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) contains, 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 four open reading frames (ORFs) that show homology to the transcription factors of the interferon regulatory factor (IRF) family. In this study we demonstrate that one of these ORFs (vIRF-2) encodes a protein with mobility of 18 kd which has distinct pattern of expression and properties from the cellular IRFs and the previously characterized vIRF-1.


METHODS
We cloned vIRF-2 by polymerase chain reaction (PCR) and studied its expression by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Biologic activities were tested by chloramphenicol acetyltransferase (CAT) assay in transiently transfected mammalian cells. We characterized its DNA binding specificity by electrophoretic mobility shift analysis (EMSA) and its protein-protein interactions by in vitro pull-down assay.


RESULTS
Although low levels of vIRF-2 mRNAs can be detected in the HHV-8-positive BCBL-1 tumor cell line, 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment does not stimulate expression of vIRF-2 gene together with primary lytic cycle genes. Recombinant vIRF-2, which can form homodimers, does not bind specifically to the oligodeoxynucleotide repeats corresponding to the 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 RelA (p65) activation domain stimulates transcriptional activity of HIV LTR, which contains two NF-kappa B sites, but does not stimulate the interferon-beta (IFNB) promoter, which contains only one NF-kappa B site. Interaction between 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 and 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 transcription factors and cofactors, vIRF-2 may modulate the expression of the early inflammatory genes and potentially deregulate the immune system.
Burysek
L
L
Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Yeow
W S
WS
Pitha
P M
PM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050881:E1A-Associated p300 Protein; D017930:Genes, Reporter; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D016325:HIV Long Terminal Repeat; D019288:Herpesvirus 8, Human / Q000235:genetics; D006801:Humans; D050836:Interferon Regulatory Factor-2; D016899:Interferon-beta / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D015534:Trans-Activators / Q000378:metabolism; D051996:Transcription Factor RelA; D014157:Transcription Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015699:HIV Envelope Protein gp120; C494229:IRF2 protein, human; D050836:Interferon Regulatory Factor-2; C494230:Irf2 protein, mouse; D016328:NF-kappa B; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D051996:Transcription Factor RelA; D014157:Transcription Factors; D016899:Interferon-beta; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse