RESUMEN
The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis. We developed a modification of the transient cotransfection replication assay wherein both lytic (oriLyt) and latent (terminal repeat) DNA replication are evaluated simultaneously. We now show that LANA represses origin-dependent lytic DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression was overcome by increasing amounts of a K-bZIP expression plasmid in the cotransfection mixture or by dominant-negative inhibition of the interaction of LANA with K-bZIP by the overexpression of the K-bZIP-LANA binding domain. Chromatin immunoprecipitation assays show that LANA interacts with oriLyt in the absence of K-bZIP expression, suggesting that suppression of lytic replication by LANA is mediated by direct binding. The interaction of K-bZIP with oriLyt was dependent upon the expression of LANA; however, LANA interacted with oriLyt independently of K-bZIP expression. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication and that K-bZIP facilitates lytic DNA replication and modulates the switch from the latent phase of the virus.
Asunto(s)
Antígenos Virales/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Ciclinas/metabolismo , ADN Viral/biosíntesis , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Proteínas Nucleares/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Línea Celular , Inmunoprecipitación de Cromatina/métodos , Humanos , Unión Proteica , Transfección/métodosRESUMEN
Kaposi's sarcoma-associated herpesvirus origin-dependent DNA replication requires the core replication proteins plus K-Rta and K-bZIP. To determine which K-bZIP protein domains contribute to oriLyt-dependent DNA replication and facilitate suppression of K-Rta-mediated transcriptional activation, we generated a series of deletion constructs and site-directed mutations within the K-bZIP ORF. Mutation of key leucine residues within the putative leucine zipper (LZ) motif eliminated the ability of the protein to homodimerize and complement oriLyt-dependent DNA replication. Deletion of the basic amino acid region (BR) or LZ domain did not affect the ability of K-bZIP to bind to K-Rta indicating that either region contributes to heterodimerization with K-Rta. However, deletions or mutations introduced into both the LZ and BR resulted in elimination of the suppressive activity of K-bZIP even in the presence of a K-bZIP-K-Rta interaction. Interestingly, mutants that lacked the ability to suppress K-Rta transactivation were still capable of complementing oriLyt-dependent DNA replication, indicating that this activity does not contribute to the DNA synthesis-related activity of K-bZIP.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Replicación del ADN/fisiología , Herpesvirus Humano 8/fisiología , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Represoras/fisiología , Transactivadores/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/fisiología , Animales , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular , Chlorocebus aethiops , Cartilla de ADN/genética , Replicación del ADN/genética , ADN Viral/biosíntesis , ADN Viral/genética , Dimerización , Genes Virales , Prueba de Complementación Genética , Herpesvirus Humano 8/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Leucina Zippers/genética , Leucina Zippers/fisiología , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Transactivadores/genética , Transactivadores/fisiología , Activación Transcripcional , Células Vero , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP. This UTPase activity was linear with respect to enzyme concentration and slightly enhanced by the addition of nucleic acid substrates. UL84 UTPase was the highest at low salt concentrations, a pH of 7.5, and a temperature of 45 degrees C. The enzyme preferred Mg2+ as the divalent cation but was also able to catalyze the UTPase reaction in the presence of Mn2+, Ca2+, and Zn2+ albeit at lower levels. The evidence presented here suggests that the UL84 UTPase activity may be part of an energy-generating system for helicase activity associated with the initiation of HCMV DNA replication.