Genetic Organization and Hypoxic Activation of the Kaposi's Sarcoma-Associated Herpesvirus ORF34-37 Gene Cluster

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We previously reported that hypoxia activates KSHV lytic replication and that the promoter for open reading frame 34 (ORF34) contains a functional hypoxia-responsive element (HRE). ORF34 is part of a cluster of lytic genes (ORF34-37) that includes ORF36, a phosphotransferase, and ORF37, a shutoff exonuclease. Rapid amplification of cDNA ends analysis revealed that they share a common polyadenylation signal but have two start sites. Two transcripts were identified, one 3.4 kb encoding ORF35-37, and the other 4.2 kb encoding ORF34 and also having coding potential for ORF35-37. Exposure of PEL cell lines to hypoxia induced messages of lengths consistent with those of these transcripts. Reporter assays with Hep3B cells showed activation of both transcripts by hypoxia. The ORF34-37 promoter region has six consensus HREs. Sequential deletion, site-directed mutagenesis experiments, and Northern blot analysis of RNA produced by constructs indicated that the second HRE (HRE-2) plays a critical role in the hypoxic activation of both RNA transcripts. The ORF35-37 transcript was upregulated by cotransfected hypoxia-inducible factor (HIF). Electrophoretic mobility shift assays demonstrated that HRE-2 and ancillary sequences bind and compete for HIF with hypoxic Hep3B nuclear extract. The activation of this gene cluster by hypoxia may have implications for the pathogenesis of PEL and KS. Moreover, the activation of ORF36 by hypoxia might be exploited to develop targeted therapy for PEL, which arises in a hypoxic environment (pleural effusions).

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

“…In K-Rta-inducible BCBL-1 cells, the 4.2-kb transcript, encoding ORF34 to ORF38, was sensitive to PAA; this finding differs from an earlier report (18). We also detected two additional transcripts (2.0 kb and 0.6 kb) that had not previously been reported (18). These differences in transcription patterns might be caused by the different method for reactivation, i.e., overexpression of the transactivator, K-Rta.…”

“…Other investigators using microarray analysis, coupled with the use of an inhibitor of viral DNA synthesis, also showed that the vPK gene was expressed early in the viral lytic cycle (31). In K-Rta-inducible BCBL-1 cells, the 4.2-kb transcript, encoding ORF34 to ORF38, was sensitive to PAA; this finding differs from an earlier report (18). We also detected two additional transcripts (2.0 kb and 0.6 kb) that had not previously been reported (18).…”

“…The two vPK encoding transcripts were coterminal with transcripts encoding ORF37 and ORF38 (see also reference 18). Precise transcription initiation sites were recently reported at position 54566 for the 4.2-kb transcript and at position 55567 for the 3.6 kb transcript (18). To aid in the study of vPK, a highly specific antibody was produced by immunizing rabbits with purified recombinant vPK protein.…”

“…However, a recent study detected vPK RNA at early time points and in the presence of an inhibitor of viral DNA synthesis (31). Detailed analysis of KSHV RNA revealed two polycistronic transcripts encoding vPK that are initiated from promoters that are active in the early stage of the viral life cycle and inducible by hypoxic conditions (18). With respect to potential therapeutic significance, vPK was shown to phosphorylate the antiherpesvirus drug ganciclovir (10).…”

A viral kinase mimics S6 kinase to enhance cell proliferation

“…This wide repertoire of targets results from vPK localizing within both the cytoplasmic and nuclear compartments (12). Within the KSHV genome, vPK is located within the ORF 34-37 cluster, which contains a hypoxia-inducible factor (HIF) response element within the promoter region (13,14). Thus, vPK expression may be induced in hypoxic environments, independent of lytic replication.…”