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Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

ABSTRACT: Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways.HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

SUBMITTER: Arora P

PROVIDER: S-EPMC5397368 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

Arora Payal P Basu Amartya A Schmidt M Lee ML Clark Geoffrey J GJ Donninger Howard H Nichols Daniel B DB Calvisi Diego F DF Kaushik-Basu Neerja N

Hepatology (Baltimore, Md.) 20170330 5

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, whic ...[more]

PMID: 28090674

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Project description:Simple Summary The loss of function of the BRCA1 tumor suppressor is common in breast cancer, but experimentally, the BRCA1 inactivation promotes cell senescence. The overstimulation of RAS signaling via Her2 upregulation is also common in breast cancer, yet it can also lead to senescence. A significant percent of primary breast tumors exhibit both defects. So how do they overcome the senescence response to become successful tumors? Here we show that the RAS senescence effector NORE1A can complex with BRCA1 and that loss of NORE1A abrogates the senescence-inducing effects of Her2 and BRCA1 dysregulation. Database analysis shows that NORE1A loss of expression is common in primary breast tumors and correlates with BRCA1 loss in Her2+ but not Her2− cases. Abstract BRCA1 is a tumor suppressor with a complex mode of action. Hereditary mutations in BRCA1 predispose carriers to breast cancer, and spontaneous breast cancers often exhibit defects in BRCA1 expression. However, haploinsufficiency or suppression of BRCA1 expression leads to defects in DNA repair, which can induce DNA damage responses, leading to senescence. Activating mutation or overexpression of the Her2 oncoprotein are also frequent drivers of breast cancer. Yet, over-activation of Her2, working through the RAS oncoprotein, can also induce senescence. It is thought that additional defects in the p53 and Rb tumor suppressor machinery must occur in such tumors to allow an escape from senescence, thus permitting tumor development. Although BRCA1 mutant breast cancers are usually Her2 negative, a significant percentage of Her2 positive tumors also lose their expression of BRCA1. Such Her2+/BRCA1− tumors might be expected to have a particularly high senescence barrier to overcome. An important RAS senescence effector is the protein NORE1A, which can modulate both p53 and Rb. It is an essential senescence effector of the RAS oncoprotein, and it is often downregulated in breast tumors by promotor methylation. Here we show that NORE1A forms a Her2/RAS regulated, endogenous complex with BRCA1 at sites of replication fork arrest. Suppression of NORE1A blocks senescence induction caused by BRCA1 inactivation and Her2 activation. Thus, NORE1A forms a tumor suppressor complex with BRCA1. Its frequent epigenetic inactivation may facilitate the transformation of Her2+/BRCA1− mediated breast cancer by suppressing senescence.

Project description:Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Project description:Background & Aims:Uncontrolled liver proliferation is a key characteristic of liver cancer; however, the mechanisms by which this occurs are not well understood. Elucidation of these mechanisms is necessary for the development of better therapy. The oncogene Gankyrin (Gank) is overexpressed in both hepatocellular carcinoma and hepatoblastoma. The aim of this work was to determine the role of Gank in liver proliferation and elucidate the mechanism by which Gank promotes liver proliferation. Methods:We generated Gank liver-specific knock-out (GLKO) mice and examined liver biology and proliferation after surgical resection and liver injury. Results:Global profiling of gene expression in GLKO mice showed significant changes in pathways involved in liver cancer and proliferation. Investigations of liver proliferation after partial hepatectomy and CCl4 treatment showed that GLKO mice have dramatically inhibited proliferation of hepatocytes at early stages after surgery and injury. In control LoxP mice, liver proliferation was characterized by Gank-mediated reduction of tumor-suppressor proteins (TSPs). The failure of GLKO hepatocytes to proliferate is associated with a lack of down-regulation of these proteins. Surprisingly, we found that hepatic progenitor cells of GLKO mice start proliferation at later stages and restore the original size of the liver at 14 days after partial hepatectomy. To examine the proliferative activities of Gank in cancer cells, we used a small molecule, cjoc42, to inhibit interactions of Gank with the 26S proteasome. These studies showed that Gank triggers degradation of TSPs and that cjoc42-mediated inhibition of Gank increases levels of TSPs and inhibits proliferation of cancer cells. Conclusions:These studies show that Gank promotes hepatocyte proliferation by elimination of TSPs. This work provides background for the development of Gank-mediated therapy for the treatment of liver cancer. RNA sequencing data can be accessed in the NCBI Gene Expression Omnibus: GSE104395.

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Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

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Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

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