Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

Abstract

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.Hepatitis C virus (HCV) is a major cause of chronic hepatitis around the world (1, 7). Chronic infection with HCV results in liver cirrhosis and may lead to hepatocellular carcinoma (53, 54). HCV is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae. The HCV RNA genome is ϳ9.6 kb in length and consists of a 5Ј nontranslated region (NTR), a large open reading frame, and a 3Ј NTR. The 5Ј NTR contains an internal ribosome entry site, which mediates the translation of a single polyprotein of ϳ3,000 amino acid (aa) residues (61, 64). This polyprotein is cleaved by host and viral proteases into at least 10 different products (33). At the amino terminus of the polyprotein are the core protein, E1, and E2, followed by p7, a hydrophobic peptide with unknown function, and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The 3Ј NTR consists of a short variable sequence, a poly(U)-poly(UC) tract, and a highly conserved X region and is critical for HCV RNA replication and HCV infection (13,29,69,71).HCV is unique among positive-strand RNA viruses in that it causes persistent and chronic infections. In addition, the high mutation rate of the gene encoding the E2 protein allows it to escape host immune surveillance, which is strongly associated with chronic inflammation of the liver (19,23,66,67). As a result...

Nucleolin Is Required for Efficient Nuclear Egress of Herpes Simplex Virus Type 1 Nucleocapsids

“…Furthermore, viral DNA that accumulates in cells infected with the UL12-null mutant virus has been shown to have an aberrant structure compared to that in cells infected with wild-type HSV-1 (29). Since nucleolin has been reported to interact with and regulate a variety of cellular and viral enzymes (e.g., simian virus 40 large tumor-antigen DNA helicase [54], RNA-dependent RNA polymerase activity of the hepatitis C virus NS5B protein [56], telomerase [22], and recombinase Rad51 [4]) and to play multiple roles in DNA metabolism (9,34), nucleolin might interact with UL12 and modify its ability to process viral DNA in infected cells. However, we should note that nucleolin knockdown cannot completely account for the role of UL12 in nuclear egress of nucleocapsids, based on the observation that the HSV-1 UL12-null mutant virus produced a 100-to 1,000-fold decrease in virus titer in Vero cells (55), whereas in the present study nucleolin knockdown produced only a modest (1.5-to 3.2-fold) decrease in Vero cells.…”

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells

“…These include factors that affect immune responses (interferons and their related genes [8,9], RIG-I, TLRs [10]), cell proliferation (BMP7 [11], TGF-beta [12], nucleolin [13]), molecular chaperone function (cyclophilin [14], ER-stress proteins [15], FKBP [16], HSP27 [17], HSP90 [18]) and lipid metabolism (cholesterol, sphingolipid [19]). However, it is often difficult to determine whether these genes are changed by HCV replication or the changes are essential for HCV replication in the host cells.…”

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

“…NCL colocalizes with hepatitis D virus delta antigen and interacts with herpes simplex virus 1 protein US11, cytomegalovirus UL44, white-spot syndrome virus VP15, influenza virus (H3N2) NS1, and the NS5B protein of hepatitis C virus. These interactions have been shown to mediate a number of different processes, including protein synthesis, RNA replication, release of virions, and trafficking of viral proteins (48)(49)(50)(51)(52)(53)(54). In addition, NCL has been shown to interact with the untranslated regions (UTR) of several other viruses, including tombusvirus, feline calicivirus, Norwalk virus, and poliovirus, where it may play roles in virus replication (55)(56)(57).…”