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

Daniel G. Cordek, Tayler J. Croom-Perez, Jungwook Hwang, Michele R.S. Hargittai, Chennareddy V. Subba-Reddy, Qingxia Han, Maria Fernanda Lodeiro, Gang Ning, Thomas S. McCrory, Jamie J. Arnold, Hasan Koc, Brett D. Lindenbach, Scott A. Showalter, Craig E. Cameron

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)24397-24416
Number of pages20
JournalJournal of Biological Chemistry
Volume289
Issue number35
DOIs
StatePublished - 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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