Characterizing the turnover of viral nonstructural proteins of the hepatitis C virus
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Abstract
After infection of the host cell, the RNA of the hepatitis C virus (HCV) is translated to the polyprotein followed by cleavage into the structural and nonstructural proteins. The structural proteins are released as viral particles from the host cell, while the equimolar amount of produced nonstructural proteins remain in the cell. Since there is no accumulation observed, a degradation of excess nonstructural proteins can be assumed. Autophagy is an important machinery for degradation and recycling and induced by HCV for replication and release. In this study, the focus was on autolysosomal degradation to investigate the fate of the HCV nonstructural proteins NS5B and NS4B and mainly NS3 and NS5A with consideration of a protein turnover by the proteasome.
The half-life of NS3, NS5A, NS5B and NS4B in untreated and treated cells was determined. Therefor, cells were treated to modulate autophagy and the proteasome and the de novo protein synthesis was prevented. Along with increased half-life after inhibition of proteasomal degradation in HCV-positive cells, nonstructural proteins had a higher half-life when autophagy was inhibited. Confocal immunofluorescence microscopy of immunostained cells revealed, that nonstructural proteins were found to colocalize with the lysosome marker protein LAMP2. The autophagosomal protein LC3 was overexpressed to enhance the autophagic flux. In cells with a stable LC3 overexpression, NS3 and NS5A were decreased, which indicates an upregulated autolysosomal degradation. Vice versa, a knockdown and knock out of LAMP2 impaired the protein turnover and showed an increase of nonstructural proteins. After isolation of autolysosomes as well as the HCV replicon complex, NS3 and NS5A were detected together with autophagosomal and lysosomal structures in the same fractions. This further confirms the relevance of autophagy in the fate of nonstructural proteins. The two populations of NS5A, distinguished by their phosphorylation form, differ with respect to their turnover of each form by autophagy in a time-dependent manner. Taken together, NS3 and NS5B were mainly affected by the crosstalk between autophagy and the proteasome, while degradation of NS5A and NS4B is favoured via the autolysosomal pathway. Therefore, beside HCV replication and release the autophagosomal machinery plays a crucial role in the turnover of excess HCV nonstructural proteins.