RESUMO
Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8) ORF50 encodes a transactivator, K-Rta, which functions as the switch from latent to lytic virus replication. K-bZIP interacts with K-Rta and can repress its transactivation activity for some viral promoters. Both K-Rta and K-bZIP are required for origin-dependent DNA replication. To determine the role of K-bZIP in the context of the viral genome, we generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a deletion in the K-bZIP open reading frame. This BACmid, BAC36DeltaK8, displayed an enhanced growth phenotype with respect to virus production and accumulation of virus-encoded mRNAs measured by real-time PCR when K-Rta was used to induce the virus lytic cycle. Conversely, induction of the virus lytic cycle using tetradecanoyl phorbol acetate/n-butyrate resulted in no virus production and an aberrant gene expression pattern from BAC36DeltaK8-containing cells compared to wild-type (wt) BAC. This null virus phenotype was efficiently complemented by the expression of K-bZIP in trans, restoring virus production to wt BAC levels. Immunofluorescence staining revealed that subcellular localization of K-Rta was unchanged; however, a disruption of LANA subcellular localization was observed in cells harboring BAC36DeltaK8, suggesting that K-bZIP influences LANA localization. Coimmunoprecipitation experiments confirmed that K-bZIP interacts with LANA in BCBL-1 cells and in cotransfection assays. Lastly, the chromatin immunoprecipitation assay revealed that, in an environment where K-Rta is overexpressed and in the absence of K-bZIP, K-Rta binds to CAAT enhancer binding protein alpha sites within oriLyt, suggesting that it is K-Rta that supplies an essential replication function and that K-bZIP may serve to augment or facilitate the interaction of K-Rta with oriLyt.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Deleção de Genes , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Regulação para Cima , Proteínas Virais/metabolismo , Replicação Viral , Antígenos Virais/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular , Cromossomos Artificiais Bacterianos/genética , Regulação Viral da Expressão Gênica , Teste de Complementação Genética , Herpesvirus Humano 8/classificação , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Nucleares/metabolismo , Fenótipo , Recombinação Genética , Proteínas Repressoras/genética , Transativadores/genética , Transfecção , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Replicação do DNA/fisiologia , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Repressoras/fisiologia , Transativadores/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular , Chlorocebus aethiops , Primers do DNA/genética , Replicação do DNA/genética , DNA Viral/biossíntese , DNA Viral/genética , Dimerização , Genes Virais , Teste de Complementação Genética , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transativadores/genética , Transativadores/fisiologia , Ativação Transcricional , Células Vero , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP).