Hijacking the cytoskeleton: association of HCV polymerase with α/β-tubulin and its potential relation to the viral replication and cell proliferation.

Hepatitis C virus (HCV) manipulates host cellular pathways to create favourable conditions that support its replication and persistence. Identifying virus-host interactions helps to prevent disease progression and supports the development of host-directed antiviral therapies (HDTs) with a reduced risk of drug resistance due to viral mutations. In this study, a proteomic approach using in vitro pulldown assay and mass spectrometry identified α- and β-tubulin as novel cellular proteins physically associated with the viral RNA-dependent RNA polymerase (NS5B). This association was validated in hepatic (Huh7) and non-hepatic (HEK293T) cells. Further analysis confirmed that the interaction between NS5B and α/β-tubulin is an indirect interaction mediated by unidentified protein(s). Domain mapping analysis using NS5B-deletion mutants localized the tubulin-interacting region of NS5B to its N-terminal domain. Nocodazole, a known inhibitor of α- and β-tubulin polymerization, significantly reduced the association between NS5B and α-tubulin in vitro and in vivo settings, but had no notable impact on the NS5B/β-tubulin interaction. Additionally, nocodazole treatment markedly inhibited RNA replication of HCV subgenomic replicon in Huh7 cells in a dose-dependent manner. These results suggest an association between tubulin proteins and HCV RNA replication, potentially involving the interaction with NS5B. In addition, NS5B expression was associated with increased cell proliferation, indicating a possible link between NS5B/tubulin interaction, microtubule dynamics, and cellular transformation. Further studies are required to determine whether these associations reflect direct functional roles in HCV RNA replication, trafficking, virus assembly and/or cellular transformation.