hnRNP L and NF90 Interact with Hepatitis C Virus 5′-Terminal Untranslated RNA and Promote Efficient Replication

Abstract

The 5=-terminal sequence of the hepatitis C virus (HCV) positive-strand RNA genome is essential for viral replication. Critical host factors, including a miR-122/Ago2 complex and poly(rC)-binding protein 2 (PCBP2), associate with this RNA segment. We used a biotinylated RNA pulldown approach to isolate host factors binding to the HCV 5= terminal 47 nucleotides and, in addition to Ago2 and PCBP2, identified several novel proteins, including IGF2BP1, hnRNP L, DHX9, ADAR1, and NF90 (ILF3). PCBP2, IGF2BP1, and hnRNP L bound single-stranded RNA, while DHX9, ADAR1, and NF90 bound a cognate double-stranded RNA bait. PCBP2, IGF2BP1, and hnRNP L binding were blocked by preannealing the single-stranded RNA bait with miR-122, indicating that they bind the RNA in competition with miR-122. However, IGF2BP1 binding was also inhibited by high concentrations of heparin, suggesting that it bound the bait nonspecifically. Among these proteins, small interfering RNA-mediated depletion of hnRNP L and NF90 significantly impaired viral replication and reduced infectious virus yields without substantially affecting HCV internal ribosome entry site-mediated translation. hnRNP L and NF90 were found to associate with HCV RNA in infected cells and to coimmunoprecipitate with NS5A in an RNA-dependent manner. Both also associate with detergent-resistant membranes where viral replication complexes reside. We conclude that hnRNP and NF90 are important host factors for HCV replication, at least in cultured cells, and may be present in the replication complex. IMPORTANCEAlthough HCV replication has been intensively studied in many laboratories, many aspects of the viral life cycle remain obscure. Here, we use a novel RNA pulldown strategy coupled with mass spectrometry to identify host cell proteins that interact functionally with regulatory RNA elements located at the extreme 5= end of the positive-strand RNA genome. We identify two, primarily nuclear RNA-binding proteins, hnRNP L and NF90, with previously unrecognized proviral roles in HCV replication. The data presented add to current understanding of the replication cycle of this pathogenic human virus.

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

“…For generation of the infectious virus, HCV-JFH1 RNA was in vitro transcribed and transfected into Huh7.5 cells. Supernatant from the transfected cells was concentrated and used for viral infection as described previously (19). Uninfected Huh7.5 cells were used as a mock control.…”

“…In a recent study, RNA binding proteins (RBPs) interacting with the HCV 5= UTR were identified through affinity pulldown experiments. Among them, hnRNPL and NF90 were found to affect HCV replication (19). La and PCBP2 proteins have been shown to interact with both of the UTRs and link the 5= and 3= ends of the viral genome, facilitating replication (22,23).…”

“…The most well-studied miRNA in the context of HCV is miR-122, a microRNA preferentially expressed in the liver cells that stimulates HCV translation and replication and alters its stability (12,13). Likewise, viral replication is also dependent on several host proteins, including cyclophilins (14,15), human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33) (16), phosphatidylinositol-4-kinase III alpha (PI4KIII-␣) (17), oxysterol-binding protein 1 (OSBP1) (18), NF90 and hnRNPL (19), etc. The importance of studying the role of these host proteins in the viral life cycle is underlined by the fact that many pharmaceutical agents directed against them, such as cyclophilin antagonists (20) and miR-122 inhibitors (21), are in clinical trials.…”

HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

“…Particularly, some factors are found not only to regulate RNA translation but also to modulate its replication (16,(18)(19)(20). Recently, hnRNP L and NF90 have been confirmed as the HCV RNA-binding proteins and have been found to coimmunoprecipitate with NS5A in an RNA-dependent manner (47). Depletion of hnRNP L and NF90 in HCV-infected cells impairs viral replication but does not affect HCV IRES translation (47).…”

“…Recently, hnRNP L and NF90 have been confirmed as the HCV RNA-binding proteins and have been found to coimmunoprecipitate with NS5A in an RNA-dependent manner (47). Depletion of hnRNP L and NF90 in HCV-infected cells impairs viral replication but does not affect HCV IRES translation (47). In the present study, we found a novel HCV RNA-binding factor, HMGB1, a host nucleic acid-binding protein, using co-IP and IP-RT-PCR techniques (Fig.…”