Affinity Capture and Identification of Host Cell Factors Associated with Hepatitis C Virus (+) Strand Subgenomic RNA

Abstract

Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequencespecific biotinylated peptide nucleic acid ( The hepatitis C virus, a blood-borne pathogen that causes chronic hepatitis, is the primary reason for liver transplantation in the United States. HCV 1 preferentially replicates in liver tissue without any direct cytopathic effect and thus is able to maintain long term, persistent infection. More than 50% of HCV-infected patients do not respond to treatment; instead, the majority of patients develop chronic hepatitis C, which leads to progressive liver fibrosis, cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The hepatitis C virus is a positive single-stranded RNA virus of a 9.6-kb genome. After its entry into cells, the (ϩ) strand RNA first serves as a messenger RNA for the translation of viral proteins. Newly synthesized HCV replicase (NS5B) then copies the (ϩ) strand RNA genome into the (Ϫ) strand RNA, which serves as a template for the production of the viral genome. The conserved 5Ј-and 3Ј-nontranslated (5ЈNTR and 3ЈNTR) regions of the HCV genome have multiple regulatory elements that are essential for replication of HCV and translation of viral proteins. Although the 5ЈNTR of HCV contains the internal ribosomal entry site, which is required for capindependent translation of (ϩ) strand HCV RNA (1-4), it is also the 3Ј region of the (Ϫ) strand RNA, which functions as the initiation site for replication of the (ϩ) strand HCV RNA genome. The 3Ј regions of both (Ϫ) and (ϩ) strand HCV RNAs are highly structured and serve as the initiation sites for viral replication (5). Various cellular proteins have been shown to interact with 5ЈNTR of HCV RNA; these include La autoantigen (6) nuclear factors NF90, NF110, NF45, and RNA helicase A (7), as well as the polypyrimidine tract-binding protein (8 -10). Recently, we affinity-captured different cellular proteins interacting with HCV 3ЈNTR and identified them by LC/MS/ MS; some of these proteins were found to be essential for HCV replication as confirmed by siRNA (11).Another recent study using sequence-specific gene silencing of the RNAi screen has identified 26 human genes encoding proteins that physically interact with HCV RNA or protein and modulate HCV replication (12). A more direct approach would be to capture the replicating HCV RNA genome in situ under physiological conditions and then identify all the cellular From the §Department of Biochemistry and Molecular Biology and Centre for the Study of Emerging and Re-emerging Pathogens,

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

“…ILF2, ILF3, DDX3, DDX6, hnRNPA1, and DHX9 (45,46,48,49,61,62,69,70), suggesting that these proteins could be part of common functional virus-host RNP complexes. Hence, the data described in these studies could be reconciled with a proposed YB-1-containing RNP complex (44).…”

Entangled in a membranous web: ER and lipid droplet reorganization during hepatitis C virus infection

“…Heterologous nuclear ribonucleoprotein K (HNRNPK) is a poly(C)-binding protein exerting anti-HCV effects through localization near the ER and LDs [75]. Silencing of HNRNPK reduced HCV entry/replication steps, but enhanced virus assembly/release steps, suggesting complex involvement of the factor in HCV infection [75–78]. One model is that HNRNPK is redistributed to sites of viral particle production where it sequesters HCV RNA, blocking HCV assembly [75].…”

Landscape of post-transcriptional gene regulation during hepatitis C virus infection

“…Several comprehensive studies identified novel RBPs targeting the HCV 3′ UTR by using RNA capture and subsequent mass spectrometry [48-50]. In the following section we will discuss some RBPs relevant for host immune responses during HCV infection.…”