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Journal of Virology, May 2009, p. 5181-5191, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.00090-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Phosphorylation of the U_L31 Protein of Herpes Simplex Virus 1 by the U_S3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

Fan Mou, Elizabeth Wills, and Joel D. Baines^*

Department of Microbiology and Immunology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14850

Received 14 January 2009/ Accepted 26 February 2009

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U_L31 and U_L34 gene products. While it is clear that the viral kinase encoded by the U_S3 gene regulates the localization of pU_L31/pU_L34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU_L31 is indispensable for the protein's normal function and contains up to six serines that are phosphorylated by the U_S3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U_S3 kinase, changing the serines to alanine caused the pU_L31/pU_L34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U_S3 kinase-dead mutant. (iv) The replacement of the six serines of pU_L31 with glutamic acid largely restored the smooth distribution of pU_L34/pU_L31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U_S3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU_L31 by pU_S3 represents an important regulatory event in the virion egress pathway that can account for much of pU_S3's role in nuclear egress. The data also suggest that the dynamics of pU_L31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14850. Phone: (607) 253-3391. Fax: (607) 253-3384. E-mail: jdb11{at}cornell.edu

Published ahead of print on 11 March 2009.

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Journal of Virology, May 2009, p. 5181-5191, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.00090-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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