Fu, B, Kuang, E, Li, W, Avey, D, Li, X, Turpin, Z, Valdes, A, Brulois, K, Myoung, J and Zhu, F (2014) Activation of p90 Ribosomal S6 Kinases (RSKs) by ORF45 of Kaposi Sarcoma-Associated Herpesvirus is Critical for Optimal Production of Infectious Viruses. Journal of Virology .

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 ERK. We have now 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. 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 was also accompanied by noticeably decreased viral gene expression during lytic reactivation. The ORF45-F66A mutant consequently 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. IMPORTANCE STATEMENT: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment, and facilitate efficient production of progeny viral particles. We have 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

Item Type:	Article
Additional Information:	NIBR author: Myoung, J institute: NIBR contributor address: Department of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USADepartment of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USANovartis Institutes for Biomedical Research, Emeryville, CA 94608, USADepartment of Biological Science, Florida State University, Tallahassee, FL 32306, USA fzhu@bio.fsu.edu
Date Deposited:	13 Oct 2015 13:11
Last Modified:	13 Oct 2015 13:11
URI:	https://oak.novartis.com/id/eprint/24251