New reporter gene-based replication assay reveals exchangeability of replication factors of porcine circovirus types 1 and 2

Abstract

Two types of porcine circovirus (PCV), which differ in their pathogenicity, are known. PCV type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome in swine, while PCV1 has not yet been linked to a disease. Corroborating earlier observations in PCV1, transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep' and that the simultaneous expression of Rep and Rep' proteins is essential for initiation of replication of PCV2. The interchangeability of the replication factors of PCV1 and PCV2 was examined. The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa. To investigate the competence of the Rep/Rep' proteins to initiate replication at the heterologous origin, a new replication assay was developed. It measures the expression of a luc reporter gene present on a plasmid carrying the origin of the investigated replicon. Replication is initiated by expression of the appendant replicase from a second plasmid and results in replication of the origin plasmid coupled with an increase in the Luc activity. Using this method to compare replication of PCV1 and PCV2 in cell culture, it was shown that the Rep/Rep' protein of PCV2 initiated replication at the origin of PCV1, as did the reciprocal combination. Our results indicate that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable.

FIG. 1.

Map of PCV1 and PCV2. A linear map of the circular genomes of PCV1 and PCV2 is shown. The origin of replication is located between the divergently transcribed cap and rep gene (gray shaded boxes). The origin is enlarged for both viruses and shows the characteristic stem-loop element and the adjacent 6-bp and 5-bp repeats (open boxes and ovals). Sequence deviations between the origins of PCV1 and PCV2 are indicated by bold letters, the minimal binding sites of the Rep and Rep′ protein in PCV1 is marked by black arrows. A stippled line indicates the rep and rep′ transcript and the position of the splice junction in PCV1 and PCV2 (bold).

FIG. 2.

Correlation between copy number of the origin and replicase plasmids with the Luc/Gal activity. To verify whether the Luc/Gal assay can be used to quantify replication rate of a particular plasmid, a variation of the copy number of the origin plasmid pRL16 (A) and the replicase-expressing plasmid pORF4 (B) was performed. (A) Increasing amounts of DNA of the plasmid pRL16 were supplemented to 175 ng with vector DNA pGL3 promoter and cotransfected with 50 ng of pORF4A and 25 ng of pRSV-βGal into PK-15 cells. A Luc/Gal assay was performed. (B) A Luc/Gal assay was performed using increasing amounts of DNA of the plasmid pORF4A were supplemented to 175 ng with vector DNA pSVL, cotransfected with 50 ng of pRL16 plus 25 ng of pRSV-βGal into PK-15 cells. SLU, standardized Luc units; error bars, standard error of the mean.

FIG. 3.

Analysis of rep gene products in PCV2-infected cells. (A) The result of an RT-PCR is shown. RNA was isolated from mock- or PCV2-infected cells and copied into cDNA. The cDNA was amplified with the primer pair F410/B411 hybridizing up- and downstream of the putative splice site of the rep transcript (lanes 2 and 3). The primer pair F412/B413 binds downstream of the splice donor and only the full-length rep transcript can be amplified (lanes 4 and 5). A sketch of the two differentially spliced rep transcripts is given in Fig. 1. (B) Expression of Rep and Rep′ in vitro. The ORF for Rep (lane 1) and Rep′ (lane 2) were cloned into plasmid pGEM3Zf(+) and expressed with the TNT wheat germ extract system using [³⁵S]methionine. Mark 12 wide-range protein standard (showing molecular mass in kilodaltons [lane M]) was stained with amido black.

FIG. 4.

Expression of Rep and Rep′ proteins in PCV-infected cells. PCV-infected PK-15 cells were probed with antisera raised against the C-terminal regions of Rep and Rep′ of PCV1. (A) Mock-infected PK-15 cells/α-Rep′(120-168); (B) mock/α-Rep(120-312)PCV1; (C) PCV1-infected PK-15 cells/α-Rep′(120-168); (D) PCV1/α-Rep(120-312)PCV1; (E) PCV2-infected PK-15 cells/α-Rep′(120-168); (F) PCV1/α-Rep(120-312)PCV1.

FIG. 5.

Replication of PCV2. A Luc/Gal replication assay was performed in which the origin of PCV2 (plasmid pRL16.2) was combined with plasmids expressing the Rep and or the Rep′ protein. Plasmid pGL3b in column 1 contains a promoter-less luc gene and represents endogenous Luc activity. Plasmid pGL3p was cotransfected with pSVL to serve as an indicator for basal Luc expression in a nonreplicated system (column 2). Influence of the origin fragment exerted upon Luc activity was tested by combination of pRL16.2 with pSVL (column 3). To test reciprocally the influence of Rep and Rep′ on expression of Luc, plasmids pGL3p was cotransfected with pSVL-rep(PCV2) (column 4). The origin of replication of PCV2 is combined with the rep gene of PCV2 expressing Rep and Rep′ in column 5, pRL16.2+pSVL-rep(PCV2). The rep* gene (column 6) is encoded in plasmid pSVL-rep*(PCV2) and contains an engineered version of rep expressing only the Rep protein. pSVL-rep′(PCV2) leads to the production of Rep′ (column 7). For reconstitution of the replication activity (column 8), the proteins are supplied separately from plasmids pSVL-rep*(PCV2) and pSVL-rep′(PCV2). SLU, standardized Luc units; error bars, standard error of the mean.

FIG. 6.

Replication proteins of PCV1 and PCV2 bind to the heterologous origin in vitro. FLAG-tagged Rep and Rep′ proteins of PCV1 (lanes 1 to 7 and 22 to 28) and PCV2 (lanes 8 to 21) were expressed in vitro and incubated with the ds EMSA substrates F229/B265 comprising the origin of PCV1 (lanes 1 to 14) or with F462/B463 carrying the origin of PCV2 (lanes 15 to 28). The fragments carry the putative stem-loop element plus the adjacently located hexamer and pentamer repeats. In lanes 1, 8, 15, and 22, an unprogrammed extract has been used as a negative control (u). In lanes marked with a +, the specificity of the binding reaction has been examined by induction of a supershift with the α-FLAG antibody. The letter R points out the use of the Rep protein, R′ indicates application of the Rep′ protein, while the mixture of Rep and Rep′ is given by R+R′. UO designates the unbound oligonucleotides; the band shifts are marked by BSI, BSII, and BSIII; and the supershift is indicated by SS.

FIG. 7.

Replication factors of PCV1 and PCV2 can be exchanged. A Luc/Gal replication assay was performed in which the origin of PCV1 was replicated by its cognate replicase proteins or by the heterologous rep gene products of PCV2 and vice versa. Plasmid pGL3b in column 1 contains a promoterless luc gene and serves as a negative control. pRL16 carries the origin of PCV1 and is combined with the vector pSVL (column 2). This experiment indicates the Luc activity of plasmid pRL16 without replication. The replication activity of ori(PCV1) is measured with cotransfected plasmids pORF4 expressing the rep gene of PCV1 (column 3) or pSVL-rep(PCV2) (column 4). The reciprocal approach with pRL16.2, carrying the origin of PCV2, is shown in the next three experiments. Column 5 indicates the Luc activity of pRL16.2 without supplementation of a rep gene. Combination of the origin of PCV2 with its rep gene is shown in column 6, while the cotransfection of ori(PCV2) with rep(PCV1) is given in column 7. SLU, standardized Luc units; error bars, standard error of the mean.