title: Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.
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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Cordek
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Tayler J
authors.initials: TJ
authors.lastname: Croom-Perez
authors.affiliation: the Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimri-ro, Seongdong-gu, Seoul, 133-791, Korea.
authors.forename: Jungwook
authors.initials: J
authors.lastname: Hwang
authors.affiliation: the Department of Chemistry, Saint Francis University, Loretto, Pennsylvania 15940.
authors.forename: Michele R S
authors.initials: MR
authors.lastname: Hargittai
authors.affiliation: the Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, and.
authors.forename: Chennareddy V
authors.initials: CV
authors.lastname: Subba-Reddy
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Qingxia
authors.initials: Q
authors.lastname: Han
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Lodeiro
authors.affiliation: the Huck Institutes of the Life Sciences, and.
authors.forename: Gang
authors.initials: G
authors.lastname: Ning
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: McCrory
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.affiliation: the Department of Pharmaceutical Science and Research, Marshall University School of Pharmacy, Huntington, West Virginia 25755.
authors.forename: Hasan
authors.initials: H
authors.lastname: Koc
authors.affiliation: the Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, and.
authors.forename: Brett D
authors.initials: BD
authors.lastname: Lindenbach
authors.affiliation: the Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Showalter
authors.affiliation: From the Department of Biochemistry and Molecular Biology, cec9@psu.edu.
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D020543:Proteome; D012367:RNA, Viral / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D064267:Intrinsically Disordered Proteins; D020543:Proteome; D012367:RNA, Viral; D014764:Viral Proteins; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Hepatitis C Virus (HCV); Intrinsically Disordered Protein; NMR; Non-structural Protein 5A; Phosphorylation; RNA Virus; Viral Replication