Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

Abstract

Edited by Charles E. SamuelHepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased RRM2 mRNA and protein expression levels. Moreover, siRNA-mediated silencing of RRM2 suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. RRM2 knockdown reduced NS5B expression, which depended on the protein degradation pathway, as NS5B RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that RRM2 silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.Hepatitis C virus (HCV) 3 is an enveloped positive-strand RNA virus belonging to the Flaviviridae family (1). HCV infects and replicates within quiescent hepatocytes, establishing a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. Although effective anti-HCV drugs have been developed, their low barrier to viral resistance and inability to prevent hepatocellular carcinoma development represent major clinical challenges (2).The HCV life cycle proceeds exclusively in the cytoplasm of host cells. Upon decapsidation, the viral genome is released into the cytoplasm, where it is translated into a large polyprotein that is processed by cellular and viral proteases to yield mature structural (core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (3). Following translation, these proteins become associated with a membranous web derived from the endoplasmic reticulum, in which viral genome replication takes place. NS3 through NS5B constitute the replication complex, and the positive-strand RNA genome serves as a template for the RNA-dependent RNA polymerase NS5B to produce the negative-sense replicative intermediate necessary for the generation of new positive-sense RNA genomes. These are in turn used as templates for further RNA replica...

Sorafenib Inhibits Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) in Hepatocellular Carcinoma Cells

“…It has been found that the HBV induces RRM2 expression via activating DNA damage response and targeting RRM2 by small molecules could inhibit HBV replication [58,59]. Similarly, RRM2 is also upregulated by the HCV and promotes viral RNA replication [60]. These studies suggest that RR-or RRM2-targeting agents may serve as potential antiviral agents for HBV/HCV infection and HBV/HCV-related HCC.…”

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits

“…For example, RNRs are almost ubiquitous among large double-stranded DNA (dsDNA) viruses, such as herpesviruses, poxviruses, and tailed phages (caudovirales), presumably due to high dNTP requirements during DNA replication [35][36][37]. On the other hand, most small dsDNA viruses and single-stranded DNA viruses do not encode RNRs and instead rely on host-encoded RNRs for deoxyribonucleotide production [38,39]. In addition to ribonucleotide reductase activity, some viral RNRs have been shown to engage in non-catalytic activities that result in proviral phenotypes.…”

Screening and Identification of Significant Genes in Hepatocellular Carcinoma via Bioinformatical Analysis and Validation in Clinical Tissue

“…Ribonucleotide reductase M2 (RRM2), a critical rate-limiting enzyme for DNA synthesis and repair, plays an important role in cell division, proliferation and differentiation by promoting cells proliferation and inhibiting cellular apoptosis. Host RRM2 and NS5B can inhibit RNA replication of hepatitis B virus, thereby inhibiting the conversion of hepatitis B to liver cancer (20). RRM2 may be a key gene for liver cancer transformation from cirrhosis via P53 pathway(21), Gao(22) suggested in his study that RRM2 was a candidate target for HCC therapy as suppression of RRM2 markedly suppressed proliferation of HCC cells and RRM2 expression was higher in HCC than in non-HCC tissue.…”