Elsevier

Journal of Hepatology

Volume 47, Issue 6, December 2007, Pages 768-776
Journal of Hepatology

Heterogeneity of CK2 phosphorylation sites in the NS5A protein of different hepatitis C virus genotypes

https://doi.org/10.1016/j.jhep.2007.07.020Get rights and content

Background/Aims

The hepatitis C virus NS5A protein is phosphorylated by several cellular kinases, including casein kinase 2 (CK2). Little is known about CK2 phosphorylation of NS5A from different HCV genotypes and clinical isolates.

Methods

NS5A from patients with HCV-1a (24 cases), HCV-1b (9) or HCV-3 (16) was analyzed by direct sequencing and CK2 phosphorylation sites were defined using a well-validated prediction rule. In vitro phosphorylation assays were performed using recombinant CK2 and synthetic peptides or full-length NS5A. In vivo phosphorylation by endogenous CK2 of NS5A expressed in hepatoma cells was also investigated.

Results

The mean number of CK2 sites within full-length NS5A, was significantly higher in HCV-3 compared to HCV-1a (P < 0.01) and HCV-1b (P < 0.01). The number of CK2 sites was more homogeneous in HCV-3 variants compared to HCV-1a and HCV-1b variants (P < 0.05). The number of predicted CK2 sites correlated with the degree of in vitro and in vivo phosphorylation of NS5A by CK2.

Conclusions

CK2-dependent phosphorylation of NS5A is heterogeneous among different HCV genotypes and clinical isolates. This might have an influence on virus biology and pathogenicity.

Introduction

The hepatitis C virus (HCV), one of the major causes of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide, expresses several structural and non-structural proteins. Among different HCV proteins, the non-structural 5A protein (NS5A) has a key role in the virus life cycle being involved in viral RNA replication [1]. NS5A has perinuclear cytoplasmic location and associates with the endoplasmic reticulum where, with other non-structural viral proteins, it forms the replicative complex [1], [2], [3].

NS5A and its truncated forms [4], [5], [6], [7], [8], have been implicated in the modulation of several cellular mechanisms. Some of the truncated forms migrate to the nucleus [9] where they act as a transcriptional activator with consequences on cell growth, apoptosis, and lipid metabolism [10], [11]. NS5A can also alter major cellular pathways by directly interacting with cellular proteins [12], [13], [14]. Finally, NS5A can hamper the antiviral treatment response by blocking interferon effectors including PKR [4] and 2′–5′ OAS [15].

Many NS5A functions depend on protein phosphorylation [16], being NS5A the main phosphoprotein of HCV. Two phosphorylated forms of NS5A, p56 and p58, have been described [17]. Different cellular kinases are responsible for NS5A phosphorylation and some of them belong to the CMGC kinase family [18], including CK2 [19]. It has been suggested that a hierarchical phosphorylation process is responsible for the whole NS5A phosphorylation where CK2 might be the primary kinase while, other kinases phosphorylate NS5A after CK2 [20]. CK2 is an ubiquitous, constitutively active Ser/Thr protein kinase with more than 300 known substrates implicated in many cellular functions [21]. CK2 phosphorylates a growing list of viral proteins leading to important changes in their biological activity [22], [23], [24], [25], [26], [27], [28], [29]. To date, CK2 recognition sites have been only partially mapped within full-length NS5A of HCV and data are available only for HCV-1b.

Here we describe analyses of CK2 recognition sites in natural NS5A variants from patients infected with different HCV genotypes. In vitro and in vivo CK2 phosphorylation of NS5As with different predicted sites was also investigated using synthetic peptides and full-length proteins expressed in transfected cells.

Section snippets

Patients

HCV-RNA was obtained from sera of 49 patients infected with HCV-1a (24 cases), HCV-1b (9 cases) and HCV-3 (16 cases). All patients had evidence of chronic hepatitis on liver biopsy.

NS5A sequencing

HCV-RNA was extracted, reverse transcribed and amplified by nested PCR with genotype-specific primers (see Table 1) as previously described [30]. PCR products were sequenced (BMR-Genomics, Italy) and sequences were corrected (Chromas2.23v; http://www.technelysium.com.au/chromas.html), aligned with SeqManII (DNASTAR,

NS5A sequence analyses

Forty-nine full-length NS5A sequences, from patients infected with different HCV genotypes, were analyzed. As expected, the NS5A protein was 448 aa long in HCV-1a, 447 aa in HCV-1b, and 452 aa in HCV-3. Potential CK2 phosphorylation sites, identified according to the consensus sequence (see Section 2) are described in Fig. 1. CK2 phosphorylation sites were identified in all 49 NS5As but their frequency was variable, with a mean number of 10.3 ± 1.0 sites (range: 8–11) for HCV-3, of 7.4 ± 1.4 sites

Discussion

The primary aim of this study was to map CK2 recognition sites within the NS5A from several clinical HCV isolates belonging to the most common genotypes.

CK2 plays a key role in the life cycle of many viruses [22], [23], [24], [25], [26], [27], [28], [29]. A previous study [19] showed that CK2 is indeed able to phosphorylate HCV-NS5A but this issue was not further analyzed in detail.

Our results show that the number and localization of CK2 sites are heterogeneous among HCV isolates with major

Acknowledgments

We thank Dr. Stefania Sarno and Dr. Chiara Romualdi at the University of Padova, for providing us the recombinant CK2 heterotetramer and for statistical analyses support, respectively. This work is part of the activities of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a Grant (LSHM-CT-2004-503359) from the Priority 1 “Life Sciences, Genomics and Biotechnology for Health” programme in the 6th Framework Programme of the EU. This work was also supported by

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    The authors declare that they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

    These authors contributed equally to this work.

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