RESUMO
The replication and transcription activator (RTA) of gamma-2 herpesvirus is sufficient to drive the entire virus lytic cycle. Hence, the control of RTA activity should play an important role in the maintenance of viral latency. Here, we demonstrate that cellular poly(ADP-ribose) polymerase 1 (PARP-1) and Ste20-like kinase hKFC interact with the serine/threonine-rich region of gamma-2 herpesvirus RTA and that these interactions efficiently transfer poly(ADP-ribose) and phosphate units to RTA. Consequently, these modifications strongly repressed RTA-mediated transcriptional activation by inhibiting its recruitment onto the promoters of virus lytic genes. Conversely, the genetic ablation of PARP-1 and hKFC interaction or the knockout of the PARP-1 gene and activity considerably enhanced gamma-2 herpesvirus lytic replication. Thus, this is the first demonstration that cellular PARP-1 and hKFC act as molecular sensors to regulate RTA activity and thereby, herpesvirus latency.
Assuntos
Gammaherpesvirinae/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Gammaherpesvirinae/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Humanos , Modelos Biológicos , Poli(ADP-Ribose) Polimerases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Replicação ViralRESUMO
Epstein-Barr virus (EBV) EBNA2 and Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) are recruited to their responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jkappa. In particular, RTA and EBNA2 interactions with RBP-Jkappa are essential for the lytic replication of KSHV and expression of B-cell activation markers CD21 and CD23a, respectively. Here, we demonstrate that like EBV EBNA2, KSHV RTA strongly induces CD21 and CD23a expression through RBP-Jkappa binding sites in the first intron of CD21 and in the CD23a core promoter, respectively. However, unlike EBV EBNA2, which alters immunoglobulin mu (Igmu) and c-myc gene expression, RTA did not affect Igmu and c-myc expression, indicating that KSHV RTA targets the Notch signal transduction pathway in a manner similar to but distinct from that of EBV EBNA2. Furthermore, RTA-induced expression of CD21 glycoprotein, which is an EBV receptor, efficiently facilitated EBV infection. In addition, RTA-induced CD23 glycoprotein underwent proteolysis and gave rise to soluble CD23 (sCD23) molecules in B lymphocytes and KSHV-infected primary effusion lymphocytes. sCD23 then stimulated primary human lymphocytes. These results demonstrate that cellular CD21 and CD23a are common targets for B lymphotropic gammaherpesviruses and that KSHV RTA regulates RBP-Jkappa-mediated cellular gene expression, which ultimately provides a favorable milieu for viral reproduction in the infected host.
Assuntos
Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/genética , Receptores de Complemento 3d/genética , Receptores de IgE/genética , Transativadores/genética , Proteínas Virais/genética , Antígenos CD/genética , Sequência de Bases , Linhagem Celular , Primers do DNA , Regulação da Expressão Gênica , Genes Reporter , Herpesvirus Humano 8/fisiologia , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Ativação Transcricional , Transfecção , Replicação ViralRESUMO
An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. In order to study the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This system uses Flp-mediated efficient recombination and tetracycline-inducible expression. The Rta transcriptional activator, which acts as a molecular switch for lytic reactivation of KSHV, was efficiently integrated downstream of the Flp recombination target site, and its expression was tightly controlled by tetracycline. Like stimulation with tetradecanoyl phorbol acetate (TPA), the ectopic expression of Rta efficiently induced a complete cycle of viral replication, including a well-ordered program of KSHV gene expression and production of infectious viral progeny. A striking feature of Rta-mediated lytic gene expression was that Rta induced KSHV gene expression in a more powerful and efficient manner than TPA stimulation, indicating that Rta plays a central, leading role in KSHV lytic gene expression. Thus, our streamlined gene expression system provides a novel means not only to study the effects of viral gene products on overall KSHV gene expression and replication, but also to understand the natural viral reactivation process.