title: Activation of p90 ribosomal S6 kinases by ORF45 of Kaposi's sarcoma-associated herpesvirus is critical for optimal production of infectious viruses.
abstract: We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838-1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.
  
  
  OBJECTIVE
  KSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Bishi
authors.initials: B
authors.lastname: Fu
authors.affiliation: Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China.
authors.forename: Ersheng
authors.initials: E
authors.lastname: Kuang
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Wenwei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Denis
authors.initials: D
authors.lastname: Avey
authors.affiliation: Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Turpin
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Valdes
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Brulois
authors.affiliation: Novartis Institutes for Biomedical Research, Emeryville, California, USA.
authors.forename: Jinjong
authors.initials: J
authors.lastname: Myoung
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, USA fzhu@bio.fsu.edu.
authors.forename: Fanxiu
authors.initials: F
authors.lastname: Zhu
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D019288:Herpesvirus 8, Human / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D017874:Immediate-Early Proteins; D050505:Mutant Proteins; C456286:ORF45 protein, Kaposi's sarcoma-associated herpesvirus; D038744:Ribosomal Protein S6 Kinases, 90-kDa