Porcine circoviruses—Small but powerful

Virus Research 143 (2009) 177–183

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Review

Porcine circoviruses—Small but powerful Tim Finsterbusch, Annette Mankertz ∗ Division of Viral Infections (FG12), Robert Koch-Institute, 13353 Berlin, Germany

a r t i c l e

i n f o

Article history: Available online 26 February 2009 Keywords: PCV1 PCV2 Porcine circovirus PMWS

a b s t r a c t When porcine circovirus type 1 (PCV1) was isolated more than 40 years ago as a non-pathogenic contaminant of a porcine kidney cell line, enthusiasm and curiosity kept within reasonable limits. Virologists became more interested, when a second variant was isolated and termed PCV2, because PCV2 is linked to postweaning multisystemic wasting disease (PMWS), a new emerging multifactorial disease in swine. Both PCV1 and PCV2 are small and rather simply organized and express only few proteins. Therefore, it was expected that the factor(s) triggering PMWS should be easily identified, but more than one decade of PCV research has not yet singled out a molecule inducing the disease onset. Unravelling the molecular features of PCV and the channels through which the virus interacts with its host are key to manage, prevent and treat PMWS and other PCV-associated diseases. Since we have learned many aspects of the molecular biology of PCV in the last years, it is time for a résumé! © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8.

The virus and the disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The viral genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The viral proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCV transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Replication of PCV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host–virus interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. The virus and the disease Porcine circoviruses belong to the genus Circovirus of the family Circoviridae, which comprises porcine viruses as Porcine circovirus type 1 (PCV1) and type 2 (PCV2) but also avian viruses as Psittacine beak and feather disease virus (BFDV), Pigeon circovirus (PiCV), Canary circovirus (CaCV) and Goose circovirus (GoCV) (Todd et al., 2005). Typical for all circoviruses is the single-stranded (ss) and circular DNA genome with a rather small size (1759–2319 nts) and the non-enveloped spherical virus particles, which display a diameter of 16–18 nm.

∗ Corresponding author at: Division of Viral Infections (FG12), Robert KochInstitut, Nordufer 20, 13353 Berlin, Germany. Tel.: +49 30 18754 2516; fax: +49 30 18754 2598. E-mail address: [email protected] (A. Mankertz). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.02.009

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PCV1 does not induce a disease in swine (Tischer et al., 1995), while PCV2 is the etiological agent of Postweaning Multisystemic Wasting Syndrome (PMWS), a new emerging and multifactorial disease in swine. PMWS was first recognized in North America in 1991 (Clark, 1997; Harding, 1996). Since then, this scourge has devastated almost every pig-producing area of the world. Although clinical signs are highly variable, characteristic PMWS symptoms are wasting, respiratory dysfunction, enlargement of superficial inguinal lymph nodes, diarrhoea, and a generalized depletion of lymphocytes (Allan and Ellis, 2000; Segales et al., 2005). Secondary infections with opportunistic organisms are often seen, suggesting that the response of the immune system is hampered in PMWSaffected animals. The fact that PCV1 and PCV2 are ubiquitous viruses hinders the diagnosis of PMWS, which must meet three criteria: (i) the presence of compatible clinical signs, (ii) the presence of moderate to severe characteristic microscopic lymphoid lesions, and (iii) the

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presence of moderate to high amounts of PCV2 within these lesions. On affected farms, mortality can be decreased by implementation of special management plans as the Madec principle (Madec 2008). In the beginning of experimental reproduction, a puzzling variety of results were observed; as either no symptoms at all, only minor lesions or a full-blown PMWS were seen. Later, it became evident that the activation of the immune system is a pivotal step in induction of PMWS (Krakowka et al., 2001). PMWS is a multifactorial disease; the onset of the disease and the severity of the symptoms are influenced by intrinsic factors such as the status of the immune system and genetic predisposition and by practical aspects such as vaccination policy and feeding stuff.

rep and cap gene and a larger one between their 5 -ends, the latter comprising the origin of viral genome replication. Similar genomic structures are found in members of the families Geminiviridae and Nanoviridae. The origin of replication is characterized by a putative stem–loop structure with a nonamer in its apex (Fig. 1). This sequence and structure element is conserved in viruses, plasmids and bacteriophages. Several of these replicons are known to replicate via rolling-circle replication (RCR) (Mankertz et al., 1997). Adjacent to the stem–loop, hexamer motifs serve as binding site for the replicases.

3. The viral proteins 2. The viral genome PCVs contain a circular ssDNA molecule with a size of 1759 in case of PCV1 and 1768 for PCV2. They are the smallest mammalian viruses yet known and encode only two major open reading frames, rep and cap, which perform the two most elementary functions of a virus, copying and the successive packaging of the viral genome. Circoviruses can therefore be regarded as a fascinating paradigm not only for induction of a complex pathogenesis but also for the reduction of the molecular equipment to the absolute necessities. The rep and cap genes are orientated divergently resulting in an ambisense genome organization. This arrangement creates two intergenic regions, a shorter one between the 3 -ends of the

Two major ORFs are encoded by the genomes of PCV1 and PCV2 (Fig. 1): The largest ORF of the ambisense-organized circoviruses is located on the viral plus-strand (rep gene or ORF1). From ORF1 two proteins are expressed, which are both necessary for viral replication. Rep and Rep are produced from differentially spliced transcripts. The Rep protein is translated from the full-length transcript (PCV1: 312 aa; PCV2: 314 aa); a spliced transcript encodes truncated and C-terminal frame-shifted Rep (PCV1: 168 aa; PCV2: 178 aa). Rep and Rep display three motifs in their common Nterminus, which are characteristic for initiator proteins of the RCR mode. Moreover, Rep contains a dNTP-binding domain, which is absent from Rep . Phylogenetic analyses suggest that circovirus Rep proteins may have evolved by a recombination event between the

Fig. 1. A map of PCV. A linear map of the PCV genome indicates the two major ORF, rep and cap. Three motifs conserved in RCR enzymes (I–III) and a dNTP-binding domain (P) indicated within the rep gene. The ORF3 differing in length in PCV1 and PCV2 is indicated in grey boxes. The lower part shows a comparison of the two origin regions of PCV1 and PCV2. Sequences have been aligned, and the hexamer repeats 1–4 are marked by open boxes. The conserved nonamer sequence within the single-stranded loop of the hairpin is indicated by a grey box, the nicking site by an arrow.

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Rep protein of nanoviruses and an RNA-binding protein encoded by picorna-like viruses or a helicase of prokaryotic origin. Interestingly, the frameshift observed in Rep coincides with the suggested point of recombination (Gibbs and Weiller, 1999). Synthesis of more than one Rep protein variant by splicing is a well-established feature in other ssDNA viruses, but in contrast to e.g. Maize streak virus, both Rep and Rep are necessary to initiate replication of PCV (Mankertz and Hillenbrand, 2001). Interestingly, replication via the PCV-origin did occur only in the presence of Rep, when replication of PCV-based plasmids was analysed in Escherichia coli (Cheung, 2006). ORF2 encodes the major structural protein Cap (PCV1: 232 aa; PCV2: 233 aa), which is also the main antigenic determinant of the virus. Cap displays a basic N-terminus rich in arginine residues, which is putatively involved in binding to viral DNA. After expression of the Cap protein in bacteria and insect cells, the protein assembled into virus-like particles (VLP) when viewed by electron microscopy. A recent finding links pathogenicity with certain alterations of the sequence of the Cap protein. A PCV2 isolate serially passaged 120 times in PK-15 cells (VP120) replicated more efficiently than the original VP1. The sequences of VP120 revealed two mutations located in the capsid gene, a proline at position 110 of the capsid protein changed to an alanine and a change from an arginine to a serine at position 191. When PCV2 VP1 and VP120 were inoculated into specific-pathogen-free pigs, viremia and histopathologic lesions were less pronounced in pigs inoculated with VP120 indicating that the P110A and R191S mutations in the Cap protein enhanced the growth ability in vitro and attenuated the virus in vivo (Fenaux et al., 2004b). Several smaller ORFs have been found, but with the exception of ORF3 of PCV2, their expression has not been demonstrated. ORF3 is comprised in PCV1 and PCV2 within ORF1, but transcribed counterclockwise. Interestingly, this ORF differs in sequence and size between the two PCV variants: a longer coding region of 621 nt is expressed in PCV1, while it is truncated to 315 nt in PCV2. This renders it an interesting candidate for the induction of pathogenesis. Unfortunately, up to now, only the PCV2encoded protein has been studied. Its expression is not essential for PCV2 replication, expression of a GFP-fused ORF3 variant induced apoptosis via caspase 8 and caspase 3 (Liu et al., 2005); therefore, an apoptotic activity has been hypothesized. Wild-type PCV2 and a mutant virus, in which expression of ORF3 was disabled, were studied in vivo. Seroconversion was seen in both cases, but the viral load of the wild-type PCV2 in serum was higher and the mutant failed to induce microscopic lesions typical for PMWS. This suggests that ORF3 may contribute to induction of PMWS. However, it does not seem likely to be the major determinant of PCV2 pathogenesis, since another study using chimeric viruses did not support this hypothesis. Fenaux et al. (2004a) produced chimeric viruses from PCV1 and PCV2 by inserting the cap gene of PCV1 into the PCV2 backbone, PCV2(cap PCV1), and vice versa PCV1(cap PCV2) by replacing the capsid gene of non-pathogenic PCV1 with that of PCV2. After inoculation of pigs with these constructs, seroconversion but no pathogenic effects were observed with either of the chimeric viruses. Since ORF3(PCV2) is comprised in the construct PCV2(cap PCV1), neither ORF3 nor Cap seem to be the only factor contributing to PCV2-induced pathogenesis. Rep and Rep co-localize in the nucleoplasm of infected cells, leaving the nucleoli blank (Finsterbusch et al., 2005). This particular localization did not appear to be altered during the infection cycle. Both Rep and Rep carry three nuclear localization signals (NLSs) in their identical N-termini, of which NLS1 and NLS2 mediate the nuclear accumulation, whereas NLS3 serves as an enhancer of the nuclear import of Rep and Rep . In contrast, the localization of the Cap protein was variable. In plasmid-transfected cells, Cap resided in the nucleoli throughout the cell cycle. In an early stage of infection, Cap was localized in the nucleoli, while later on migration

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to the nucleoplasm and export into the cytoplasm was observed, indicating that the Cap protein shuttles between distinct cellular compartments during the infection cycle. Since all PCV-encoded proteins are chiefly located in the nucleus, DNA replication and encapsidation of the circular ssDNA genome will probably occur in the nucleus and not in cytoplasmic compartments. The localization of Cap to the nucleoli in early infections may be promoted by interaction with nucleolar proteins (see below). Nucleolar localization has been described for proteins of many viruses (Hiscox, 2002) and it has been proposed that virus proteins enter the nucleoli to support viral transcription or influence the cell cycle, but the effect of this phenomenon for PCV life cycle is not understood at the moment.

4. PCV transcription The start of the rep transcript of PCV1 has been mapped to nucleotide 767 ± 10. The promoter of rep, Prep, overlaps the origin of replication (nucleotides 640–796). Prep is negatively regulated by the Rep protein, which binds to hexamers H1 and H2. Interestingly, Rep also binds to H1/H2, but does not repress the activity of Prep. Binding of Rep and Rep to these sequence elements is also an essential prerequisite for initiation of replication. Since mutagenesis of H1/H2 decreases but does not completely abolish Prep transcription, putative interaction of Rep protein with inhibitors of transcription seem to contribute to repression of Prep. In contrast to Prep, the promoter of the cap gene of PCV is not located in the intergenic region but has been mapped to a fragment within the rep gene (pos. 1168–1428) and Rep, Rep and Cap did not influence transcription initiated at Pcap (Mankertz and Hillenbrand, 2002). The cap transcript starts at nucleotide 1238 with an untranslated leader sequence of 119 nt (1238–1120) joined to exon 2 of the ORF1 transcript at nucleotide 737, immediately adjacent to the start point of translation. Processing of this RNA may have evolved to avoid synthesis of another protein initiated at an internal start codon in the intron (Mankertz et al., 1998). Little is known about the cellular events triggered by infection with PCV2 in PMWS. By cDNA differential display (DDRT-PCR) (Bratanich and Blanchetot, 2006), several porcine genes were found to be upregulated not only in lymph node tissue but also in PK-15 cells: (i) a gene similar to the hyaluronan-mediated motility receptor (RHAMM; NM 012485), which induces the Ras- and ERK-signalling pathways after binding of hyaluron. RHAMM is an oncogene that is regulated by growth-promoting factors such as TGF-␤1, ␤1 integrins and PKC and interacts with proteins of the cytoskeleton and DNA repair genes; (ii) a RNA splicing factor (SPF30, NM 005871) essential for assembly of the ribonucleoproteins into the spliceosome; (iii) a RNA helicase comprising a DEAD box (XM 537542); (iv) PACSIN2 (CR456536), also called Syndapin 2 belonging to the Src-homology 3 (SH3)-domain-containing proteins. These proteins interact with dynamin and several other proteins implicated in vesicle traffickingand is involved in the regulation of tubulin polymerisation, endocytosis and actin dynamics; (v) and a human kynurenine 3-monooxygenase enzyme (AL591898), a mitochondrial enzyme in the of tryptophan degradation pathway that has been linked directly to the pathophysiology of Huntington disease in humans. In a similar study performed in our group, we have found several genes up- and downregulated after infection with PCV1 and PCV2 (C. Adlhoch, pers. communication), most of them were involved in immune modulation, signal transfer, and intracellular vesicle transport. Recently, it has been demonstrated that PCV2 induces the activation of NFkB by IkBa phosphorylation and degradation and subsequent translocation of NFkB p65 from the cytoplasm to the nucleus. Treatment of cells with an inhibitor of NFkB reduced

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PCV2-encoded protein expression and production of progeny virus (Wei et al., 2008). All these findings are informative steps towards an understanding of the changes induced in the host cell after PCV infection. Interestingly, many results point towards intracellular signalling and endocytotic pathways. Thus, these aspects seem to deserve our increased attention. 5. Virus import PCVs are the smallest viruses that autonomously replicate in mammalian cells. In vivo, viral DNA and antigens are found in monocytic, epithelial and endothelial cells, but no substantial replication was identified in monocytic cells. In contrast, replication of PCV2 infection was detected in the endothelial cell line PEDSV.15, aortic endothelial cells, gut epithelial cells, and fibrocytes by an increase in the levels of Cap and Rep protein (Steiner et al., 2008). Interestingly, variations in endocytic activity, virus binding, and uptake did not relate to the replication efficiency in a particular cell and replication did not correlate to cell proliferation. The glycosaminoglycans heparin, heparan sulfate and chondroitin sulfate B, but not chondroitin sulfate A and keratan sulfate, serve as attachment receptors for PCV2 (Misinzo et al., 2006), but another molecule may be needed as a specific receptor (Fig. 2). When binding to and entry of VLPs into the porcine monocytic cell line 3D4/31 were studied, PCV2 bound quickly to all cells but the VLPs were internalized slowly via clathrin-mediated endocytosis into only some of the cells (Misinzo et al., 2005). Viruses enter the host cell by several common pathways (Marsh and Helenius, 2006; Vincent et al., 2005) as e.g. clathrin-mediated endocytosis, macropinocytosis, caveolin-mediated endocytosis and clathrinand caveolin-independent pathways. PCV2 infection of 3D4/31 cells was decreased significantly by inhibitors that specifically blocked actin-dependent processes, clathrin-mediated endocytosis, and cytosol acidification, while inhibition of macropinocytosis and caveolae-dependent endocytosis did not reduce PCV2 infection. Taken together, PCV2 enters 3D4/31 cells via clathrin-mediated endocytosis and requires an acidic environment. After internalization, PCV2 was localized in endosomes. As the endosomes progress into the inner cell, the luminal pH of these vesicles drops to acidic values. Use of inhibitors made evident that a serine protease is essential for the release of PCV2 from the endosome (Misinzo et al., 2005), suggesting that this process may induce a proteolytic cleavage of the Cap protein. A similar experiment using endosomal–lysosomal acidification inhibitors astonishingly revealed increased PCV2 infection rates in PK-15 epithelial cells (Misinzo et al., 2008), suggesting that serine protease-mediated

PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibiting endosomal–lysosomal system acidification. Immunofluorescence analysis had revealed that PCV2 co-localized with clathrin during internalization, but not with caveolin. Unexpectedly, inhibition of clathrin-mediated endocytosis resulted in an increase in the number of PCV2infected PK-15 cells, indicating that this pathway does not represent the main internalization route in epithelial cells. Further analysis demonstrated that also macropinocytosis, dynamin-dependent internalization and import via lipid rafts do not play the predominant role in PCV2 entry, while it could be ascertained that PCV import relies upon small GTPases and actin polymerization. In summary, a dynamin- and cholesterol-independent, but actin- and small GTPase-dependent pathway allows PCV2 internalization into epithelial cells and leads to infection, while clathrin-mediated PCV2 internalization in epithelial cells is not followed by a full replication. 6. Replication of PCV Upon infection, the viral ssDNA genome is converted by host cell factors into a dsDNA replicative form that serves as template for viral DNA replication (Fig. 2). The origin of replication is located within the non-coding region between the ORFs of rep and cap and overlaps with the promoter of the rep gene. A characteristic nonanucleotide sequence (5 -(A/T)AGTATTAC-3 in PCV2 and PCV1, respectively) is conserved in all circoviruses and flanked by an inverted repeat (palindrome) of 11 nucleotides (Fig. 1). Adjacent to this structure, direct hexamer and pentamer repeats are found. By in vitro experiments, the minimal binding site (MBS) for the PCV replication proteins Rep and Rep was mapped to the 3 -part of the inverted repeat plus the two inner hexamers H1 and H2 (Steinfeldt et al., 2001). Binding of Rep and Rep to the origin destabilizes and unwinds the dsDNA and induces the exposure of the nonamer sequence as ssDNA, which is subsequently recognized and cleaved by Rep/Rep . Sequence homologies at the origin and similarities among the Rep proteins suggest that PCVs may replicate via the rolling-circle cruciform model described for plant gemini- and nanoviruses (Hanley-Bowdoin et al., 2000; Ilyina and Koonin, 1992; Koonin and Ilyina, 1993; Palmer and Rybicki, 1998; Stenger et al., 1991). This model postulates extrusion of a cruciform or stem–loop structure in which the inverted repeats form a stem with the nonanucleotide exposed in the apex. Recently, an alternative replication mechanism, the rolling-circle melting-pot replication model was proposed for PCVs (Cheung, 2004a, 2004b). In contrast to the rolling-circle cruciform model, binding of Rep/Rep to the origin induces a sphere of instability instead of a stem–loop

Fig. 2. Life cycle of PCV. PCV uses glycosaminoglycans as attachment receptors. The ssDNA genome is transported into the nucleus and converted by host enzymes into a dsDNA intermediate. The rep and cap mRNAs are transcribed, and the proteins are synthesized and imported from the cytoplasm. Rep/Rep bind to the dsDNA and initiate RCR by introduction of a nick that serves as primer. Elongation of the primer by host enzymes leads to replication. Meanwhile the Rep protein is covalently bound to the DNA and terminates the reaction by introduction of a second cleavage reaction via Tyr-93. Events leading to assembly and release of virions have not yet been studied.

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Table 1 Viral protein

Cellular interacting protein

Ontology of cellular protein

Identified by

Rep protein

Syncoilin c-myc ZNF265 TDG VG5Q

Transport processes Transcriptional regulation Alternative splicing DNA repair, transcriptional regulation Angiogenesis

Bacterial two-hybrid Bacterial two-hybrid Yeast two-hybrid Yeast two-hybrid Yeast two-hybrid

Cap protein

P-selectin C1qB gC1qR MKRN1 Par-4 NAP1 Hsp40 NPM1 DDE-like transposase

Cell adhesion molecule Complement factor C1qb receptor, multifunctional E3 ubiquitin ligase Apoptosis, cell mobility, transport Transport, chaperonin Chaperonin Ribosome biogenesis, transport Transposase

Bacterial two-hybrid Bacterial two-hybrid Yeast two-hybrid Yeast two-hybrid Yeast two-hybrid Yeast two-hybrid Yeast two-hybrid Yeast two-hybrid Bacterial two-hybrid

ORF3 protein

Membrane interacting protein RGS16 pPirh2

Cell signalling E3 ubiquitin ligase, p53-induced apoptosis

Bacterial two-hybrid Yeast two-hybrid

structure. Within this destabilized environment, all four strands of the inverted repeats are in a “melted” state resulting in a four-stranded tertiary structure. In consequence, both minusand plus-DNA strands are available as templates during initiation as well as termination of DNA replication. Based upon sequence comparisons, three conserved RCR-motifs (RCR-I (FTLN), RCR-II (HxQ) and RCR-III (YxxK)) have been identified within Rep and Rep plus a GKS box (P-loop) for dNTP-binding (Ilyina and Koonin, 1992; Steinfeldt et al., 2006; Vega-Rocha et al., 2007). Mutations in all these four motifs interfered with PCV replication in cell culture (Mankertz and Hillenbrand, 2001; Steinfeldt et al., 2007). The role of RCR-I in the catalytic activity of Rep/Rep is unknown, while RCR-II mediates the coordination of bivalent metal cations, required for the nicking of the DNA (Hafner et al., 1997; Koonin and Ilyina, 1992; Laufs et al., 1995; Steinfeldt et al., 2006; Vega-Rocha et al., 2007). Tyr-93 within RCR-III of Rep/Rep performs the cleavage of the phosphodiester bond by a nucleophilic attack and subsequently becomes attached to the 5 -end of the 3 -cleavage product via a tyrosylester (Steinfeldt et al., 2007; Vega-Rocha et al., 2007). The so-generated 3 -hydroxyl group serves as a primer for DNA synthesis, while the Rep proteins remain covalently attached to the 5 -phosphate of the cleavage product (Steinfeldt et al., 2007; Vega-Rocha et al., 2007). After one round of replication, the newly synthesized DNA strand is cleaved again within the regenerated origin and the 5 -phosphate is ligated to the novel 3 hydroxyl group. This process results in the release of ss unit-length monomers. 7. Host–virus interaction Due to their small genome size and highly limited coding capacity, the life cycle of PCV relies predominantly on host cell factors. Recently, several porcine proteins interacting with the viral proteins Rep/Rep , Cap and the ORF3 protein were identified either by an yeast or a bacteria-based two-hybrid assay (Finsterbusch et al., 2009; Liu et al., 2007; Timmusk et al., 2006). Almost all interaction partners (summarized in Table 1) are annotated in the literature as proteins with multiple functions but there is no indication of a common ontology. They can be associated with manifold aspects of viral replication such as transcriptional regulation and intracellular transport processes. Timmusk et al. (2006) have identified two cellular Rep interacting porcine proteins, an intermediate filament protein similar to the human syncoilin and the transcriptional regulator protein c-myc. Syncoilin has been associated with redistribution of desmin from filaments and into the cytoplasm (Newey, 2001), and potentially, syncoilin is involved in the transport or sequestering of viral DNA. Moreover, three porcine

homologues of ZNF265, TDG and VG5Q were identified to bind to Rep (Finsterbusch et al., 2009). ZNF265 is an alternative component of the spliceosome, which can replace the essential constituent SF2/ASF (Adams et al., 2001). A Rep–ZNF265 complex may therefore influence transcription and alternative splicing, and, strengthening this hypothesis, co-expression in HEK293 cells led to their accumulation in punctiform structures within the nucleus, which resembled transcriptional compartments. VG5Q and TDG were also described as linked to transcriptional regulation. TDG was initially described as a DNA repair protein (Neddermann et al., 1996), but it also interacts with a number of transcriptional activators and coactivators as CBP/p300 (Tini et al., 2002), estrogen receptor alpha (Chen et al., 2003) and the thyroid transcription factor-1 TTF1 (Missero et al., 2001) indicating its role in transcriptional regulation. Six cellular proteins (MKRN1, gC1qR, Par-4, NAP1, NPM1 and Hsp40) were found with the yeast system to bind Cap (Finsterbusch et al., 2009). MKRN1 is a member of the E3 ubiquitin ligase family and has been reported to modulate telomere length homeostasis by ubiquitination and proteasome-mediated degradation of the telomerase subunit hTERT (Kim et al., 2005). The observation that co-expression of Cap and MKRN1 leads to reduced levels of Cap expression implies that MKRN1 may promote the degradation of Cap. Hsp40 is a chaperone known to prevent aggregation and misfolding of proteins (Hartl, 1996). Moreover, it affects the replication of several viruses in different ways. Hsp40 suppresses hepatitis B virus replication (Sohn et al., 2006), whereas human immunodeficiency virus type 1 (HIV-1) gene expression and replication is enhanced (Kumar and Mitra, 2005). The complement receptor for C1q binds several viral proteins including the HIV-1 Tat and Rev (Berro et al., 2006), the rubella capsid protein (Beatch et al., 2005) and the core proteins of adenovirus (Matthews and Russell, 1998), thereby promoting viral transcription, replication, capsid assembly and protein transport. It serves as the receptor for Hantaan virus (Choi et al., 2008) and is known to modulate innate immunity by inhibiting RIG-I and MDA5-dependent antiviral signalling (Xu et al., 2009). Interestingly, the bacterial two-hybrid assay revealed that the complement factor C1qB interacts with the Cap protein (Timmusk et al., 2006), the ligand of gC1qR. One is tempted to speculate, whether a ternary complex between Cap, C1qB and gC1qR is formed. Par-4 was first identified as a pro-apoptotic gene product upregulated in prostate cancer cells undergoing apoptosis (Sells et al., 1994) and interacting with Wilms’ tumor suppressor protein WT1 (Johnstone et al., 1996). Recently, it was described that Par-4 targets the zipper interacting protein kinase to the actin cytoskeleton of smooth muscle cells (Vetterkind and Morgan, 2008). The association of Cap and Par-4 might play a role for the processing

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or transport of viral proteins or particles. The Cap interacting proteins NAP1 and NPM1 are also involved in intracellular transport processes, since both serve as chaperones and bind to histones and other basic proteins. NPM1 is a nucleolar phosphoprotein involved in ribosome biogenesis (Savkur and Olson, 1998), protein transport (Szebeni et al., 1995), duplication of centrosomes (Okuda et al., 2000) and has chaperone protein characteristics (Szebeni and Olson, 1999). The ORF3 protein interacts with another porcine ubiquitin E3 ligase pPirh2 (porcine p53-induced RING-H2), and facilitates p53 expression in viral infection. ORF3 expression suppressed pPirh2 expression in PCV2-infected cells and led to an increase in p53 expression. Phosphorylation of p53 at Ser-46, which is linked to p53-induced apoptosis, was also observed in PCV-infected and ORF3-transfected cells (Liu et al., 2007). 8. Conclusion After more than one decade of research addressing the molecular biology of PCV1 and PCV2, many questions concerning these two viruses are not yet answered. Although we have caught a first glimpse on the entry of PCVs into the cells, we do not know yet where the virus is assembled and how it is exported from the cell. Moreover, one would like to obtain more information about the virus–host interaction and the processes that lead to the onset of the disease. Therefore, we have to sort out, which of the several connections between cellular and PCV-encoded proteins are really relevant for induction of pathogenesis and which are employed to support the viral life cycle but do not harm the organism. The fact, that PMWS is a multifunctional disease, does not render this task easier, but we are optimistic that the studies summarized in this review are a sound basis to continue the research leading to a better knowledge of PCV and PMWS. References Adams, D.J., van der Weyden, L., Mayeda, A., Stamm, S., Morris, B.J., Rasko, J.E., 2001. ZNF265—a novel spliceosomal protein able to induce alternative splicing. J. Cell Biol. 154 (1), 25–32. Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. J. Vet. Diagn. Invest. 12 (1), 3–14. Beatch, M.D., Everitt, J.C., Law, L.J., Hobman, T.C., 2005. Interactions between rubella virus capsid and host protein p32 are important for virus replication. J. Virol. 79 (16), 10807–10820. Berro, R., Kehn, K., de la Fuente, C., Pumfery, A., Adair, R., Wade, J., Colberg-Poley, A.M., Hiscott, J., Kashanchi, F., 2006. Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J. Virol. 80 (7), 3189–3204. Bratanich, A., Blanchetot, A., 2006. A gene similar to the human hyaluronanmediated motility receptor (RHAMM) gene is upregulated during Porcine Circovirus type 2 infection. Virus Genes 32 (2), 145–152. Chen, D., Lucey, M.J., Phoenix, F., Lopez-Garcia, J., Hart, S.M., Losson, R., Buluwela, L., Coombes, R.C., Chambon, P., Schar, P., Ali, S., 2003. T:G mismatch-specific thymine-DNA glycosylase potentiates transcription of estrogen-regulated genes through direct interaction with estrogen receptor alpha. J. Biol. Chem. 278 (40), 38586–38592 (Epub 2003 Jul 21). Cheung, A.K., 2004a. Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus. J. Virol. 78 (8), 4268–4277. Cheung, A.K., 2004b. Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model. J. Virol. 78 (17), 9016–9029. Cheung, A.K., 2006. Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli. J. Virol. 80 (17), 8686–8694. Choi, Y., Kwon, Y.C., Kim, S.I., Park, J.M., Lee, K.H., Ahn, B.Y., 2008. A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection. Virology 381 (2), 178–183. Clark, E.G., 1997. Post-weaning multisystemic wasting syndrome. In: Proceedings of the American Association of Swine Practitioners, 28th Annual Meeting, pp. 499–501. Fenaux, M., Opriessnig, T., Halbur, P.G., Elvinger, F., Meng, X.J., 2004a. A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs. J. Virol. 78 (12), 6297–6303. Fenaux, M., Opriessnig, T., Halbur, P.G., Elvinger, F., Meng, X.J., 2004b. Two amino acid mutations in the capsid protein of type 2 porcine circovirus (PCV2) enhanced

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