An ISRE sequence 5′-CTGAAAACGAAAGA-3′ which is precisely conserved among the different gene-types of PCV2 locates at nt 1737–1751 in the PCV2 Rep promoter, Previous study  found that when the PCV2 ISRE sequence present in the context of intact virus but not in isolation influences the interferon-mediated enhancement of PCV2 replication in vitro, they also found that mutating the ISRE sequence did not significantly affect viral replication in vitro, but our research demonstrated that PCV2 ISRE sequence is a functional motif in and out of the whole viral genome and it can affect the PCV2 replication in vitro and in vivo.
Porcine IFN-α could enhance the transcription of PCV2 ISRE reporter construct significantly. Even though the absolute lucifease expression of all reporter constructs include pGL3-promoter were significantly decreased in the presence of IFN-α treatment due to systemic inhibition effect of translation by IFN-α on host cell gene (data not shown). The relative lucifease activity (firefly luciferase/Renila luciferase) of the pGm-PCV2-ISRE (3) increased by two fold in the presence of 100 U/mL porcine IFN-α. The findings are somewhat different from the results by S. Ramamoorthy et al , they concluded IFN-α or IFN-γ treatments of 3D4/31 cells transfected with the reporter gene constructs carrying the wildtype ISRE sequence did not increase the reporter gene expression. It is possible that the cells, vectors and the rep promoter gene fragments we used were different. They used 3D4/31 cells and pGL3-basic vectors, whereas we used PK-15 cells and pGL-mini vectors, the gene fragments they used were relatively intact rep promoter, but what we used were only three copies of ISRE in this part.
The obvious functional experiment was to test whether Rep promoter can be activated upon IFN treatment. However, the full-length Rep promoter reporter construct did not respond to porcine IFN-α treatment significantly. This result might be due to negative regulatory elements in the promoter or the basal level of luciferase activity was high after transfection. For the transfection procedure itself activated interferon expression(data not shown). Interferon deficiency cell line suitable for porcine circovirus is now not available. In order to solve this problem, Rep promoter was further characterized.
Computer-assisted sequence analysis showed that PCV2 Rep promoter does not contain any recognizable TATA or CAAT elements. However, besides a sequence, spanning nt -80 to nt -67 that is homologous to the ISRE present in the promoter of ISGs, there are GC box like sequences at four different sites before Rep gene, which are known to implicate in transcription.
Interestingly, from the promoter activity assay using various promoter constructs, we found that promoter construct lacking GC box at −192 to −84 significantly reduced luciferase activity, suggesting that Rep expression might be partially controlled by a GC box binding protein such as transcription factor Sp-1 [18, 19]. Sp1 is a ubiquitous transcription factor which is responsible for activating many cellular and viral genes. Like PCV2 Rep, many eukaryotic genes with TATA-less promoters contain multiple Sp1-binding motifs arranged in tandem and in close proximity within the proximal promoter . Sp1 can activate many of these promoters synergistically by Sp1–Sp1 interactions and these interactions are an important mechanism for modulating the expression of this class of genes .
Further study indicated that Prep is composed of two independent mini promoters, the two mini promoters are both able to initiate the expression of luciferase gene and GFP gene in PK-15 cells (pictures not shown). With the proximal Rep promoter, mutation of the ISRE was shown to abrogate IFN-α mediated up-regulation of PCV2 proximal Rep promoter driven reporter gene expression. This suggested a crucial role of the ISRE in IFN-α induced enhancement transcription. Based on the responsiveness to IFN-α, and the viral ISRE sequence similarity to consensus ISRE, we concluded that Rep promoter has a functional ISRE sequence. With the distal Rep promoter, decreased luciferase transcription was observed in the presence porcine IFN-α treatment. There are three GC box like sequences which predicted to be SP-1 binding site in the computer-assisted scanning. Within the Sp-family transcription factors, Sp-1 and Sp-3 are ubiquitously expressed in mammalian cells. Sp-3 is structurally similar to Sp-1, with similar affinities for the Sp-1-binding site . Previous study suggested different roles for Sp-1 and Sp-3, with Sp-1 being involved in basal transcription and Sp-3 having a role in the interferon inducible transcription of the PKR gene . Whether GC box like sequence is the binding site of Sp-1 or Sp-3 and contribute to the regulation of Rep promoter has yet to be determined.
The luciferase transcription activity of the full-length Rep promoter is higher than the value of distal Rep promoter with proximal Rep promoter, so we conclude the two mini-promoters have a synergistic effect. However, luciferase transcription activity of the ISRE mutant full-length Rep promoter is even lower than the distal Rep promoter, indicating the PCV2 ISRE sequence plays an important role in the activity of Rep promoter.
PCV2 Rep gene promoter is known to support the synthesis of Rep and Rep’, Rep’ is truncated, and the C terminus is expressed in a different reading frame. Two products of the Rep gene are indispensable for PCV replication . The Rep-complex recognizes and binds the direct H tandem repeats and the right-arm of the palindrome destabilizes and unwinds the Ori sequence, and then nicks the octanucleotide sequence between T6 and A7 to generate a free 3′-OH end for initiation of plus-strand DNA replication . Besides its role in replication, the Rep protein is a transcriptional repressor of Rep gene expression. Previous study indicated that the full-length Rep protein could repression the Prep by binding to the two inner hexamers, H1 and H2, located in the origin of PCV1 . So the transcriptional activity of Prep is controlled by both viral and cellular factors. Since the Rep and Rep’ are essential for PCV DNA replication, their expression regulation is expected to affect progeny virus production.
The results of viral mutational analysis in vitro demonstrated that the ISRE is non-essential for virus replication, for the progeny PCV2mut with function abrogated ISRE was readily recovered from cultures transfected with the excised and recircularized double-stranded mutant genomes. However, PCV2mut decreased replication competence and lower sensitivity to IFN-α were observed demonstrated a significant role of ISRE in the viral replication efficiency and regulation of IFN-α-mediated PCV2 replication on PK-15 cells.
The data of animal experiments showed that the mutation of PCV2 ISRE sequence not only affect the replication efficiency in PK-15 cells, but also had negative effects in vivo, the ISRE mutant PCV2 infection elicited a lower antibody response and replication efficiency than the wild type PCV2 infection. Therefore, we conclude that the PCV2 ISRE is an important motif in the viral transcription start in vivo and in vitro.
The interaction of viral ISRE located in the Rep promoter of PCV2 with host cell transcription factor regulated by IFN may be anther underlying mechanism for enhanced virus production. Previous study has reported that up-regulation of PCV2 in PK-15 cells by IFN-γ is due to enhanced internalizing of viral particles . Besides ISGF3, IRFs are another important factor induced by type I and II IFNs. Since the sequence of IRF binding site termed IRF-E overlaps with ISRE, IRF also binds to ISRE sequence and activates the interferon-inducible gene transcription, but in part, functionally different . So which transcription factor binds the PCV2 ISRE? Further research should clarify this issue.