1. Expanding the Proteome of an RNA Virus by Phosphorylation of an Intrinsically Disordered Viral Protein.
- Author
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Cordek, Daniel G., Croom-Perez, Tayler J., Hwang, Jungwook, Hargittai, Michele R. S., Subba-Reddy, Chennareddy V., Qingxia Han, Lodeiro, Maria Fernanda, Gang Ning, McCrory, Thomas S., Arnold, Jamie J., Koc, Hasan, Lindenbach, Brett D., Showalter, Scott A., and Cameron, Craig E.
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PROTEIN transport , *PROTEIN synthesis , *METABOLISM in RNA viruses , *PHOSPHORYLATION kinetics , *VIRAL proteins , *PHYSIOLOGY - Abstract
The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline- II motifs are often located near sites of phosphorylation. Wehave used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus nonstructural 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 15N-, 13C-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 hepatitisCvirus 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. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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