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Journal of Virology, February 2009, p. 1602-1610, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01590-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

ISG15 Arg151 and the ISG15-Conjugating Enzyme UbE1L Are Important for Innate Immune Control of Sindbis Virus{triangledown}

Nadia V. Giannakopoulos,1 Elena Arutyunova,2 Caroline Lai,1,3 Deborah J. Lenschow,1,3 Arthur L. Haas,2* and Herbert Whiting Virgin1*

Department of Pathology and Immunology, Department of Molecular Microbiology,1 Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110,3 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana 701122

Received 25 July 2008/ Accepted 2 December 2008

Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-{alpha}βR–/–) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-{alpha}βR–/– mice or prolong survival of ISG15–/– mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.

* Corresponding author. Mailing address for Arthur L. Haas: Department of Biochemistry and Molecular Biology, LSUHSC, 1901 Perdido Street, New Orleans, LA 70112. Phone: (504) 568-3004. Fax: (504) 568-3370. E-mail: ahaas{at}lsuhsc.edu. Mailing address for Herbert Whiting Virgin: Department of Pathology and Immunology, Washington University, 660 S. Euclid Ave, Box 8118, St. Louis, MO 63110. Phone: (314) 362-9223. Fax: (314) 362-4096. E-mail: virgin{at}wustl.edu

{triangledown} Published ahead of print on 10 December 2008.

Journal of Virology, February 2009, p. 1602-1610, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01590-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hsiang, T.-Y., Zhao, C., Krug, R. M. (2009). Interferon-Induced ISG15 Conjugation Inhibits Influenza A Virus Gene Expression and Replication in Human Cells. J. Virol. 83: 5971-5977 [Abstract] [Full Text]  


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