The Effect Of Response Elements In HHV-8 ORF57 Promoter On RTA Transactivation And IRF-7 Repression Of Gene Expression

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of Kaposi's sarcoma, the most common neoplasm in AIDS patients. Like other herpesviruses, HHV-8 goes through lytic and latent phases of infection, and the switch from latency to lytic replication is governed by the viral replication and transcription activator (RTA). RTA, encoded by HHV-8 open reading frame 50 (ORF50) gene, can activate promoters of a number of downstream viral genes through direct binding to RTA responsive element (RRE), these downstream genes include PAN, K2, K9, K14, ORF37, ORF52, ORF56 and ORF57. ORF57 encodes a viral early nuclear protein named mRNA transcript accumulation protein (MTA), which controls the switch from the early stage to the late stage of viral lytic replication.A 40 bp region encompassing nt 81904 to 81943 in the ORF57 promoter has been identified to be necessary for RTA transactivation. Two AT-rich RREs were found within this region, namely RRE1 and RRE2. RRE1 shares homology with the promoter sequence of viral K8 gene; whereas RRE2 binds not only RTA, but also interferon regulatory factor7 (IRF-7), a key regulator of type-I interferon-dependent innate immune response in host cells. Moreover, the competition between RTA and IRF-7 for binding with RRE2 affects RTA transactivation and modulates viral activation. Between RRE1 and RRE2, a 7 bp recognition site for recombination signal-binding protein RBP-Jκhas been identified, which could be important to mediate the interaction between RTA and RRE.To characterize the response elements within the 40 bp region in the ORF57 promoter that are responsive to both RTA transactivation and IRF-7 repression, and to search for novel RRE outside this region, ORF57 promoter fragments were amplified using forward primers 1-5 and reverse primer 6 or 7 and then inserted into the pGL3-basic vector to generate deletion reporters S1-S5 and L1-L5. Reporters S1-S5 contained promoter regions spanning from nt 81556,81904,81918,81930,81944, respectively, to the first transcription start site (nt 82003), and reporters L1-L5 contained the promoter regions extending to the second transcription start site (nt 82081). Reporters C1-C8 were also constructed to investigate the effect of the number of and the distance between response elements on RTA transactivation and IRF-7 suppression. 293T cells were co-transfected with each of the S1-S5 ORF57 promoter-derived luciferase reporters, with or without RTA and IRF-7 expression plasmid, and luciferase activity was measured. Our results demonstrated that the RRE1 was critical for the responsiveness to RTA transactivation, since RTA could barely activate the reporter when RRE1 was deleted. Transfection experiments with L1-L5 reporters were also conducted in a similar way. Surprisingly, when RRE1 was deleted, the reporter retained responsiveness to RTA and IRF-7. Further deletion of the RBP-Jκsite abolished the responsiveness. Comparing the transfection results from S and L reporters, it was shown that the 78 bp region between nt 82003 and 82081 might exist a novel functional RRE which mediated RTA transactivation and could complement the loss of RTA activation resulting from the deletion of RRE1. This novel RRE was named as RRE4, its position and the activation mechanism need to be investigated further. Co-transfection of IRF-7 expression plasmid indicated that IRF-7 could suppress not only the RRE 1-mediated, but also the RRE4-mediated RTA transactivation, suggesting that the repression by IRF-7 of the adjacent RRE was orientation-independent.Addition of an extra RRE1 did not notably affect the RTA transactivation. However, addition of two more RBP-Jκsites enhanced the RTA transactivation, which could not be suppressed by IRF-7. Besides, deletion of the RBP-Jκsite abolished the responsiveness to RTA, indicating that the RBP-Jκsite was very critical for the RTA transactivation. Slightly separating the RRE1 from the RBP-Jκsite did not affect RTA transactivation and IRF-7 repression, suggesting that the 40 bp region can be divided. However, the distance between the RBP-Jκsite and RRE2 was crucial for RTA transactivation, since alteration of the distance between the RBP-Jκsite and RRE2 abolished the responsiveness to RTA.This study provides insights into how viral gene expression is affected by the number of and the distance between responsive elements in the viral gene promoter, and will benefit the elucidation of how viral gene expression is precisely regulated by viral and host proteins.Dimerization of IRF-7 was also observed in native-PAGE.