Rep and Rep′ protein of Porcine circovirus type 1 bind to the origin of replication in vitro

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. (C) 2001 Elsevier Science.