Localization determines function: N-terminally truncated NS5A fragments accumulate in the nucleus and impair HCV replication

https://doi.org/10.1016/j.jhep.2008.11.024Get rights and content

Background/Aims

The Hepatitis C Virus (HCV) nonstructural protein 5A (NS5A) is an essential part of the ER-localized HCV–replicon complex. Although NS5A harbours a conserved NLS in its C-terminal domain, NS5A is associated with the cytoplasmic face of the ER by an amphipathic helix close to its N-terminus.

Methods

Intracellular distribution of NS5A in HCV replicating cells was analyzed by confocal microscopy and subcellular fractionation. The effect on HCV replication was analyzed using the JFH-1-based infection/replication system.

Results

During viral life cycle N-terminally truncated NS5A fragments are caspase-dependent formed that lack the ER-attachment signal and are localized within the nucleus. These N-terminally truncated fragments inhibit HCV replication. If their formation is blocked by inhibition of caspases HCV replication is increased.

The C-terminal domain of NS5A binds to c-Raf and thereby localizes it to the replicon complex. This interaction is essential for HCV replication. The N-terminally truncated NS5A fragments are still able to bind c-Raf. However, due to their nuclear localization they withdraw c-Raf from the replicon complex into the nucleus resulting in an impaired HCV replication.

Conclusions

Formation of N-terminally truncated NS5A fragments could represent a mechanism to regulate HCV replication by withdrawal of essential factors from the replicon complex.

Introduction

Hepatitis C virus (HCV) is a prevalent pathogen associated with a high subsequent risk of progression to liver cirrhosis and hepatocellular carcinoma (HCC) [1], [2], [3]. The HCV genome is a single-stranded positive-sense RNA molecule of approximately 9600 bases. The viral RNA codes for one large polyprotein of approximately 3100 amino acids that is posttranslationally processed by cellular and viral proteases [4]. The N-terminus encompasses the structural proteins core, E1 and E2, the C-terminus encompasses the p7 protein and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B [4], [5]. The mature NS5A protein is generated by the action of the NS3/NS4A serine protease. NS5A is a phosphoprotein that exists in a basal or in a hyperphosphorylated state (p56 and p58). Via an amphipathic α-helix, NS5A is associated with the cytoplasmic face of the ER and is an integral part of the replication complex [4], [6], [7], [8], [9]. Although NS5A harbours a classic nuclear localization signal (NLS) in its C-terminal domain, intact NS5A is predominantly found in the extranuclear compartment due to its ER-association mediated by the N-terminal ER-attachment signal. N-terminal deletion mutants of NS5A are preferentially localized within the nucleus and are reported to exert a function as transcriptional regulator. Recent reports describe in detail that NS5A can be cleaved by caspases 3 and 6 and by Ca2+-dependent calpain proteases which results in the formation of N-terminally truncated NS5A fragments [10], [11], [12].

As an integral part of the HCV replicon complex NS5A is able to interfere with a variety of viral and cellular proteins [13], [14], [15], [16], [17], [18], [19], [20], [21]. Some of these interaction partners seem to be relevant for triggering a deregulation of host cell signal transduction, such as Grb2, PI3K, p53 or c-Raf. In case of c-Raf it was shown that the interaction of c-Raf with NS5A is mediated by the C-terminal domain of NS5A. Moreover, it was shown that integrity of c-Raf is crucial for HCV replication [16]. This study was performed mainly in the JFH-1-based HCV replication system. This enables the analysis of the complete life cycle of infectious viral particles with the aim to analyze: (i) whether truncated NS5A fragments are generated in HCV-replicating cells and (ii) to characterize their potential relevance for viral life cycle.

Section snippets

Plasmids

All plasmids were generated using standard technology. To generate the NS5A genotype 1b-specific fragments by PCR the sequence 5′-gcgtatctagaagcttcttcagcagcagac-gatgtcgtc-3′ was used as backward primer. The forward primers were: for NS5A 1–449, gcgcgtcgacgcgggatccgtatgggctccggctcgtggcta, for NS5A 31–449, gcgcgtcgacgcgggatccgtatgc-cgaaattgccgggagtc, for NS5A 105–449, gcgcgtcgacgc-gggatccgtatgccgaactattccagggcg, for NS5A 221–449, gcgcgtcgacgcgggatccgta-tgacagcagagacggctaaa and for NS5A 302–449,

During viral life cycle nuclear-localized N-terminally truncated NS5A fragments are generated

The hepatitis C virus protein NS5A is localized on the ER despite the presence of a functional nuclear localization signal (NLS) in its C-terminal region (amino acids (aa) 354–362) [26], [27]. An amphipathic α-helix localized close to the N-terminus mediates the attachment to the ER. The question was whether proteolytic processing of NS5A occurs in HCV-replicating cells and where the resulting fragments are localized.

Western blot analysis of cellular lysates derived from HCV-infected cells

Discussion

We were able to show that during the life cycle of HCV genotype 2a N-terminally truncated fragments of the NS5A protein are formed. These N-terminally truncated fragments have the capability to translocate into the nucleus. Inhibition of caspases resulted in the absence of nuclear localized NS5A fragments. To investigate the physiological significance of these fragments for the viral life cycle, N-terminally truncated fragments were overproduced in HCV-replicating cells. The overexpression of

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

    These authors contributed equally to this work.

    Present address: University Medical Center Hamburg – Eppendorf, Clinic of Radiotherapy and Radiooncology, D-20246 Hamburg, Germany.

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