The Casein Kinase 2‐Dependent Phosphorylation of NS5A Domain 3 from Hepatitis C Virus Followed by Time‐Resolved NMR Spectroscopy

Abstract

Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A-D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2-dependent phosphorylation sites, we monitored the reaction between NS5A-D3 and CK2 in vitro by time-resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly-and in the modulation of the viral activity-at the molecular level.

Overall Structural Model of NS5A Protein from Hepatitis C Virus and Modulation by Mutations Confering Resistance of Virus Replication to Cyclosporin A

“…Globally in the NS5A-D1D2D3 sample, the phosphorylation levels are 33%, 100%, 62%, 77%, 63%, 67%, 67%, 67% and 64% for the residues Ser 401 , Ser 408 , Ser 412 , Ser 415 , Ser 429 , Ser 432 , Ser 434 , Thr 435 , and Ser 437 respectively. Our results are in line with a recent study showing that in vitro the casein kinase 2 quickly phosphorylates S408 in NS5A-D3, whereas S401, S429, and S434 are more slowly and less efficiently modified . Despite the presence of numerous serine and threonine residues in LCS1-domain2 and despite the fact that phosphorylations have already been reported in these regions, , we did not detect any phosphorylation here by NMR spectroscopy.…”

“…From a structural point of view, the present NMR analyses revealed that phosphorylations do not change the disordered nature of D2 and D3 domains but multiply the number of conformers due to partial phosphorylations. In line with our results, Secci et al have concluded that the phosphorylation of pS401, pS408, pS429, and pS432 in NS5A-D3 do not influence its secondary structure content . Differential phosphorylation patterns could give rise to a huge number of NS5A phosphovariants probably serving various functions .…”

“…Our results are in line with a recent study showing that in vitro the casein kinase 2 quickly phosphorylates S408 in NS5A-D3, whereas S401, S429, and S434 are more slowly and less efficiently modified. 61 Despite the presence of numerous serine and threonine residues in LCS1-domain2 and despite the fact that phosphorylations have already been reported in these regions, 62,63 we did not detect any phosphorylation here by NMR spectroscopy. In fact, as reported in the following section, phosphorylated residues were indeed identified in LCS1-Domain2 by mass spectroscopy in the NS5A-D1D2D3 sample (Table 2 and Figure 1), indicating that their NMR signals were broadened beyond detection.…”

“…In line with our results, Secci et al have concluded that the phosphorylation of pS401, pS408, pS429, and pS432 in NS5A-D3 do not influence its secondary structure content. 61 Differential phosphorylation patterns could give rise to a huge number of NS5A phosphovariants probably serving various functions. 91 Phosphorylations could regulate the interactions of NS5A with other viral or host factors both by influencing the folding of its molecular recognition elements and by the destruction of intermolecular interactions, or conversely by the formation of new interaction sites.…”

Quo Vadis Biomolecular NMR Spectroscopy?

“…By exploiting the non-destructive and quantitative nature of such NMR measurements, we illustrated another analytical advantage of this approach: The ability to directly follow PTM reactions in a time-resolved fashion in order to deduce site-specific modification rates [97,98]. Indeed, protein phosphorylation studies by time-resolved NMR spectroscopy became very popular and proved essential for delineating mechanistic insights into diverse sets of signaling reactions [99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135].…”