Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli - PubMed (original) (raw)
Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli
Andrew K Cheung. J Virol. 2006 Sep.
Abstract
A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.
Figures
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 coordinates that indicate the last nucleotide of each respective exon. The coding sequence of each transcript is shaded, and the nucleotide coordinates are indicated below each RNA. Locations of the PCV1 Rep mutation (nt 727A) and the PCV1 _Rep_′ mutation (nt 812C) are indicated. (B) Schematic representation of the PCV1 and PCV2 plus-strand Ori, indicating potential base-pairing of the flanking palindrome. The genomic sequences of PCV1 (1,759 nt) and PCV2 (1,768 nt) with respect to the presumed nick site (AGTATT↓AC) present in the octanucleotide (O-nt) and the D-nt of the loop are in bold letters and enclosed in boxes. The nucleotide coordinates 1, 2, 3, etc. are based on the actual genomic sequence, and the nucleotide coordinates 3′, 4′, 5′, etc. are arbitrarily assigned to show nucleotide complementarity of the flanking palindromic sequences. The Ori nucleotide sequences to the left of the nick site of PCV1 and PCV2 are designated Ori-1L and Ori-2L, and sequences to the right of the nick site are labeled Ori-1R and Ori-2R. The 6-nt tandem H repeat sequence (CGGCAG, CGGCTG, and CAGCAG) and 5-nt common sequence (CACCT) between PCV1 and PCV2 are enclosed in square and oval boxes, respectively. Nucleotide differences between PCV1 and PCV2 are shaded, and the initiation codons for Rep and CR are in black boxes.
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 is in the open box. The restriction enzyme sites relevant to this study are denoted. The Xho1 site is present only in the pSK+ sequence, and the Stu1 site is present in both the PCV1 and PCV2 genomes. The BamH1 and Sph1 sites were engineered into the constructs for cloning purposes.
FIG. 3.
Agarose gel electrophoresis of plasmid DNA recovered from E. coli TOP10 cells transformed with pChi-6 and pChi-7 (a) and plasmid DNA recovered from various E. coli strains transformed with pChi-6 (b). The predominant closed-circular ds molecules are labeled U, L, and Q.
FIG. 4.
Restriction enzyme digestion and Southern blot analysis of pChi-6. (a) Agarose gel stained with ethidium bromide (Et-Br) and blots hybridized with nick-translated probes pSK+ or 5′-end-labeled probes (c1703F and c887R) are indicated on the top of each panel. Uncut and restriction enzyme cut DNA is indicated on the top of each lane. (b) Total bacterial or plasmid DNA was hybridized with the nick-translated JEco probe. The closed-circular ds molecules are labeled U, L, and Q, and the linearized DNA species are indicated with the prefix lin-.
FIG. 5.
The role of Rep, Rep′, and Cap in the generation of L and Q from U. (a) Mutational analysis of Rep and Rep′ of pChi-7 in E. coli. (c) Mutational analysis of Rep and Rep′ of pChi-7 in PK15 cells. (d) Mutational analysis of the Cap gene of pChi-6 in E. coli. The DNA constructs used in each experiment are indicated on top of each lane.
FIG. 6.
Mutational analysis of selected _cis_-acting elements essential for L and Q generation from U. (a) Mutation of the Oc8 motif. The position of each mutation is indicated at the top of each lane. (b) Mutation of the H1/H2 tandem repeat of PCV1, PCV2, or both.
FIG. 7.
Model for the generation of Q and L from pChi constructs (or U) by the RCR copy-release mechanism (adapted from reference 35). Expression of the PCV Rep genes in E. coli results in the production of active Rep protein (▪) that nicks the Oc8 nucleotide within the Ori and attaches itself to the 5′ end of the nicked DNA covalently. Extension of the 3′ end of the nicked DNA by host polymerase (▴) results in displacement of the plus-strand DNA. When the plus-strand DNA containing the second Ori is displaced, Rep nicks the second Oc8 sequence while simultaneously ligating the ends of the ss molecule to reconstitute a new Oc8 sequence. For the production of Q that contains the composite Ori-2R/1L, Rep initiates DNA replication at the PCV1 Ori and terminates at the PCV2 Ori. Production of the ds replication intermediate by host enzymes may involve an unidentified minus-strand Ori and an initiation primer. For the production of L that contains the composite Ori-1L/2R, Rep initiates DNA replication at the PCV2 Ori and terminates at the PCV1 Ori. The newly synthesized DNA is dotted, and the displaced strand is black.
References
- Cheung, A. K. 2003. Comparative analysis of the transcriptional patterns of pathogenic and non-pathogenic circoviruses. Virology 310:41-49. - PubMed
- Cheung, A. K. 2003. The essential and non-essential transcription units for viral protein synthesis and DNA replication of porcine circovirus type 2. Virology 313:452-459. - PubMed
- Cheung, A. K. 2004. Identification of the essential and non-essential transcription units for protein synthesis, DNA replication and infectious virus production of porcine circovirus type 1. Arch. Virol. 149:975-988. - PubMed
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