Rolling-Circle Replication of an Animal Circovirus Genome in a Theta-Replicating Bacterial Plasmid in Escherichia coli

Porcine circovirus (PCV) is a member of the genus Circovirus of the Circoviridae family, which includes a group of diverse animal viruses with small, closed-circular, single-stranded (ss) DNA that replicate their genomes through double-stranded (ds) intermediates (28, 34). The animal circoviruses are closely related to the plant circoviruses, now renamed nanoviruses (27, 29). Nucleotide sequence analysis showed that the PCV genome is intermediate between the genomes of plant geminivirus and nanovirus (30). Based on analysis of replication initiator protein (Rep) sequences, it has also been suggested that the PCV genome was the result of a recombination event between a nanovirus and an animal picorna-like RNA virus (13). Two genotypes of PCV have been identified, PCV type 1 (PCV1) and PCV2, and their genomes share 68 to 76% sequence homology. PCV has an ambisense circular genome that encodes proteins both by the encapsidated viral DNA and by the minus-strand genome of the replication intermediate synthesized in the host. Two coding regions of opposite polarity, the Rep protein on the right and the capsid protein (Cap) on the left, are separated at their 5′ ends by the origin of DNA replication (Ori) intergenic region (IR) of approximately 80 nucleotides (Fig. ​(Fig.1).1). Sequence and structural motif similarities suggest that PCV replicates via a rolling-circle replication (RCR) mechanism in a manner similar to the Geminiviridae family (see reviews in references 14, 16, and 32) with modifications at the Ori proposed by the RCR “melting pot” model (3, 4), which is consistent with the four-stranded DNA structures detected among short inverted repeat sequences when examined by electron and atomic force microscopy (20, 21, 31). These similarities include the following: (i) a Rep protein that contains three conserved RCR motifs (RCR-I, -II, and -III) and an nucleoside triphosphate-binding (P-loop) core homologous to the Rep proteins of other prokaryotic and eukaryotic RCR systems (18, 23) and (ii) the Ori-IR of PCV and geminivirus contain a similar nonanucleotide sequence (TAGTATTAC for PCV1, AAGTATTAC for PCV2, and TAATATT↓AC for geminivirus [the ↓ indicates a nick site]) flanked by a pair of inverted repeat (palindromic) sequences capable of forming a stem-loop structure during DNA replication. It has been demonstrated that the nonanucleotide of geminivirus is cleaved at the indicated nick site by the virus-encoded Rep to initiate plus-strand DNA replication (15, 17, 22, 23). FIG. 1. Schematic representation of the origins of DNA replication of PCV1 and PCV2. (A) Transcription patterns of the major PCV1 RNAs (1). Capsid RNA (CR) is transcribed leftward. Rep and Rep′ are transcribed rightward. The RNAs are annotated with nucleotide ... The minimal Ori of PCV1 has been mapped to a 111-bp fragment (24) which includes the 83-nucleotide (nt) Ori-IR (Fig. ​(Fig.1).1). The current model for PCV DNA replication postulates that the closed-circular ssDNA genome is first converted to a superhelical dsDNA replication intermediate. Interestingly, replication of geminivirus or nanovirus DNA in plant cells requires just one multifunctional initiator Rep protein, while replication of PCV DNA in mammalian cells requires two virus-encoded proteins, Rep and Rep′ (the Rep complex) (2, 5, 25). The Rep′ RNA is derived from the Rep RNA via internal splicing. The spliced Rep′ RNA codes for a protein identical to Rep in the amino portion but different in the carboxyl portion because splicing changes the translational frame. The Rep complex recognizes and binds the direct hexanucleotide (H) tandem repeats and the right arm of the stem-loop structure (38), destabilizes and unwinds the Ori sequence, and then nicks the octanucleotide A1G2T3A4T5T6↓A7C8 (designated Oc8, or O nucleotides) (6, 7) within the nonanucleotide sequence between T6 and A7 to generate a free 3′-OH end for initiation of plus-strand DNA replication. It has been demonstrated that the Oc8 motif sequence (A1x2T3A4x5T6↓A7C8) and the hexanucleotide tandem repeat (H1/H2) abutting the stem-loop structure are essential for PCV DNA replication (6-9). After nicking Oc8, the Rep complex is attached to the 5′ end of the displaced genomic strand via the conserved tyrosine residue of motif RCR-III. It is not clear whether Rep or Rep′ mediates the nicking function or which protein is attached to the displaced DNA. The Rep complex is not expected to possess any DNA polymerase activity and, therefore, the actual polymerization of the nascent viral genome is carried out by cellular enzymes. Upon completion of the first round of DNA synthesis, the Rep complex cleaves Oc8 between the nascent and original DNA. Concomitantly, the Rep complex mediates joining the ends of the displaced genome to reconstitute the original Oc8 and release the Rep complex and a ss viral genome. Previous work with geminiviruses showed that plasmid constructs containing greater-than-unit-length viral DNA accumulate viral replicative-form DNAs in bacteria indistinguishable from those produced in infected plants (35, 37), and accumulation of the viral DNA species depends on the presence of two Oris in the plasmid constructs. In this study, we examined two constructs, pChi-6 and pChi-7 (Fig. ​(Fig.2),2), each consisting of 1.75 copies of PCV1/PCV2 DNA with two PCV Oris (specific for the RCR mechanism) inserted into the pBluescript SK(+) (pSK+) bacterial plasmid containing the colicin E1 (ColE1) Ori (specific for a unidirectional theta-replication mechanism) (19, 40). PCV1/PCV2 heterologous tandem constructs were chosen because the components essential for PCV1 or PCV2 DNA replication (the Ori-IR and the Rep complex) are interchangeable (6, 26) and the two viral genomes are different enough to be easily discernible. The results demonstrated that three distinct DNA species of different molecular sizes were generated from each engineered construct utilizing two different replication mechanisms in Escherichia coli. In comparison to the homologous geminivirus systems used in previous studies (35, 37), the heterologous tandem constructs employed in this work allowed easier identification of the mechanism for generation of the novel DNA species. FIG. 2. (a) Schematic representation of pChi-6, pChi-7, and the U, L, and Q DNA species. (b) Locations of oligonucleotides. See Materials and Methods for construction of pChi-6 and pChi-7. PCV1 DNA is indicated in black, PCV2 DNA is shaded, and pSK+ DNA ...