Background/Aim: The state of cellular hydration changes under the influence of hormones, cumulative substrate uptake and oxidative stress resulting in cellular shrinkage or swelling. We aimed at investigating whether anisoosmolarity has an effect on HCV replication.
Methods: Huh9–13 cells carrying a subgenomic hepatitis C replicon, were cultured in either hypoosmotic (220 mosmol/l), isoosmotic (320 mosmol/l) or hyperosmotic media (420 mosmol/l) for 48h. Cytotoxicity was measured via XTT assay. Levels of genomic virus RNA were measured by RT-PCR and alterations in viral protein expression were measured by western blotting. Gene expression profiling was performed via cDNA microarray analysis and differentially expressed proteins were identified by 2D-DiGE analysis and subsequent ESI-Qq-TOF MS.
Results: With no cytotoxicity measured after 48h, hypoosmolarity reduced replicon RNA levels by 36% (p<0,05) and hyperosmolarity led to a 70% rise (p<0,05). Similar results were achieved using the uncharged and membrane impermeable osmolyte raffinose instead of NaCl. Changes were not only induced in replicon RNA levels but also in HCV protein expression levels. Furthermore hyperosmolarity partially reversed the antiviral effect of interferon-α2a. Whilst incubation with IFN-α (1000 IU/ml) markedly reduced replicon RNA levels by 90% (p<0,001) as expected, hyperosmolarity raised them by a 4-fold. cDNA microarray analysis showed regulation of 62 transcripts under hypoosmotic vs. 218 transcripts under hyperosmotic conditions. 64 transcripts were regulated by anisotonicity in general. Proteomics revealed 45 differential spots in 2D-DiGE analysis yielding 19 non-redundant differentially expressed proteins after ESI-Qq-TOF MS.
Conclusions: HCV replication in the replicon system is osmo-sensitive. We are currently evaluting all data in order to identify host factors and to further clarify the link between sensitivity to anisoosmolarity and HCV replication.
Anisotonicity - Cell shrinkage - Cell swelling - Hepatitis C - subgenomic Replicon
