title: Functional characterization of Kaposi's sarcoma-associated herpesvirus open reading frame K8 by bacterial artificial chromosome-based mutagenesis.
abstract: The open reading frame K8 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a basic leucine zipper (bZip) protein that binds to the origin of viral DNA replication and is an integral component of viral lytic DNA replication complex. Moreover, K8 physically interacts with replication and transcription activator (RTA) and represses its transactivation activity on several viral promoters. To investigate the role of this protein in viral life cycle, we constructed two K8-null recombinant mutant viruses (BAC-ΔK8 and BAC-stopK8) by using a bacterial artificial chromosome (BAC) system. Latent viral infection can be reconstituted in 293T and BJAB cells with wild-type and the K8-null recombinant viruses by introducing the cloned viral genomes into the cells. When the cells carrying these viruses were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, no significant difference was seen in overall viral gene expression between wild-type and K8-null viruses, with lytic DNA replication still active in the latter. However, 293T cells harboring K8-null mutant viruses, either BAC-ΔK8 or BAC-stopK8, displayed lower copy numbers of latent KSHV genome in comparison with wild-type viruses. Furthermore, although K8 deficiency appeared to not affect infectivity when K8-null viruses were used to infect 293T, primary human microvascular dermal endothelial and human foreskin fibroblast cells, they exhibited much lower viral genome copy numbers in all types of cell compared to wild-type viruses. Taken together, these data suggest a possible role of K8 in abortive lytic DNA replication occurring in early stages of de novo infection or in the maintenance of latent viral genomes.
authors.affiliation: Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Narayanan
authors.initials: N
authors.lastname: Sathish
authors.forename: Charles
authors.initials: C
authors.lastname: Hollow
authors.forename: Yan
authors.initials: Y
authors.lastname: Yuan
mesh_terms: D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D006566:Herpesviridae Infections / Q000821:virology*; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D014764:Viral Proteins