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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.

Author information

1
From the Department of Biochemistry and Molecular Biology.
2
the Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul, 133-791, Korea.
3
the Department of Chemistry, Saint Francis University, Loretto, Pennsylvania 15940.
4
the Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, and.
5
the Huck Institutes of the Life Sciences, and.
6
the Department of Pharmaceutical Science and Research, Marshall University School of Pharmacy, Huntington, West Virginia 25755.
7
the Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802.
8
From the Department of Biochemistry and Molecular Biology, cec9@psu.edu.

Abstract

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.

KEYWORDS:

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

PMID:
25031324
PMCID:
PMC4148867
DOI:
10.1074/jbc.M114.589911
[Indexed for MEDLINE]
Free PMC Article

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