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Journal of Virology, December 2009, p. 12462-12472, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.01546-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells{triangledown}

Xiaoxing Wang,1,{dagger} Ying Liao,2,{dagger} Pei Ling Yap,1 Kim J. Png,1 James P. Tam,2 and Ding Xiang Liu1,2*

Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673,1 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore2

Received 26 July 2009/ Accepted 15 September 2009

A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the {alpha} subunit of eukaryotic initiation factor 2 (eIF-2{alpha}). In this report, we show that phosphorylation of eIF-2{alpha} was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2{alpha}, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2{alpha}, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2{alpha}, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.

* Corresponding author. Mailing address: Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Singapore. Phone: (65) 65869581. Fax: (65) 67791117. E-mail: dxliu{at}imcb.a-star.edu.sg

{triangledown} Published ahead of print on 23 September 2009.

{dagger} These authors contributed equally.

Journal of Virology, December 2009, p. 12462-12472, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.01546-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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