title: Identification of direct transcriptional targets of the Kaposi's sarcoma-associated herpesvirus Rta lytic switch protein by conditional nuclear localization.
abstract: Lytic reactivation from latency is critical for the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta's direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.
authors: Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey/New Jersey Medical School, Newark, New Jersey, USA.
authors: Wei
authors: W
authors: Bu
authors: Diana
authors: D
authors: Palmeri
authors: Raghu
authors: R
authors: Krishnan
authors: Roxana
authors: R
authors: Marin
authors: Virginie M
authors: VM
authors: Aris
authors: Patricia
authors: P
authors: Soteropoulos
authors: David M
authors: DM
authors: Lukac
mesh_terms: D002467:Cell Nucleus / Q000737:chemistry; D019288:Herpesvirus 8, Human / Q000502:physiology*; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals; D011960:Receptors, Estrogen / Q000235:genetics; D011993:Recombinant Fusion Proteins; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D017384:Sequence Deletion; D013629:Tamoxifen / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*; D014775:Virus Activation
chemical_list: D017874:Immediate-Early Proteins; D019913:Nuclear Localization Signals; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C417604:Rta protein, Human herpesvirus 8; D020845:Selective Estrogen Receptor Modulators; D015534:Trans-Activators; D014764:Viral Proteins; D013629:Tamoxifen