insect vector. A later recombination with a sequence from an ssRNA virus, such as a calicivirus, would explain the similarity of another part of the PCV genome with RNA viruses (Gibbs & Weiller, 1999).
The PCV2 genome contains four main open reading frames (ORFs) (Meehan et al., 1998), of which three have been characterized in detail. ORF1 (314 aa) encodes replicases, which are required for virus replication (Cheung, 2003b; Mankertz et al., 2003). Two different replicases (Rep and Rep') are produced from ORF1 by splicing of the Rep' transcript. The Rep and Rep' of PCV1 associate with each other and recognize the potential stem–loop structure at the origin of replication (Steinfeldt et al., 2001). ORF2 (233 aa) encodes the immunogenic capsid (Cap) or coat protein (Nawagitgul et al., 2000), which forms the viral capsid. The N-terminal part of Cap displays a nuclear localization signal, which is required for the proper localization of Cap during the viral cycle (Cheung & Bolin, 2002; Liu et al., 2001). Cap contains one cysteine residue, surrounded by a well-conserved 12–16 aa region, which may be responsible for dimer formation between coat protein subunits or for interactions with other proteins. The newly characterized protein encoded by ORF3 (105 aa) appears to contribute to virus-induced apoptosis of the host cell (Liu et al., 2005). Other ORFs are present in the PCV2 genome, such as the 59 aa cysteine-rich ORF4, but their expression and function are still unknown.
Studies in vitro have shown that PCV2 Rep proteins are localized in the nucleus of infected PK15A cells, whereas Cap can be detected in the nucleus and cytoplasm (Gilpin et al., 2003). The same pattern emerged in PCV1-infected cells, where the Rep and Rep' proteins co-localized in the nucleus, whereas the Cap protein was present in the nucleoli at an early stage of infection and in the nucleoplasm and cytoplasm later (Finsterbusch et al., 2005). To understand the infection biology of PCV2 further, a systematic screen for cellular partners of PCV2 proteins was performed using a bacterial two-hybrid approach, the BacterioMatch system. In a first construct, PCV2 proteins derived from ORFs 1–4 (the bait) were fused to the full-length bacteriophage 
C1 protein in pBT. In parallel, a porcine expression cDNA library generated from PK15A cells (the target) was fused to the N-terminal domain of the 
-subunit of RNA polymerase in pTRG. Bacteria were co-transformed with the two plasmid preparations and bacterial colonies in which a target and a bait protein interacted were selected through their activation of the transcription of reporter genes that made the bacteria carbenicillin (ampicillin) resistant. A second reporter gene, 
-galactosidase, was expressed from the same promoter, providing an additional mechanism to validate the bait and target interaction. The strength of the interactions was therefore assessed from the intensity of the blue colour of growing colonies. Interactions between viral proteins were also analysed by this approach and several potential interactions appeared. Three of these interactions, binding of the PCV2 Rep protein to the Cap protein, to an intermediate filament-like protein and to c-myc, were confirmed by GST pull-down assay. Furthermore, a Cap–Cap interaction was demonstrated by GST pull-down assay.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and primer sequences are given in Table 2. Modifications and handling of the material were carried out according to standard protocols (Sambrook & Russell, 2001).
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. In order to generate the target plasmid, the cDNA was cloned unidirectionally into the EcoRI/XhoI sites of the target vector pTRG, which fuses the cDNA with the RNA polymerase activation domain. The cDNA library was then transformed into electrocompetent E. coli XL-Blue bacteria and purified plasmid DNA was extracted directly from the plated library. The titre of the library was 5.3x108 c.f.u. ml–1 and 2x106 recombinants were used per screening.
For generation of bait plasmids, the four ORFs of PCV2 (ORF1, 2, 3 and 4) were amplified from DNA purified from PCV2 Stoon (PCV2-St; GenBank accession no. AF055392
[GenBank]
) using forward and reverse primers as specified in Table 2. For comparison, PCV2 ORF1 and PCV2 ORF2 were also amplified using DNA extracted from a lymph node recovered from a Swedish pig with PMWS (PCV2-Swe/N1; S. Timmusk and others, unpublished) as template. The PCR products were cloned in fusion with the bacteriophage C1 repressor gene into the bacterial two-hybrid plasmid (pBT) using the EcoRI/BamHI sites for ORFs 1, 3 and 4 and the HindIII/BamHI sites for ORF2.
Bacterial two-hybrid screening procedure.
The screening procedure was performed as described in the manual (Stratagene) but, in order to increase the screening efficiency, the transformations were carried out in two steps. In brief, E. coli BL21 was first transformed with a pBT bait plasmid containing viral gene inserts as specified (Table 1). The bacteria were then made electrocompetent and co-transformed with the expression cDNA library in the target pTRG plasmid. Bacterial colonies in which target and bait proteins interacted were revealed by their activation of the transcription of reporter genes, which rendered the bacteria carbenicillin (ampicillin) resistant and activated the -galactosidase gene. Positive interactions were selected after growth for 36 h at 30 °C on agar plates containing four antibiotics (tetracycline, chloramphenicol, kanamycin and carbenicillin). Colonies growing on the four-antibiotic plates were transferred to plates containing three antibiotics (tetracycline, chloramphenicol and kanamycin) and were then replated on these three antibiotics in combination with -galactose. The strength of the interactions was assessed by comparing the intensity of the blue colour to internal controls during this second screen for interaction. All blue clones growing on the selective plates were then grown in Luria–Bertani broth with tetracycline, the DNA was prepared and the sequence of the interacting target was determined.
Purification of recombinant proteins.
For generation of GST-fusion proteins, the entire coding region of PCV2-St ORF1 was inserted into the EcoRI/XhoI sites of plasmid pGEX-5T, which adds a 7 aa (GSEASNP) insertion between the GST part and the ORF1 part. The entire PCV2-St ORF2 coding region was inserted into the HindIII/XhoI sites of plasmid pGEX-5T, which gives a 5 aa (GSEAS) insertion between the GST part and the ORF2 part. E. coli BL21 harbouring GST-fusion protein plasmids was cultivated at 37 °C with vigorous shaking and protein expression was induced by the addition of 0.4 mM IPTG for 4 h at 30 °C. The cells were disrupted by the addition of lysozyme, sonicated to shear the DNA and cleared by centrifugation for 15 min at 10 000 g. GST-fusion proteins were purified in one step by passing the cell-free lysate over a glutathione–agarose (Sigma-Aldrich) column, eluted with glutathione and dialysed against PBS. The purity and stability of the fusion proteins were assessed by PAGE. For expression in the coupled transcription translation system (TNT; Promega), the genes were cloned in the eukaryotic expression vector pcDNA3 (Invitrogen Life Technologies). The luciferase gene was included as an expression control in the expression system (Promega). Proteins of interest (Cap, syncoilin and c-myc) and an internal control (luciferase) were expressed as 35S-labelled proteins from plasmids in the TNT system (Promega). The lengths of the TNT-translated proteins corresponded to 233 aa of Cap (full size), 176 aa of syncoilin (C-terminal part) and 212 aa of c-myc (N-terminal part).
To verify that genes were cloned in-frame, the sequence of the insertion junction and most of the ORFs was determined by routine methods at the Department of Animal Breeding and Genetics, SLU, Uppsala, Sweden. Sequences similar to candidate genes were searched for and identified using BLAST programs (at http://ncbi.nlm.nih.gov, http://tigrblast.tigr.org/tgi and http://www.sanger.ac.uk/Projects/S_scrofa/).
Pull-down experiment.
The GST pull-down assay was performed as described previously (Berg & Stenlund, 1997). In brief, dilutions of GST-fusion proteins were mixed with 35S-labelled proteins, both the protein of interest and an internal negative control (luciferase), in phosphate buffer. The proteins were incubated together for 30 min at room temperature. The volume was then increased to 100 µl and GST–Sepharose beads (Pharmacia Biotech) were added to the mixture. The GST-fusion protein was recovered by a brief centrifugation of the GST–Sepharose beads. The pellet was washed four times in phosphate buffer. The beads plus fusion protein were boiled in SDS-loading buffer and analysed directly on SDS-PAGE. As negative controls, unrelated GST-fusion proteins (GST–p23 or GST–p40) were used (Berg et al., 1998).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Results from sequence analysis were compared to general databases using BLAST programs and revealed that the sequence of the protein was highly similar to an intermediate filament protein, syncoilin. This sequence was also similar to the syncoilin of different mammals and probably represented the swine homologue of syncoilin (Fig. 2). This protein was selected twice when using the Rep protein of PCV2-St as bait and once with the Rep protein of PCV2-Swe/N1, indicating that it was probably a cellular partner of the Rep protein and suggesting that the interaction was not dependent on viral genotype/origin. To get a longer sequence of swine syncoilin, the sequence selected by the BacterioMatch assay was subjected to a systematic comparison of EST databases. An EST sequence (GenBank accession number AJ683762
[GenBank]
) was then identified, which confirmed that this candidate was a cellular swine protein and completed the sequence of the ORF.
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The capsid protein interacts with the complement factor C1qB
To get insights into the cellular partners of the PCV2 Cap protein, the same approach was followed using Cap from PCV2-St as bait. In this context, several clones expressing a protein with high sequence similarity to porcine complement factor C1qB were selected. C1q is a subunit of the C1 enzyme complex, which activates the complement cascade and possesses a small N-terminal globular domain, a collagen-like central part and a conserved C-terminal region. Clones encoding C1qB were selected when using viral Cap from both PCV2-St and PCV2-Swe/N1. Another potential partner of Cap protein identified when using PCV2-St Cap as bait encoded P-selectin, a protein that is rapidly expressed at the surface of activated or injured cells.
ORF3 protein interacts with several proteins with unknown significance
The protein encoded by PCV2 ORF3 has been associated with the induction of apoptosis by the virus. The BacterioMatch system selected three different potential candidates for binding to ORF3 (Table 3). Of these, the identification of a sequence similar to the RGS16 (regulator of G-protein signalling 16) protein appeared most relevant for the immunomodulatory effects suggested for PCV2. The selected clone encoded a partial protein that was highly similar to murine and human RGS16. This sequence was used for BLAST searches in EST databases and identified a porcine EST. The sequence combining the EST (GenBank accession number CF181051
[GenBank]
) and the BacterioMatch clone was identical to the porcine homologue of RGS16, suggesting that the ORF3 protein may interfere with the G signalling of infected cells.
Other interacting proteins of unknown significance were also found. One clone contained a 960 bp ORF, but did not show significant similarity with any known protein. The second interacting protein to ORF3 was highly similar to a Tn10 transposase sequence found in many species including swine.
The protein encoded by PCV2 ORF4 was also used as bait in the same bacterial two-hybrid screening, but no interacting partners could be identified.
The viral proteins Rep and Cap interact with each other
To study the potential interactions of PCV2 proteins further, the BacterioMatch system was used to test the binding of viral proteins to each other. As indicated in Table 4, the only significant binding involved Rep and Cap proteins. This result was observed both when PCV2-St ORF1 and ORF2 sequences were cloned as bait and target and in the opposite configuration. Weak interactions between Rep and proteins encoded by ORF3 or ORF4 were barely detectable, but were also observed in both assays using Rep as bait or as target. Neither Rep nor Cap protein interacted with itself in this assay.
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, lanes 1 and 2). This experiment confirmed that syncoilin was captured by Rep–GST, validating the interaction suggested by the BacterioMatch assay. Luciferase was used as an internal control for the specificity of the Rep–syncoilin interaction and was never recovered. Another viral GST-fusion protein encoding the p23 protein of Borna disease virus (Berg et al., 1998) was used as a negative control for the syncoilin interaction (Fig. 3b, lanes 3 and 4) and further confirmed the specificity of the interaction. The same strategy was used to confirm the interaction between Rep and Cap viral proteins. 35S-labelled Cap protein was incubated with different amounts of Rep–GST fusion protein and the complexes were precipitated (Fig. 3a, lanes 1 and 2). The same negative control (GST–p23) was used as for the syncoilin assay (Fig. 3a, lanes 3 and 4). The fusion protein captured the radioactive target in a dose-dependent manner, which confirmed the interaction between PCV2 Rep and Cap proteins and further validated the BacterioMatch system.
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, lanes 1–3). Another viral GST-fusion protein encoding the p40 protein of Borna disease virus (Berg et al., 1998) was used as a negative control in this experiment (Fig. 3d, lanes 4 and 5). This pull-down experiment confirmed the specificity of the interaction between Rep and c-myc.
Finally, to lend further support to the validation of the BacterioMatch assay and to show that the viral proteins were in a native form, a GST pull-down assay using GST–Cap and in vitro-expressed Cap was performed. In this case, the Cap protein was expressed in a eukaryotic expression system and GST–Cap in a bacterial one. As shown in Fig. 3c (lanes 1–3), these proteins interacted with each other as expected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Association of syncoilin and desmin links intermediate filament proteins to the dystrophin-associated protein complex (Poon et al., 2002). Viruses often use cytoskeletal filaments for transport of viral proteins or particles to defined subcellular locations (Suikkanen et al., 2003; Sodeik et al., 1997; Smith & Enquist, 2002). In this respect, the interaction between PCV2 Rep and syncoilin is interesting. It is possible that Rep is sequestered in the cytoplasm to regulate some as-yet undefined part of the viral life cycle.
Another possibility is that Rep is transported to the nucleus via syncoilin-type intermediate filaments. Syncoilin filaments are distributed throughout the whole cytoplasm, but are bundled adjacent to the nucleus, which would support this hypothesis (Poon et al., 2002). Furthermore, intermediate filaments are in close association with the nucleus in many cells, and their components may affect the shape of the nucleus (Sarria et al., 1994). It has also been shown that DNA can be transported via intermediate filaments (Hartig et al., 1998). Thus, since Rep is known to bind viral DNA, transport or sequestering of viral DNA could also be a possible function. For example, it has been shown that dendritic cells harbour large quantities of inactive viral DNA (Vincent et al., 2003). The mechanism behind this observation could be explained by our finding that Rep binds both syncoilin and DNA.
The observation that Rep interacted with the multifunctional c-myc was potentially interesting as well. The expression of this transcription factor correlates with cell proliferation and is especially high in various tumours. c-myc is involved in the regulation of many cellular pathways, including apoptosis. At this stage, we cannot correlate this observation with a viral function, but one possibility is that Rep is involved in processes of cell regulation and apoptosis, as has been shown with the plant circoviruses (Xie et al., 1995). In the case of PCV2 infections, however, it is notable that c-myc regulates the expression of Nramp1 which, at least in mice, is important for resistance to intracellular pathogens that reside in cells of the monocyte/macrophage lineage (Lapham et al., 2004). Another hypothesis would be that Rep mediates the activation of genes that are important for viral DNA replication through its interaction with c-myc.
It was also demonstrated that Rep binds the viral protein Cap. Localization studies revealed that PCV2 Rep proteins were localized in the nucleus in infected PK15A cells, whereas Cap could be detected in the nucleus and cytoplasm (Gilpin et al., 2003). Multiplication of circoviral DNA occurs essentially by rolling circle replication (RCR), similarly to other ssDNA viruses. The end products of RCR accumulate as both double-stranded (ds) and ssDNA circular viral DNA. It has been shown for geminiviruses that the absence or inactivation of coat protein results in a reduced level of viral ssDNA without a reduction in the level of dsDNA (Briddon et al., 1989). Disruption of coat protein synthesis resulted in a drastic reduction in ssDNA accumulation and a three- to fivefold increase in dsDNA accumulation (Padidam et al., 1999). The reduction in ssDNA accumulation has been ascribed to the loss of nuclear localization of coat protein (Qin et al., 1998). Thus, the coat protein may play a role in controlling the copy number of viral DNA. This interference is not very surprising, since coat protein is expressed late in the infection cycle and might be expected to influence early events including DNA synthesis. Finsterbusch et al. (2005) also showed that PCV1 Cap and Rep locate in different compartments of the nucleus during the early phase of PCV1 infection. Localization of Cap in the nucleoli and Rep in the nucleoplasm was followed by co-location of both proteins in the nucleoplasm. This may indicate that the RCR of PCV is controlled and stopped via Cap–Rep interactions.
One of the proteins that was found to interact with Cap was the complement factor C1qB. The physiologically most important activation of C1 is initiated by binding of the globular domains of C1qB to IgG or IgM molecules that have bound to antigen. C1q also has an important role in clearance of apoptotic cells through phagocytosis by macrophages (Navratil et al., 2001). During active PCV2 infection, organs that have intensive capillary networks are usually damaged. It is therefore possible that interaction between PCV2 and C1qB via Cap could contribute to immune complex-mediated lesions. Another possibility is that C1q facilitates the uptake of PCV2 by phagocytic cells. To strengthen the observation that Cap interacts with C1qB, the N1 genotype also tested positive for this interaction. The amino acid sequences are 93 % identical between these two genotypes (16 different amino acids among 233). However, Cap proteins from both viral strains interacted with C1qB, indicating that the differences in amino acid sequences were not reflected in their respective capacity to interact with C1qB. The interaction between Cap and C1qB and its possible implications in PCV2-induced disease remain to be determined.
Bacterial two-hybrid screening revealed that the ORF3 protein interacted with a porcine homologue of an RGS factor. RGS factors are involved in the negative regulation of signalling through heterotrimeric G protein-coupled receptors (GPCRs). Divergent isoforms of RGS proteins have been identified, several of which downregulate chemokine signalling in haematopoietic cells. RGS16, for example, has been described as a negative regulator of stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signalling. The signalling pathway through CXCR4 is involved in many activities such as B lymphopoiesis, myelopoiesis, homing of haematopoietic stem/progenitor cells (Kawabata et al., 1999) and interaction of immature megakaryocytes with the microenvironment (Berthebaud et al., 2005). RGS16 has also been shown to inhibit IL-8 and RANTES CCR5-mediated signalling in lymphocytes (Beadling et al., 1999) and participates in inflammation-induced T-cell migration (Lippert et al., 2003). More interestingly, RGS16 is inducible by IL-2 in human T cells (Beadling et al., 1999), by engagement of TLR-3 and -4 in dendritic cells and by LPS in myocytes. Thus, an interaction of ORF3 with RGS16 may have important implications for PCV2-induced diseases and the identified interaction must be confirmed.
The putative protein from ORF4 did not interact with any protein in this screen. However, we found a weak interaction between this protein and Rep. The implication of this possible interaction is currently unknown.
As in any system using fusion proteins, the conformation of the proteins used in the present work is clearly an important issue for the validity of the results. It was somewhat surprising that we could not positively identify a Cap–Cap interaction using the BacterioMatch system. In contrast, we were able to show this Cap–Cap interaction between a GST-fusion protein and a protein produced by the TNT system (Fig. 3c), indicating that our viral proteins expressed as fusion proteins in bacteria were properly folded. The most likely explanation for the inability of the BacterioMatch system to find this protein–protein interaction is that the fusion between Cap and either the RNA polymerase activation domain or the bacteriophage C1 repressor masks the regions required for such an interaction.
With all techniques, one has to be aware of the limitations of the method applied. As the BacterioMatch system is a bacterial system, some proteins will not be in a native conformation and will not be processed like they would have been in eukaryotic cells. However, the system also has many advantages over the more commonly used yeast two-hybrid system. One if these is that the system can produce more colonies, thus providing more interactions to be screened because of the better transformation efficiency of bacteria compared with yeast cells.
It was also notable that the BacterioMatch screening preferentially selected clones with comparatively short inserts as the cellular partners for viral ORFs. This phenomenon could reflect the fact that shorter copies in the library are replicated faster and colonies that harbour short inserts therefore acquire the necessary level of resistance more quickly and are more easily selected. Nevertheless, a number of potentially important targets for the PCV2 proteins were identified, and some possible mechanisms for their involvement in the development of PCV2-associated diseases were suggested. These results can direct future studies on molecular mechanisms that support latency or activation of PCV2 infections.
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ACKNOWLEDGEMENTS |
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Received 21 December 2005;
accepted 1 July 2006.
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