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Journal of Virology, September 2009, p. 9195-9205, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00271-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5{triangledown}

Dipankar Bhattacharya,1,# Mayuri ,2,#,{dagger} S. M. Best,4 R. Perera,2 R. J. Kuhn,2 and Rob Striker1,3*

University of Wisconsin—Madison, Department of Medicine, Madison, Wisconsin 53706,1 Purdue University, Department of Biological Sciences, West Lafayette, Indiana 47907,2 W. S. Middleton Memorial Veterans Administration Hospital, Madison, Wisconsin 53706,3 Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 598404

Received 6 February 2009/ Accepted 29 June 2009

Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.

* Corresponding author. Mailing address: University of Wisconsin—Madison, Madison, WI 53706. Phone: (608) 262-2994. Fax: (608) 262-8418. E-mail: rtstriker{at}wisc.edu

{triangledown} Published ahead of print on 8 July 2009.

# These authors contributed equally.

{dagger} Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

Journal of Virology, September 2009, p. 9195-9205, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00271-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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