Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1

Abstract

The replication proteins Rep and Rep' of porcine circovirus type 1 (PCV1) are both capable of introducing and resealing strand discontinuities at the viral origin of DNA replication in vitro underlying genome amplification by rolling-circle replication. The PCV1 origin of replication encompasses the minimal binding site (MBS) of the Rep and Rep' proteins and an inverted repeat with the potential to form a stem-loop. In this study, both elements of the PCV1 origin were demonstrated to be essential for viral replication in transfected cells. Furthermore, investigation of conserved amino acid motifs within Rep and Rep' proteins revealed that the mutation of motifs I, II, and III and of the GKS box interfered with viral replication. In vitro studies demonstrated that motifs I to III were essential for origin cleavage, while the GKS box was dispensable for the initiation of viral replication. A covalent link between Rep/Rep' and the DNA after origin cleavage was demonstrated, providing a mechanism for energy conservation for the termination of replication.

FIG. 1.

(A) A linear map of the circular genome of PCV1 is depicted. ORFs are indicated by open bars, and transcripts of Rep and Rep′ with splice sites are indicated by horizontal arrows. Positions and sequences of conserved amino acid motifs I to III and the GKS box are given. (B) Conserved sequence elements within the origin of PCV1 are shown at the top of the figure. Oligonucleotides used for in vitro studies and origin plasmids tested for replication in the reporter gene assay are indicated below. Only sequences deviating from the wt origin sequence are denoted.

FIG. 2.

Purified PCV1 Rep and Rep′ recombinant fusion proteins. His-tagged proteins His-Rep, His-Rep′, His-RepmutY93, and His-Rep′mutI (A, lanes 2, 3, 4, and 5, respectively) were expressed in E. coli and subsequently purified by affinity chromatography using Ni-nitrilotriacetic 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. Rebuffered and concentrated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by staining with Coomassie brilliant blue dye. The apparent molecular mass was determined by comparison with a protein standard with the indicated molecular masses (A, lane 1; B lane 4).

FIG. 3.

Impact of conserved amino acid motifs within PCV1 Rep/Rep′ on origin cleavage. Purified fusion protein (500 ng) was incubated with 0.5 pmol oligonucleotide in the cleavage reaction in vitro. Cy5-labeled oligonucleotide F301 representing the conserved sequences of the PCV1 origin of replication was incubated with His-Rep/Rep′ (lanes 2 and 3), His-RepmutY93 (lane 4), His-Rep′mutI (lane 5), GST-Rep (lane 7), GST-RepmutII (lane 9), or GST-RepmutP (lane 10). As a negative control (n.c.), purified GST protein was used (lane 8). Samples were resolved on native polyacrylamide gels and compared to oligonucleotides of defined sizes (lanes 1 and 6). Positions and sizes of oligonucleotides are marked, and cleavage products are highlighted by arrowheads.

FIG. 4.

PCV1 Rep is covalently attached to the DNA after cleavage. The purified His-Rep fusion protein (500 ng) was incubated with 1 (lane 1 and 6), 10 (lane 2 and 7), 50 (lane 3 and 8), or 100 (lane 4 and 9) pmol of oligonucleotide F1165 comprising sequence alterations upstream of the inverted repeat and labeled with biotin at the 3′ end in the cleavage reaction in vitro. As a control, purified His-Rep was incubated without an oligonucleotide under the same conditions (lane 5). Samples were resolved on denaturing 10% polyacrylamide gels after the addition of SDS loading buffer and heating to 95°C for 5 min. Immunoblots were detected with anti-biotin antibody (lanes 1 to 4) or anti-His tag antibody (lanes 5 to 9). The molecular masses of free substrate, free protein, and the protein-DNA complex were determined by comparison with a protein standard.

FIG. 5.

Impact of conserved amino acid motifs within PCV1 Rep/Rep′ on viral replication. Replication proteins Rep and Rep′ mutated in the conserved amino acid motifs I, II, and III or the GKS box were tested for replication activity. Plasmids encoding the recombinant protein were cotransfected with pRL16 carrying the wt PCV1 origin of replication (bars 4, 5, 6, and 10) and compared to the replication activity of wt Rep/Rep′ (pORF4A and pTriEx6HN-rep [bars 3 and 9]). In the negative controls, the replication activity of wt Rep/Rep′ was determined under repression of replication (pGL3-p [bars 2 and 8]), as was the replication activity of pRL16 in the absence of trans-active replication factors Rep and Rep′ (pSVL-SV40 and pTriEx6HN [bars 1 and 7]).

FIG. 6.

ATPase assay. PCV1 replication proteins His-Rep and His-Rep′ (bars 2, 3, and 4), and mutated PCV1 proteins GST-RepmutII, His-RepmutY93, and GST-RepmutP (bars 5, 6, and 7, respectively) were incubated in the presence of [γ-³²P]ATP in vitro. After precipitation of residual [γ-³²P]ATP, ATPase activity was determined by measuring radioactive free phosphate in the supernatant and compared with that of the negative control (n.c.). Unless otherwise noted, the reaction mixture was supplemented with Mg²⁺.

FIG. 7.

Conserved origin sequences exert different impacts on viral replication. Plasmids carrying wt or mutated origin fragments were tested for replication in PK15 cells in the presence or absence of the replication proteins Rep and Rep′. The replication activity of wt (pRL16) or mutated origin plasmids (pRL16 and pRL17 derivatives) was determined by cotransfection with pORF4A (PCV1 Rep/Rep′) and compared to the replication rate of the same replicon in the absence of replication proteins (pSVL-SV40) (A, bars 2 to 15; B, bars 2 to 9). Plasmid pGL3-p encompasses the luc gene but misses the cis-active origin and represents PCV1 Rep/Rep′ replication activity under the repression of replication (A and B, bar 1).