PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication

Abstract

Background Hepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection. Methods Wild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc. Results Mass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B’delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B. Conclusions PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Emerging Roles of B56 Phosphorylation and Binding Motif in PP2A-B56 Holoenzyme Biological Function

“…The classic example is that the small T antigen (ST) of simian virus 40 (SV40) interacts with the PP2A scaffolding A by displacing regulatory B subunits, thereby inhibiting PP2A-mediated dephosphorylation of numerous substrates and ultimately promoting viral replication [ 49 ]. In particular, the great majority of viral proteins believed to engage with the regulatory subunit of PP2A exclusively target B56 [ 47 , 48 , 50 ]. For instance, Co-IP analysis verified the specific interaction between B56δ and HCV NS5B, which is essential for HCV infection in hepatoma cells by promoting HCV replication [ 50 ].…”

“…In particular, the great majority of viral proteins believed to engage with the regulatory subunit of PP2A exclusively target B56 [ 47 , 48 , 50 ]. For instance, Co-IP analysis verified the specific interaction between B56δ and HCV NS5B, which is essential for HCV infection in hepatoma cells by promoting HCV replication [ 50 ]. In recent years, the identification of B56 binding motifs LxxIxE in various viruses has provided a reasonable explanation for this phenomenon and is also conducive to understanding how viral hijacking of PP2A-B56 can produce different biochemical and phenotypic events ( Figure 4 ).…”

Research progress and application of liver organoids for disease modeling and regenerative therapy

“…Viral hepatitis is an inflammatory liver disease caused by viral infection, with five main types, among which hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most common, both primarily transmitted through bodily fluids [ 67 ]. Due to the narrow species tropism exhibited by hepatitis virus, infecting mainly humans and higher primates, suitable animal models are lacking, hindering the development and clinical testing of antiviral drugs [ 68 ], although human hepatocellular carcinoma cell lines (such as Huh7 [ 69 ], HepG2 [ 70 ], and HepaRG [ 71 ]) have been utilized to establish models of hepatitis virus infection, but prolonged extensive culturing of these 2D cancer cell lines harbors significant genetic, epigenetic, and functional alterations, severely compromising the fidelity of recapitulating virus-host interactions and evaluating antiviral drug efficacy. The establishment of human liver organoid infection models provides a unique opportunity to circumvent these contemporary challenges.…”