Differential Regulation of the Overlapping Kaposi's Sarcoma-Associated Herpesvirus vGCR (orf74) and LANA (orf73) Promoters

Abstract

Similar to that of other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) lytic replication destroys the host cell, while the virus can persist in a latent state in synchrony with the host. During latency only a few genes are transcribed, and the question becomes one of what determines latent versus lytic gene expression. Here we undertake a detailed analysis of the latency-associated nuclear antigen (LANA [orf73]) promoter (LANAp). We characterized a minimal region that is necessary and sufficient to maintain high-level transcription in all tissues tested, including primary endothelial cells and B cells, which are the suspected natural host for KSHV. We show that in transient-transfection assays LANAp mimics the expression pattern observed for the authentic promoter in the context of the KSHV episome. Unlike other KSHV promoters tested thus far, LANAp is not affected by tetradecanoyl phorbol acetate or viral lytic cycle functions. It is, however, subject to control by LANA itself and cellular regulatory factors, such as p53. This is in contrast to the K14/vGCR (orf74) promoter, which overlaps LANAp and directs transcription on the opposite strand. We isolated a minimal cis-regulatory region sufficient for K14/vGCR promoter activity and show that it, too, mimics the regulation observed for the authentic viral promoter. In particular, we demonstrate that its activity is absolutely dependent on the immediate-early transactivator orf50, the KSHV homolog of the Epstein-Barr virus Rta transactivator.Using representational difference analysis Chang et al. (6) demonstrated the presence of a novel human virus in Kaposi's sarcoma (KS) biopsy samples: Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8. KSHV has since been detected in all manifestations of KS as well as in two lymphoproliferative disorders: primary effusion lymphoma (4) and multicentric Castleman's disease (53). On the basis of the complete sequence of the 137-kbp double-stranded DNA genome, KSHV is classified as a gamma-2 herpesvirus, a member of the lymphotropic subgroup of the Herpesviridae (17,36,45).The epidemiological evidence implicating KSHV as a causative agent for KS is compelling (reviewed in reference 51). (i) KSHV DNA is found in Ͼ90% of KS biopsy samples. (ii) KSHV latent mRNAs and proteins are detectable in every KS spindle cell by in situ methods. (iii) Antibodies to KSHV exist in Ն80% of KS patients, and multiple viral antigens have been identified as targets of this response. (iv) Increases in peripheral-blood viral load as well as anti-KSHV antibody titer precede the onset of disease and correlate with increased risk for KS. These observations establish KSHV as a necessary cofactor for KS.KSHV, like all herpesviruses, displays two modes of replication: lytic replication, during which the host cell is destroyed and viral progeny are released, and latent replication, during which the viral genome persists indefinitely and no viral progeny are released. In KS, KSHV persists latently in Ն90% of...

Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus

“…The ORF 50/Rta also transactivated the promoter of vGPCR/ORF 74, which partially overlaps with the LANA 5Ј-UTR (47,62). This close proximity suggested the hypothesis that ORF 50 was involved in regulating the LANAp.…”

“…All constructs exhibited basal activity independently of the presence of other KSHV proteins. However, our studies also showed that LANAp was regulated positively by LANA and negatively by the p53 tumor suppressor protein (47). Thus, so far these two are the only known trans-acting factors that regulate LANAp.…”

“…Plasmids pDD41, pDD83, pDD104, pDD124, pDD125, pDD163, pDD274, and pDD267 were described previously (47,49). The plasmids pGL3-1 (pDD463), pGL3-2 (pDD464), pGL3-3 (pDD465), pGL3-4 (pDD466), and pGL3-5 (pDD467) were a gift from Yuan Chang (University of Pittsburg, OH) (14).…”

“…␤-Galactosidase activity was determined using o-nitrophenyl-␤-D-galactopyranoside as described (47) or the Galacto-Light Plus kit (Tropix, Bedford, MA) according to the manufacturer's recommendations.…”

“…Promoter Activity-The side-by-side comparison of the deletion clones from Sarid et al (14) with those from Jeong et al (47) allowed us to further define LANAp. Confirming previous observations, both sets of clones exhibited significant constitutive promoter activity as long as sequences up to Ϫ88 (127,968) were included.…”