title: Quantitative analysis of the bidirectional viral G-protein-coupled receptor and lytic latency-associated nuclear antigen promoter of Kaposi's sarcoma-associated herpesvirus.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) establishes sustained latent persistence in susceptible cells. This is dependent on the latency-associated nuclear antigen (LANA). Understanding how LANA transcription is regulated thus aids our fundamental understanding of KSHV biology. Two hundred ninety-four base pairs are sufficient to regulate LANA transcription in response to the viral RTA protein and RBPjκ. The same region controls K14/viral G-protein-coupled receptor (vGPCR) transcription in the opposite direction. We used a quantitative analysis in conjunction with specific nucleotide substitutions and defined gain-of-function and loss-of-function RTA mutants to dissect this region. We used a bidirectional reporter driving red and green luciferase to study the LANApi and K14p promoters simultaneously. This established that LANApi/K14p functions as a canonical bidirectional promoter. Both were TATA dependent. K14p was favored by ∼50-fold in this context. Eliminating the distal LANApi TATA box increased maximal output and lowered the induction threshold (T) of K14p even further. Two RBPjκ binding sites were independently required; however, at high concentrations of RTA, direct interactions with an RTA-responsive element (RRE) could complement the loss of one RBPjκ binding site. Intracellular Notch (ICN) was no longer able to activate RBPjκ in the viral context. This suggests a model whereby KSHV alters ICN-RBPjκ gene regulation. When the architecture of this pair of head-to-head RBPjκ binding sites is changed, the sites now respond exclusively to the viral transactivator RTA and no longer to the host mediator ICN.
authors: Curriculum in Genetics and Molecular Biology, Comprehensive Cancer Center, Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors: Isaac B
authors: IB
authors: Hilton
authors: Dirk P
authors: DP
authors: Dittmer
mesh_terms: D000956:Antigens, Viral / Q000235:genetics* / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D016679:Genome, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity / Q000502:physiology*; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000502:physiology*; D011991:Receptors, Virus / Q000235:genetics / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology; D017735:Virus Latency / Q000235:genetics / Q000502:physiology
chemical_list: D000956:Antigens, Viral; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D043562:Receptors, G-Protein-Coupled; D011991:Receptors, Virus; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; C109540:latency-associated nuclear antigen