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ISG15 conjugation system targets the viral NS1 protein in influenza A virus?infected cells (Proc Natl Acad Sci USA, abstract, edited)
ISG15 conjugation system targets the viral NS1 protein in influenza A virus?infected cells
1. Chen Zhao, 2. Tien-Ying Hsiang, 3. Rei-Lin Kuo, and 4. Robert M. Krug
1- Author Affiliations 1. Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, TX 78712
1. Edited by Alexander Varshavsky, California Institute of Technology, Pasadena, CA, and approved December 21, 2009 (received for review August 11, 2009)
Abstract
ISG15 is an IFN-α/β?induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a ?loss of function? mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.
* interferon * antiviral * dsRNA * Herc5 * importin-α
Footnotes
* 1To whom correspondence should be addressed. E-mail: rkrug@mail.utexas.edu.
* Author contributions: C.Z., R.-L.K., and R.M.K. designed research; C.Z., T.-Y.H., and R.-L.K. performed research; C.Z., T.-Y.H., R.-L.K., and R.M.K. analyzed data; and C.Z. and R.M.K. wrote the paper.
* The authors declare no conflict of interest.
* This article is a PNAS Direct Submission.
* This article contains supporting information online at https://www.pnas.org/cgi/content/ful...DCSupplemental.
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<cite cite="http://www.pnas.org/content/107/5/2253.short?rss=1">ISG15 conjugation system targets the viral NS1 protein in influenza A virus?infected cells ? PNAS</cite>
1. Chen Zhao, 2. Tien-Ying Hsiang, 3. Rei-Lin Kuo, and 4. Robert M. Krug
1- Author Affiliations 1. Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, TX 78712
1. Edited by Alexander Varshavsky, California Institute of Technology, Pasadena, CA, and approved December 21, 2009 (received for review August 11, 2009)
Abstract
ISG15 is an IFN-α/β?induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a ?loss of function? mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.
* interferon * antiviral * dsRNA * Herc5 * importin-α
Footnotes
* 1To whom correspondence should be addressed. E-mail: rkrug@mail.utexas.edu.
* Author contributions: C.Z., R.-L.K., and R.M.K. designed research; C.Z., T.-Y.H., and R.-L.K. performed research; C.Z., T.-Y.H., R.-L.K., and R.M.K. analyzed data; and C.Z. and R.M.K. wrote the paper.
* The authors declare no conflict of interest.
* This article is a PNAS Direct Submission.
* This article contains supporting information online at https://www.pnas.org/cgi/content/ful...DCSupplemental.
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