Overexpression of the kaposi's sarcoma-associated herpesvirus transactivator K-Rta can complement a K-bZIP deletion BACmid and yields an enhanced growth phenotype.
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.
Kato-Noah
Taeko
T
Department of Microbiology, Cell and Molecular Biology Program, University of Nevada School of Medicine, Reno, Nevada 89557, USA.
Xu
Yiyang
Y
Rossetto
Cyprian C
CC
Colletti
Kelly
K
Papousková
Iva
I
Pari
Gregory S
GS
D000956:Antigens, Viral / Q000378:metabolism; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D005816:Genetic Complementation Test; D019288:Herpesvirus 8, Human / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D000956:Antigens, Viral; D050976:Basic-Leucine Zipper Transcription Factors; D017874:Immediate-Early Proteins; C118265:K8 protein, Human herpesvirus 8; D009687:Nuclear Proteins; D012097:Repressor Proteins; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; C109540:latency-associated nuclear antigen