Rep and Rep′ Protein of Porcine circovirus Type 1 Bind to the Origin of Replication in Vitro

Abstract

Genome replication of Porcine circovirus type 1 (PCV1) relies upon expression of the full-length protein Rep and a spliced isoform (Rep'), and the presence of a 111-bp genomic fragment comprising the origin of replication. Using an electrophoretic mobility shift assay (EMSA), the capability of both Rep proteins to bind to partial fragments of the origin of replication of PCV1 was investigated in vitro. Both proteins formed complexes with double-stranded DNA origin fragments containing a stem-loop structure with a conserved nonamer and four hexamer repeats (5'-CGGCAG; H1 to H4). Use of truncated EMSA substrates identified minimal binding sites (MBS) for Rep and Rep' protein: The Rep binding site was mapped to the right leg of the stem-loop and the two inner hexamer repeats H1/H2, while binding of Rep' required only the presence of two hexamer repeats. Two differentially retarded complexes were observed with Rep protein, which presumably result from alternative binding to the MBS or to H3/4.

Functional Analysis of cis - and trans -Acting Replication Factors of Porcine Circovirus Type 1

“…The G 176 KS sequence is conserved in proteins mediating ATP binding and hydrolysis (28 , lanes 2, 3, 4, and 5, respectively) were expressed in E. coli and subsequently purified by affinity chromatography using Ninitrilotriacetic acid agarose beads. The GST fusion proteins GST-Rep, GST-RepmutII, and GST-RepmutP (B, lanes 1, 2, and 3, respectively) were purified using glutathione-Sepharose after expression in E. coli.…”

“…A putative helicase activity of the PCV Rep protein associated with ATP hydrolysis could lead to a further unwinding of DNA strands as a prerequisite for DNA synthesis. This hypothesis is endorsed by the identification of a putative helicase domain within the C terminus of the Rep proteins of PCV1 and PCV2 (12), similar to other viral helicases (28).…”

“…The inverted repeat and the nonamer are essential for cleavage and ligation of origin fragments in vitro (27), and cruciform extrusion from the double-stranded replicative form is assumed to provide the single-stranded DNA (ssDNA) conformation necessary for cleavage in vivo. Hexamer repeats (5Ј-CGG/TCAG-3Ј) are found adjacent to the stem-loop as a part of the minimal binding site (MBS) responsible for the recruitment of the replication proteins Rep and RepЈ (28). Similar features are also found in the Geminiviridae and Nanoviridae, ssDNA plant viruses replicating via RCR.…”

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

“…In vitro experiments showed that PCV1 Rep binds to the right arm of the presumed stem-loop, while both Rep and RepЈ bind to two adjacent, almost perfect 6-nucleotide (CGGCAG or CGT CAG) tandem direct repeats located at nucleotide 13,19,30, and 36 ( Fig. 1A) (39). However, in vivo analysis demonstrated that Rep may interact with the palindromic stem only via the C nucleotide at positions 3 and 10 (4).…”

New Reporter Gene-Based Replication Assay Reveals Exchangeability of Replication Factors of Porcine Circovirus Types 1 and 2

“…A deoxynucleoside triphosphate-binding domain has been identified in the Rep protein, but not in RepЈ (9). The rep gene products of PCV1 have been reported to bind in vitro to double-stranded DNA (ds DNA) fragments carrying the origin of replication (18). Expression of both proteins is essential for initiation of PCV1 replication (9).…”