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J Biol Chem. 2014 Aug 29;289(35):24397-416. doi: 10.1074/jbc.M114.589911. Epub 2014 Jul 16.

Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

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From the Department of Biochemistry and Molecular Biology.
the Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul, 133-791, Korea.
the Department of Chemistry, Saint Francis University, Loretto, Pennsylvania 15940.
the Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, and.
the Huck Institutes of the Life Sciences, and.
the Department of Pharmaceutical Science and Research, Marshall University School of Pharmacy, Huntington, West Virginia 25755.
the Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802.
From the Department of Biochemistry and Molecular Biology,


The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.


Hepatitis C Virus (HCV); Intrinsically Disordered Protein; NMR; Non-structural Protein 5A; Phosphorylation; RNA Virus; Viral Replication

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