Expanding the Proteome of an RNA Virus by Phosphorylation of an Intrinsically Disordered Viral Protein

• Published in: The Journal of biological chemistry 2014-08, Vol.289 (35), p.24397-24416
• Main Authors: Cordek, Daniel G., Croom-Perez, Tayler J., Hwang, Jungwook, Hargittai, Michele R.S., Subba-Reddy, Chennareddy V., Han, Qingxia, Lodeiro, Maria Fernanda, Ning, Gang, McCrory, Thomas S., Arnold, Jamie J., Koc, Hasan, Lindenbach, Brett D., Showalter, Scott A., Cameron, Craig E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Cell Line; Cyclic AMP-Dependent Protein Kinases - metabolism; DNA Primers; Hepacivirus - genetics; Hepacivirus - metabolism; Hepacivirus - physiology; Humans; Intrinsically Disordered Proteins - metabolism; Microbiology; Molecular Sequence Data; Phosphorylation; Polymerase Chain Reaction; Proteome; RNA, Viral - genetics; Tandem Mass Spectrometry; Viral Proteins - metabolism; Virus Replication
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.589911

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 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 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.