Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein [Cell Biology]

July 16th, 2014 by Cordek, D. G., Croom-Perez, T. J., Hwang, J., Hargittai, M. R. S., Subba-Reddy, C. V., Han, Q., Lodeiro, M. F., Ning, G., McCrory, T. S., Arnold, J. J., Koc, H., Lindenbach, B. D., Showalter, S. A., Cameron, C. E.

The human proteome contains myriad intrinsically disordered proteins (IDPs). Within IDPs, 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 (IDD) of hepatitis C virus (HCV) non-structural protein 5A (NS5A) on T2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A IDD 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 SH3 domain of c-Src, consistent with formation of a complex. Changing T2332 to alanine in HCV 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 T2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce pT2332-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.