Unique properties of a second human herpesvirus 8-encoded interferon regulatory factor (vIRF-2)

Objective: Human herpesvirus 8/Kaposi's sarcoma herpesvirus (HHV-8/KSHV) co ntains, in addition to genes required for viral replication, an unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8; encodes fo ur open reading frames (ORFs) that show homology to the transcription facto rs of the interferon regulatory factor (IRF) family. In this study we demon strate that one of these ORFs (vIRF-2) encodes a protein with mobility of 1 8 kd which has distinct pattern of expression and properties from the cellu lar IRFs and the previously characterized vIRF-1. Methods: We cloned vIRF-2 by polymerase chain reaction (PCR) and studied it s expression by Northern blot and reverse transcription-polymerase chain re action (RT-PCR). Biologic activities were tested by chloramphenicol acetylt ransferase (CAT) assay in transiently transfected mammalian cells. We chara cterized its DNA binding specificity by electrophoretic mobility shift anal ysis (EMSA) and its protein-protein interactions by in vitro pull-down assa y. Results: Although low levels of vIRF-2 mRNAs can be detected in the HHV-8-p ositive BCBL-1 tumor cell line, 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment does not stimulate expression of vIRF-2 gene together with primar y lytic cycle genes. Recombinant vIRF-2, which can form homodimers, does no t bind specifically to the oligodeoxynucleotide repeats corresponding to th e interferon-stimulated response element (ISRE), but it does bind to the NF -kappa B binding site. The fusion protein generated from vIRF-2 and the Rel A (p65) activation domain stimulates transcriptional activity of HIV LTR, w hich contains two NF-kappa B sites, but does not stimulate the interferon-b eta (IFNB) promoter, which contains only one NF-kappa B site. Interaction b etween recombinant vIRF-2 and cellular IRFs such as IRF-1, IRF-2, and ICSBP was detected by in vitro binding assay, but no interaction between IRF-3 a nd vIRF-2 was found. Interaction of vIRF-2 with RelA (p65) and the carboxy- terminal part of p300 was also observed. In a transient transfection assay, vIRF-2 inhibits the IRF-1- or IRF-3-mediated transcriptional activation of interferon-alpha (IFNA) gene promoter in infected cells and downmodulates RelA (p65)-stimulated activity of HIV LTR. Conclusions: These results suggest that, by interacting with cellular trans cription factors and cofactors, vIRF-2 may modulate the expression of the e arly inflammatory genes and potentially deregulate the immune system.