RESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) stabilizes hypoxia-inducible factor α (HIF-1α) during latent infection, and HIF-1α reactivates lytic replication under hypoxic stress. However, the mechanism utilized by KSHV to block lytic reactivation with the accumulation of HIF-1α in latency remains unclear. Here, we report that LANA encoded by KSHV contains a unique SUMO-interacting motif (LANA(SIM)) which is specific for interaction with SUMO-2 and facilitates LANA SUMOylation at lysine 1140. Proteomic and co-immunoprecipitation analysis further reveal that the SUMO-2 modified transcription repressor KAP1 is a critical factor recruited by LANA(SIM). Deletion of LANA(SIM) led to functional loss of both LANA-mediated viral episome maintenance and lytic gene silencing. Moreover, hypoxia reduced KAP1 SUMOylation and resulted in dissociation of both KAP1 and Sin3A repressors from LANA(SIM)-associated complex. Therefore, the LANA(SIM) motif plays an essential role in KSHV latency and is a potential drug target against KSHV-associated cancers.
Asunto(s)
Antígenos Virales/metabolismo , Herpesvirus Humano 8/fisiología , Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Virales/metabolismo , Latencia del Virus/fisiología , Secuencias de Aminoácidos , Antígenos Virales/genética , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas Nucleares/genética , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3 , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación/genética , Proteína 28 que Contiene Motivos Tripartito , Proteínas Virales/genética , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patologíaRESUMEN
Aberrant expression of Aurora A kinase has been frequently implicated in many cancers and contributes to chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. Previous studies showed that p53 is degraded by Kaposi's sarcoma herpesvirus (KSHV) encoded latency-associated nuclear antigen (LANA) through its SOCS-box (suppressor of cytokine signaling, LANA(SOCS)) motif-mediated recruitment of the EC(5)S ubiquitin complex. Here we demonstrate that Aurora A transcriptional expression is upregulated by LANA and markedly elevated in both Kaposi's sarcoma tissue and human primary cells infected with KSHV. Moreover, reintroduction of Aurora A dramatically enhances the binding affinity of p53 with LANA and LANA(SOCS)-mediated ubiquitylation of p53 which requires phosphorylation on Ser215 and Ser315. Small hairpin RNA or a dominant negative mutant of Aurora A kinase efficiently disrupts LANA-induced p53 ubiquitylation and degradation, and leads to induction of p53 transcriptional and apoptotic activities. These studies provide new insights into the mechanisms by which LANA can upregulate expression of a cellular oncogene and simultaneously destabilize the activities of the p53 tumor suppressor in KSHV-associated human cancers.
Asunto(s)
Herpesvirus Humano 8/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Antígenos Virales/genética , Antígenos Virales/metabolismo , Aurora Quinasas , Línea Celular Tumoral , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Regulación hacia ArribaRESUMEN
Kaposi's Sarcoma-associated Herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is the most recently identified human tumor virus,and is associated with the pathogenesis of Kaposi's sarcoma and two lymphoproliferative disorders known to occur frequently in AIDS patients-primary effusion lymphoma and multicentric Castleman disease. In the 15 years since its discovery, intense studies have demonstrated an etiologic role for KSHV in the development of these malignancies. Here, we review the recent advances linked to understanding KSHV latent and lytic life cycle and the molecular mechanisms of KSHV-mediated oncogenesis in terms of transformation, cell signaling, cell growth and survival, angiogenesis, immune invasion and response to microenvironmental stress, and highlight the potential therapeutic targets for blocking KSHV tumorigenesis.
Asunto(s)
Herpesvirus Humano 8/genética , Procesos Neoplásicos , Sarcoma de Kaposi/patología , Animales , Transformación Celular Neoplásica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Humanos , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virología , Transducción de SeñalRESUMEN
The hypoxic microenvironment contributes to embryonic development and tumor progression through stabilization of the potent transcriptional factor HIFalpha. In normoxia, the tumor suppressor protein VHL acts as an E3 ubiquitin ligase to target HIFalpha for proteolytic destruction. Increasing evidence shows that VHL is a multifunctional adaptor involved in inhibition of HIFalpha-dependent and independent cellular processes. However, the molecular effect of hypoxic stress on VHL functions remains elusive. Here we report that PIASy, a SUMO E3 ligase upregulated in hypoxia, interacts with VHL and induces VHL SUMOylation on lysine residue 171. Moreover, PIASy-mediated SUMO1 modification induces VHL oligomerization and abrogates its inhibitory function on tumor cell growth, migration and clonogenicity. Knockdown of PIASy by small interfering RNA leads to reduction of VHL oligomerization and increases HIF1alpha degradation. These findings reveal a unique molecular strategy for inactivation of VHL under hypoxic stress.
Asunto(s)
Hipoxia , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lisina/química , Modelos Biológicos , Proteínas de Unión a Poli-ADP-Ribosa , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
Cellular protein degradation pathways can be utilized by viruses to establish an environment that favors their propagation. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) directly functions as a component of the EC5S ubiquitin complex targeting the tumor suppressors von Hippel-Lindau (VHL) and p53 for degradation. We have characterized a suppressor of cytokine signaling box-like motif within LANA composed of an Elongin B and C box and a Cullin box, which is spatially located at its amino and carboxyl termini. This motif is necessary for LANA interaction with the Cul5-Elongin BC complex, to promote polyubiquitylation of cellular substrates VHL and p53 in vitro via its amino- and carboxyl-terminal binding domain, respectively. In transfected cells as well as KSHV-infected B lymphoma cells, LANA expression stimulates degradation of VHL and p53. Additionally, specific RNA interference-mediated LANA knockdown stabilized VHL and p53 in primary effusion lymphoma cells. Thus, manipulation of tumor suppressors by LANA potentially provides a favorable environment for progression of KSHV-infected tumor cells.
