Detection of Template Strand Switching during Initiation and Termination of DNA Replication of Porcine Circovirus

Abstract

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated invertedrepeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

Palindrome Regeneration by Template Strand-Switching Mechanism at the Origin of DNA Replication of Porcine Circovirus via the Rolling-Circle Melting-Pot Replication Model

“…Although some of the progeny viruses recovered from the right-arm deletion genomes could be accounted for by sequence duplication caused by slippage of the replication machineries (vRD3.3, vRD3.4, vRD3.5, and vDel3) (10), template strand switching during initiation of DNA replication was evident in vRD1, vRD2a, vRD2b, vRD3.1, vRD3.2, and vRD10. These results prominently display the inverted repeat correction mechanism inherent in the rollingcircle melting-pot replication model (4). The LD7 and LD11 progeny viruses provided the best examples to support this model by exhibiting template strand switching in the middle of synthesizing the left arm and unequivocally demonstrated that both the minus genome and the newly synthesized strand a N were available simultaneously to serve as templates during termination of DNA replication.…”

“…Mutant genomes with deletions of three or fewer nucleotides (RD1, RD2a, RD2b, RD3, and Del3) yielded progeny viruses consistently, while a seven-nucleotide-deletion genome (RD7) did not give any progeny virus, and a 10-nucleotide-deletion genome (RD10) yielded infectious viruses only occasionally. In comparison with a previous study (4), nucleotide substitutions engineered into the right arm, left arm, or both arms of the palindrome did not affect protein synthesis or progeny virus production. Therefore, the types of mutations and their specific locations at the origin have different effects on PCV protein synthesis, self-DNA replication, and progeny virus regeneration.…”

“…Previous work (4) showed that the C nucleotides at positions 3 and 10 (designated C 3 and C 10 ) are critical for PCV1 progeny virus regeneration. The following experiments were aimed at deleting C 3 or disrupting the spatial distance between C 3 and C 10 .…”

“…However, in vivo analysis demonstrated that Rep may interact with the palindromic stem only via the C nucleotide at positions 3 and 10 (4). The presence of the "conserved" octanucleotide flanked by inverted repeats at the origin and similarities among the Rep proteins essential for virus replication suggest that PCV DNA may replicate via the rolling-circle "cruciform" model proposed for the Mastrevirus genus of the Geminiviridae family (18, 34) with modifications specified by the recently proposed rolling-circle "melting-pot" model (4).…”

“…Therefore, two DNA strands are available as templates via template strand switching (i.e., from the minus genome to the corresponding palindromic strand) during initiation as well as termination of DNA replication. For this reason, the rolling-circle melting-pot replication model permits the use of either the left-arm or right-arm inverted sequence to regenerate the wild-type palindrome (inverted repeat correction, previously referred as gene correction) (38) or to adopt a mutilated sequence to form a new palindrome (inverted repeat conversion) in the progeny virus (4).…”