Homologous recombination plays minor role in excision of unit-length viral genomes from head-to-tail direct tandem repeats of porcine circovirus during DNA replication in Escherichia coli

Abstract

In this report, we confirmed previous work that a theta-replicating bacterial plasmid containing 1.75 copies of genomic porcine circovirus (PCV) DNA in head-to-tail tandem (HTT) [a partial copy of PCV type 1 (PCV1), a complete copy of PCV type 2 (PCV2) and two origins of DNA replication (Ori)] yielded three different double-stranded DNA species when transformed into Escherichia coli: the input construct (U), the unit-length PCV1/PCV2 genome with a composite Ori lacking the plasmid vector (Q(RC)) and the remaining left-over 0.75 copy PCV1/PCV2 genome with a different composite Ori inserted in the plasmid vector (L(RC)). Replication of U was presumably via the theta-like replication mechanism utilizing the colicin E1 Ori, while derivation of L(RC) and Q(RC) was via the rolling-circle replication (RCR) copy-release mechanism and required the presence of two PCV Oris and the virus-encoded Rep protein. Essentially, excision of a unit-length PCV1/PCV2 genome (Q(RC)) via RCR from U yielded L(RC). Furthermore, we examined whether homologous recombination may also result in excision of a different type of unit-length PCV genome (Q(H)) from identical HTT constructs to generate L(H). Whereas the generation of L(RC) is Rep-protein-dependent, the generation of L(H) is Rep-protein-independent. Accordingly, the L(RC) and Q(RC) molecules derived from RCR possess different characteristics from the L(H) and Q(H) molecules generated via homologous recombination. In one of the studies in which both L(RC) and L(H) were generated simultaneously from the same HTT construct, out of 69 samples analyzed, 66 were derived via RCR and 3 were derived via homologous recombination. Thus, in comparison with RCR, homologous recombination plays a minor role in the excision of unit-length PCV genomes from HTT constructs in Escherichia coli.

Recombination in Eukaryotic Single Stranded DNA Viruses

“…Recombination experiments in geminiviruses [192] and circoviruses [193], have shown that this genome site is a mechanistically predisposed recombination hot-spot because of the replicational release of viral genomes from genomic concatomers ( Figure 3). When these concatomers either arise following a copy-choice mediated polymerase strand switch, or break and are rescued by host double stranded break repair pathways (i.e., by recombination dependent replication [78]), recombinants that are replicationally released will have one breakpoint at the site of the strand-switch/breakage and another at the v-ori (see Section 2.2 above).…”

Molecular study of porcine circovirus type 2 circulating in Portugal

“…We also report the presence of genotype PCV2a strains from 2007 onward, in a confined area. The recent reappearance of genotype PCV2a strains have been described in other countries such as Korea (Kim et al, 2009), USA (Cheung, 2007) and China (GQ359003) and may represent a shift back of genotype PCV2b to genotype PCV2a.…”

Coreplication of the Major Genotype Group Members of Porcine Circovirus Type 2 as a Prerequisite to Coevolution May Explain the Variable Disease Manifestations

“…Furthermore, several studies presented sequence evidence for PCV2 genotype group members' recombination (18,21,26). Recombination events were observed by amplicate sequencing analysis in both major PCV2 ORFs (5,32). One may surmise that the contribution of recombination to PCV2 genomic evolution may be the critical factor in initiating PMWS in an animal's immune system already weakened by lymphopenia caused by PCV2.…”