405 HCV NS5A phosphorylation by CK2 is genotype dependent

Background and Aim: NS5A, the major phosphoprotein of the hepatitis C virus (HCV), can be phosphorylated by casein kinase 2 (CK2), a pleiotropic cellular kinase often involved in many viral functions including replication and pathogenic mechanisms. Aim of this study was to investigate the effects of a different number and localization of CK2 recognition sites observed in NS5A natural variants from patients infected with different HCV genotypes. Materials and Methods: Sequence analysis was performed on the full-length NS5A gene from 28 patients with HCV-la, 14 with HCV-lb and 21 with HCV-3. Both, synthetic peptides reproducing the 60aa at the C-terminus of the NS5A and full-length recombinant NS5A proteins were produced and assessed in in vitro CK2 phosphorylation assays. Results: The number of predicted CK2 phosphorylatable residues was highly variable both among NS5A variants from diverse genotypes and from different isolates belonging to the same genotype. Divergences were mainly localized at the C-terminus of the viral protein. The highest heterogeneity with regards to both the number and the localization of the potential CK2 sites was found among patients with HCV-la genotype (from 1 to 5 sites) while, in HCV-3 genotype infected patients, four highly conserved predicted sites were identified. Kinetic assays with peptide substrates confirmed a higher affinity of CK2 for substrates having a better consensus sequence, reflecting the number of identified potential sites. These evidences were further confirmed by the autoradiography results obtained after in vitro phosphorylation of full-length NS5A variants. Furthermore, a 45 kDa truncated fragment, endogenously generated by proteolytic cleavage when NS5A proteins were expressed in mammalian cells, was a better substrate for CK2. Western blot analysis with antibodies against the C-terminal His-tag suggests that the N-terminal of NS5A can decrease its CK2 mediated phosphorilatability. Conclusions: Naturally occurring NS5A variants contain a number of functional active CK2 phosphorylation sites that is related to the HCV genotype. Moreover, this heterogeneity in the phosphorylatability grade could represent an important post-translational regulatory mechanism conferring distinct NS5A biological activities proper of each genotype.