Replicative Intermediates of Porcine Circovirus in Animal Tissue Cultured Cells Or in Bacteria Undergoing Copy-Release Replication$

Virology 434 (2012) 38–42

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Virology

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Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication$

Andrew K. Cheung n

Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, PO Box 70, Ames, IA 50010, USA article info abstract

Article history: Porcine circovirus (PCV) has been assumed to replicate its genome via the rolling-circle replication Received 20 June 2012 (RCR) mechanism because it encodes a Rep protein that contains several amino acid motifs commonly Returned to author for revisions found in other RCR biological systems. Two proteins, Rep and Rep’, are essential for PCV DNA replication 3 August 2012 in mammalian cells. In this work, replicative intermediates of PCV-infected porcine kidney (PK15) cells Accepted 14 August 2012 or copy-release of PCV genomes from a head-to-tail tandem construct (without Rep’) in Escherichia coli Available online 29 August 2012 were examined. In PK15 cells, replicative intermediates consistent with complementary-strand Keywords: replication which converts single-stranded circular genome to double-stranded supercoiled DNA and Porcine circovirus RCR which generates single-stranded plus strand progeny genome were observed. To a lesser extent, Rolling-circle DNA replication intermediates suggestive of recombination-dependent replication were also detected. In Escherichia Copy-release DNA replication coli, copy release of the single-stranded circular PCV genome with conversion to a supercoiled molecule by complementary-strand synthesis was observed. However, replicative intermediates indicative of RCR were not detected. Published by Elsevier Inc.

Background DNA replication (Ori). Based on the fact that porcine circovirus (PCV), a member of the genus Circovirus of the Circoviridae family The rolling-circle replication (RCR) mechanism has been (McNulty et al., 2000), encodes a Rep protein that contains several demonstrated in the synthesis of circular DNAs of phages amino acid motifs (RC-I, RC-II and RC-III and a P-loop domain) (Novick, 1998), bacterial plasmids (del Solar et al., 1998; Khan, (Steinfeldt et al., 2007) present in other biological systems that 2000), plant viruses (Gutierrez, 1999; Hanley-Bowdoin et al., 2000; replicate by RCR and that Rep exhibits nicking/joining activities in Palmer and Rybicki, 1998) and animal viruses (Graham et al., 1989; concert with the viral sequences at the Ori (Steinfeldt et al., 2006), Musatov et al., 2000). The common features exhibited by these it has been theorized that PCV replicates its genome via RCR. diverse biological systems include an initiator Rep protein (Ilyina During a PCV productive infection in mammalian cells, the and Koonin, 1992) and a pair of inverted repeats or palindromic single-stranded (ss) circular genome is transported to the nucleus. sequences capable of forming a stem-loop structure at the origin of Two virus-encoded proteins, Rep and Rep’, are essential for PCV DNA replication (Cheung, 2003a,b, 2004b, 2006b; Mankertz and Hillenbrand, 2001). However, direct evidence demonstrating the $Disclaimer: Mention of trade names or commercial products in this article is replicative intermediates of PCV has not been reported. Nucleotide solely for the purpose of providing specific information and does not imply sequence analysis shows that the PCV Rep gene is intermediate recommendation or endorsement by the US Department of Agriculture. The US between the Rep genes of plant geminiviruses and nanoviruses Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where (Meehan et al., 1997; Niagro et al., 1998), which suggests that PCV applicable, sex, marital status, familial status, parental status, religion, sexual may replicate its genome in a manner similar to the Geminiviridae orientation, genetic information, political beliefs, reprisal, or because all or part of (Gutierrez, 1999; Hanley-Bowdoin et al., 2000; Palmer and Rybicki, an individual’s income is derived from any public assistance program. (Not all 1998)orNanoviridae (Gronenborn, 2004) viruses. Three DNA synth- prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, esis mechanisms have been reported for geminiviruses: audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice recombination-dependent replication (RDR), complementary- and TDD). To file a complaint of discrimination, write to USDA, Director, Office of strand replication (CSR) and RCR. RDR replicative intermediates Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or are double-stranded (ds) linear DNAs of various sizes (designated call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal hds) derived from recombination/invasion of a ds genome by a ss opportunity provider and employer. n Fax: þ515 337 7428. linear DNA (Jeske et al., 2001; Preiss and Jeske, 2003). CSR converts a E-mail address: [email protected] ss circular genome to a relaxed ds circular genome. This ss to ds

0042-6822/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.virol.2012.08.020 A.K. Cheung / Virology 434 (2012) 38–42 39

conversion requires a minus genome primer. A variety of minus 0.75 copy PCV1Cap/PCV2Rep genome with chimeric Ori-1L/2R genome primers had been reported for geminiviruses and nano- together with the pSKþ bacterial plasmid. Presumably, the gen- viruses (Donson et al., 1984; Hafner et al., 1997; Saunders et al., eration of cccL and cccQ is via RCR copy-release mechanism, 1992); however, the origin and synthesis of these primers have not which requires the presence of two PCV Oris and a functional Rep been determined. The relaxed ds circular genomes then become with no involvement of Rep’ (Cheung, 2004a, 2005a,b, 2006a). supercoiledmoleculesandserveastemplatesforRCRinthe In this work, viral replicative intermediates of PCV2-infected presence of the Rep-complex. It is expected that during productive PK15 cells, PCV1-persistently-infected PK15 cells, or copy-release of PCV infections, like geminiviruses, both CSR and RCR occur simulta- unit length PCV genome from a chimeric HTT construct in E. coli neously and intermediates specific to each process are present. were examined. Total cell DNAs were analyzed by 2-D gel/Southern Two genotypes of PCV, PCV1 and PCV2, have been described. blot analysis. The first dimension is in neutral agarose gel (separa- The PCV virion is non-enveloped, icosahedral, 16–21 nm in tion by molecular size) and second dimension is in chloroquine diameter and contains a closed-circular, ss-DNA genome agarose gel (separation by topology or physical configuration). This (McNulty et al., 2000). PCV1 [1759 nucleotides (nt)] was discov- 2-D gel system and in combination with electron microscopy had ered in 1974 in a persistently-infected porcine kidney (PK15- been used successfully in resolving the replicative intermediates of ATCC-133) cell line and it is non-pathogenic (Tischer et al., 1986; geminiviruses (Alberter et al., 2005; Erdmann et al., 2010; Jeske Tischer et al., 1974), while PCV2 (1768 nt) is associated with post- et al., 2001; Jovel et al., 2007; Preiss and Jeske, 2003). weaning multi-systemic wasting syndrome in swine (Clark, 1996; Harding, 1996). The genome organization of PCV1 and PCV2 is very similar. The cis-acting and trans-acting replication factors of Results both viruses are interchangeable for DNA replication and for chimeric virus production (Beach et al., 2010a,b; Cheung, 2003a; Immunohistochemical staining of PCV2-infected and PCV1- Fenaux et al., 2004; Mankertz et al., 2003). persistently-infected PK15 cell Previously, it was demonstrated that a unit size PCV genome can be excised from a chimeric head-to-tail (HTT) tandem DNA PCV-free PK15 cells infected with PCV2 (multiplicity of consisting of 0.8 copy of PCV type 1 (PCV1) and 0.95 copy PCV infection¼0.4) at 38 h postinfection or PK15 cells persistently- type 2 (PCV2) inserted into the pBluescript SKþ (pSKþ) bacterial infected with PCV1 (passage 152) were analyzed by immunohisto- plasmid, that replicates via the theta replication mechanism chemical staining as previously described (Cheung and Bolin, 2002). (Tomizawa et al., 1974), in the presence of a functional Rep gene The primary antiserum used for PCV1 or PCV2 was hyperimmune (no Rep’) and two Ori cis-acting elements (Cheung, 2006b). serumraisedinswine.Inbothexperiments,viralantigenswere Briefly, this HTT construct containing 1.75 copies of PCV yielded detected in less than 1% of the virus-infected cells (data not shown). three different molecular size supercoiled ds DNA species (designated cccU, cccL and cccQ) when transformed into E. coli: cccU is Replicative intermediates of PCV2 in newly-infected PK15 cells the input parent construct, cccQ is the unit-length chimeric

PCV1Rep/PCV2Cap genome with chimeric Ori-2L/1R but lacking the DNAs isolated from PCV2- infected PK15 cells at 38 h post- plasmid vector, and cccL is a molecule consisting of the remaining infection were analyzed by 2-D Southern blot analysis (Fig. 1a).

Fig. 1. 2-D Southern blot analysis of (a) PCV2-infected PK15 cells and (b) PCV1-persistently-infected PK15 cells. The following PCV DNA forms were identiﬁed: circular and linear ss-genome (1xss), ds linear (lin), monomeric open circular (oc), monomeric covalently closed circular (ccc), dimeric ss circular (2xss-c), dimeric ss linear (2xss-l), dimeric covalently closed circular (2ccc) and heterogenous ds (hds) DNA. YR and P are unknown intermediates. 40 A.K. Cheung / Virology 434 (2012) 38–42

Intermediate DNA forms representing different replication DNA forms detected in this sample were essentially identical to mechanisms (CSR, RCR, and RDR) similar to that observed with those observed with PCV2-infected PK15 cells. In this sample, the the maize streak virus (MSV) of the Geminiviridae were observed stalled intermediate P, the 2ccc DNA and the RDR intermediates (Erdmann et al., 2010). As such, the various PCV2 DNA forms were readily observed. In addition, the end product of RCR that detected were designated similarly. originated from the oc DNA displayed a discernible line that Genomic length ssDNA (1xss) that migrated farthest in the ended abruptly, which indicated the terminal point was an oc second dimension gel and away from the heterogenous hds DNA molecule linked with a monomeric length of ss DNA. arc was the most prominent species observed. Dimers of ssDNA (2xss) with resolution into circular (c) or linear (l) forms were Copy-release replicative intermediates from a PCV–HTT construct in clearly detected. A molecular species southeast of the 2xss l-form, E. coli designated P (present but not addressed in MSV), may represent a stalled intermediate. Linear ds (lin), open circular ds (oc) and A previously described chimeric PCV1/PCV2 HTT construct (pChi- topoisomers of covalently closed circular (ccc) DNAs migrated as 7Rep’) capable yielding supercoiled unit-length Q PCV genome distinct molecular species. Higher molecular weight species (cccQ) via the copy-release mechanism (Cheung, 2006b) was exam- (Z2ccc) displaying a triangular pattern and hds DNA were ined. Total DNAs isolated at mid-log and stationary growth phases present at the upper part of the Southern blot. were analyzed by 2-D/Southern blot (Fig. 2). The probe used The predominant DNA species observed was the ss template hybridizes to U- and Q- but not L-DNA species. With respect to Q, (1xss), which may be linear or circular. CSR starts with 1xss and DNA species representing 1xss, lin, oc and 1ccc DNA forms were the end products are linear ds (lin) or open-circular (oc) DNA. The observed. In the mid-log phase, 1xss was the major DNA species. In two lines connecting 1xss-lin and 1xss-oc represent different the stationary growth phase, oc and ccc were the major DNA species. proportion of ds DNA synthesis during progressive CSR on each The data suggested that most of the 1xss were converted ccc or oc by template. RCR originates from oc DNA and the intermediates CSR at the stationary growth phase. Interestingly, RCR intermediates protrude northwest as a straight line. The DNA forms indicated by that originate from the oc DNA species were not detected. YR, a straight line that runs parallel to RCR intermediates, have been described to being RCR intermediates nicked at a single site on the template strand (Erdmann et al., 2010). RDR intermediates starting at 2ccc were recognizable with detection of a faint 2ccc Discussion DNA species upon long exposure (outlined as a dotted comma). Employing the 2-D gel electrophoresis methodology estab- lished to study the replicative intermediates of plant gemini- Replicative intermediates of PCV1 in persistently-infected PK15 cells viruses, the DNA proﬁles of PCV during virus replication in animal PK15 cells and in copy-release of unit-length viral genome from a DNAs isolated from PCV1-persistently-infected PK15 cell HTT construct in E. coli were delineated. In PK15 cells, the (Fig. 2b) was analyzed by 2-D Southern blot. The various PCV1 replicative DNA proﬁles of PCV were strikingly similar to that of

Fig. 2. 2-D Southern blot analysis of (a) mid-log growth phase and (b) stationary phase E. coli transformed with pChi-7Rep’ HTT. U-DNA and Q-DNA were detected. The following Q DNA forms were identiﬁed: circular ss-genome (1xss), ds linear (lin), monomeric open circular (oc), and monomeric covalently closed circular (ccc) DNA. A.K. Cheung / Virology 434 (2012) 38–42 41

MSV (Erdmann et al., 2010). Various DNA forms and intermedi- Hybridization was carried out overnight at 421 C, washed twice in ates demonstrating that PCV undergoes CSR, RCR and RDR were 2 SSC and twice in 0.5 SSC at 601 C for 15 min each. readily recognized. Although RDR was detected, the presence of large amount of ss viral genome indicated that RCR is the predominant mechanism for PCV DNA replication. The number Acknowledgments of DNA forms and intermediates detected between the PCV freshly-infected and persistently-infected PK15 cells were essen- The author thanks Drs. M.E. Kehrli, Jr. and J.F. Ridpath for tially identical, however, there were differences among the critical reading of the manuscript, N. Otis for technical assistance, relative amounts of various DNA species. It is not clear whether and M. Marti and S. Ohlendorf for manuscript preparation. these differences were intrinsic to the virus genotype (PCV1 versus PCV2) or results of the length of virus infection (new References infection versus persistent infection). In the E. coli/HTT system, ss circular PCV genomes (1xss) were Alberter, B., Ali Rezaian, M., Jeske, H., 2005. Replicative intermediates of Tomato readily observed but RCR intermediates that originate from the oc leaf curl virus and its satellite DNAs. Virology 331 (2), 441–448. DNA species were not detected. Therefore, 1xss must have been Beach, N.M., Juhan, N.M., Cordoba, L., Meng, X.J., 2010a. Replacement of the derived from cccU directly via copy-release replication and not replication factors of porcine circovirus (PCV) type 2 with those of PCV type 1 greatly enhances viral replication in vitro. J. Virol. 84 (17), 8986–8989. from RCR of Q. For the generation of supercoiled Q, copy-release Beach, N.M., Ramamoorthy, S., Opriessnig, T., Wu, S.Q., Meng, X.J., 2010b. Novel replication generates the 1xss circular PCV genome (in which Rep chimeric porcine circovirus (PCV) with the capsid gene of the emerging PCV2b provided the nicking and joining function of the Ori sequence), subtype cloned in the genomic backbone of the non-pathogenic PCV1 is attenuated in vivo and induces protective and cross-protective immunity CSR converts 1xss to a ds relaxed molecule (using the yet to be against PCV2b and PCV2a subtypes in pigs. Vaccine 29 (2), 221–232. identified minus-genome primer), and subsequent supercoiling Cheung, A.K., 2003a. The essential and nonessential transcription units for viral by host enzymes to cccQ. Since Rep’ is not synthesized by Chi- protein synthesis and DNA replication of porcine circovirus type 2. Virology 313 (2), 452–459. 7Rep’ , whether initiation of RCR in E. coli requires contribution Cheung, A.K., 2003b. Transcriptional analysis of porcine circovirus type 2. 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