Identification of Cellular Factors Associated with the 3′-Nontranslated Region of the Hepatitis C Virus Genome

Abstract

Chronic infection by hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remains largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3-nontranslated region (3-NTR) of the (؉) strand HCV genome are essential for initiation of (؊) strand synthesis. The RNA signals within the highly conserved 3-NTR may be the site for recruiting cellular factors that mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this report, we demonstrate that an RNA affinity capture system developed in our laboratory More than a decade ago, hepatitis C virus (HCV) 1 was discovered as the major causative agent of parenteral non-A non-B hepatitis (1, 2), and the number of HCV-infected individuals worldwide is currently estimated at 170 million. Although some infected individuals are able to clear the virus without treatment, most infections persist leading in about 50% of all cases to chronic hepatitis, which may develop into chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. The main therapeutic regimen currently in clinical use is a combination treatment consisting of high doses of interferon-␣ and the nucleoside analog ribavirin, and a large percentage of patients receiving this regimen are not responsive. These stark facts underscore the importance of expediently developing new strategies to combat this viral infection.The hepatitis C virus is a positive strand RNA virus of the flaviviridae family having a genome roughly 9.6 kb in length (3). The RNA genome contains a single ORF that encodes a polyprotein of ϳ3000 amino acids that is processed by a combination of host-and virus-encoded proteases (4) into four structural proteins (core, E1, E2, and p7) and six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. Replication of HCV is initiated at the 3Ј-NTR of the RNA genome, although little is known about the mechanism of initiation or the factors required for this process. The NS5B protein is the RNA-dependent RNA polymerase, or HCV replicase, responsible for replication of the HCV genome whose structure and enzymatic activities have been well characterized (5, 6). Although it has been established that NS5B can initiate primer-independent RNA synthesis, the mechanistic details of this process remain the source of some controversy (7-9). Hong et al. (10) have proposed that a structural motif within NS5B positions the terminal nucleotides of the genome in such a way that de novo synthesis is initiated from the 3Ј-end of the genome. The initiation of HCV virus replication may very well involve the aid of cellular factors in...

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

“…Viral and host proteins interacting with these regions could play an important role in regulating the viral life cycle. The expression and binding of these proteins may not be simultaneous but instead may be dependent on cell cycle phases and the stages of the virus life cycle and disease (27). HuR is an RNA binding protein that has previously been shown to bind to the HCV 3= UTR and has also appeared in some whole-genome siRNA knockdown studies in the context of HCV (28,29,44,45).…”

“…HuR, a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress, has also been shown to bind to the HCV 3= UTR and help in HCV translation as well as replication (27)(28)(29)(30). However, the molecular mechanism of its participation in these processes is not clear.…”

“…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). Other independent studies on RNA binding proteins like hn-RNPD, hnRNPA1, HuR, Unr, hnRNPU, etc., have indicated that they interact with the viral RNA (24)(25)(26)(27). However, the molecular basis of regulation of viral replication by these RBPs remains a mystery.…”

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

“…Interestingly, the knockdown of IGF2BP1 resulted in an inhibition of the HCV IRES-mediated translation, but did not influence replication. In contrast, knockdown of RNA helicase A, DDX5, or HuR, which interact with the HCV 59UTR and/or HCV 39UTR, resulted in a reduced replication (Harris et al 2006;Isken et al 2007). For hnRNP Q, an influence on HCV IRES-mediated translation and HCV replication has been shown (Kim et al 2004;Liu et al 2009).…”

“…The proteins IGF2BP1, hnRNP C, hnRNP D, hnRNP Q, polypyrimidine tract binding protein 1, PAI-1 mRNA binding protein 1, nucleolin, NF90/NFAR, RNA helicase A, and five subunits of eIF 3 (out of nine identified) match with proteins isolated from cytoplasmic Huh7 cell extract employing an HCV 59UTR that lacks domain I and represents the HCV IRES consisting of domains II to IV (Kim et al 2004;Lu et al 2004). HnRNP C, YB-1, polypyrimidine tract binding protein 1, DDX3, NF90/NFAR, and RNA helicase A have been identified as HCV 39UTR binding proteins in Huh7 extract (Gontarek et al 1999;Harris et al 2006;Isken et al 2007). Only for some of these interactions has a functional relevance been shown.…”