Heterogeneous Nuclear Ribonucleoprotein I (hnRNP-I/PTB) Selectively Binds the Conserved 3′ Terminus of Hepatitis C Viral RNA

doi:10.1006/bbrc.1998.9949

Biochemical and Biophysical Research Communications

Volume 254, Issue 2, 19 January 1999, Pages 351-362

https://doi.org/10.1006/bbrc.1998.9949 Get rights and content

Abstract

Hepatitis C virus (HCV) is a positive-strand RNA virus whose genome is replicated by a direct RNA-to-RNA mechanism. Initiation of negative-strand RNA synthesis is believed to proceed from the 3′ end of the genomic RNA. The high conservation of the 3′ terminus suggests that this region directs the assembly of proteins required for the initiation of RNA replication. We sought to determine whether host proteins bind specifically to this RNA structure. We observed specific binding of cellular proteins to labeled 3′-terminal RNA by mobility shift analysis. UV crosslinking revealed that the predominant 3′-terminal RNA-binding protein migrates as a single, 60-kDa species that can be precipitated by monoclonal antibodies directed against heterogeneous nuclear ribonucleoprotein I, also called polypyrimidine tract-binding protein (hnRNP-I/PTB), a protein previously shown to bind to the 5′ internal ribosome entry site (IRES) of the HCV genome. Purified hnRNP-I/PTB also bound selectively to the 3′ end of the HCV genome. hnRNP-I/PTB binding requires the upstream two stem-loop structures (SL2 and SL3) but not the most 3′-terminal stem-loop (SL1). Minor alteration of either the stem or loop sequences in SL2 or SL3 severely compromised hnRNP-I/PTB binding, suggesting extremely tight RNA structural requirements for interaction with this protein. hnRNP-I/PTB does not bind to either end of the antigenomic RNA strand and binds to the 5′ IRES element of the genome at least 10-fold less avidly than to the 3′ terminus. The strong, selective, and preferential binding of hnRNP-I/PTB to the 3′ end of the HCV genome suggests that it may be recruited to participate in viral replication, helping to direct initiation of negative-strand RNA synthesis, stabilize the viral genome, and/or regulate encapsidation of genomic RNA.

References (43)

T. Tanaka et al.
Biochem. Biophys. Res. Commun.
(1995)
J.-H. Yen et al.
Virology
(1995)
M.M. Konarska et al.
Cell
(1986)
M. Niepmann
FEBS Lett.
(1996)
A.A. Haller et al.
Virology
(1995)
Q. Liu et al.
Virology
(1997)
A.L.M. Bothwell et al.
J. Biol. Chem.
(1991)
M. Houghton
Fields Virology
(1996)
M.E. Major et al.
Hepatology
(1997)
A.A. Kolykhalov et al.
J. Virol.
(1996)

T. Tanaka et al.

J. Virol.

(1996)

K. Tsukiyama-Kohara et al.

J. Virol.

(1992)

C.M. Rice

S.E. Behrens et al.

EMBO J.

(1996)

V. Lohmann et al.

J. Virol.

(1997)

H. Okamoto et al.

Jpn. J. Exp. Med.

(1990)

C.H. Lin et al.

RNA

(1995)

K.J. Blight et al.

J. Virol.

(1997)

R. Singh et al.

Science

(1995)

M.A. Garcia-Blanco et al.

Genes Dev.

(1989)

A. Ghetti et al.

Nucleic Acids Res.

(1992)

Cited by (62)

HCV infection, IFN response and the coding and non-coding host cell genome
2016, Virus Research
Citation Excerpt :
Interestingly, hnRNPA1 binds HCV NS5B and the 5′ and 3′UTR of the HCV genome and is required for viral replication (Kim et al., 2007). Similarly, PTB binds to the IRES and the 3′UTR of HCV (Chung and Kaplan, 1999) and it has been described to be required for HCV translation (Ali and Siddiqui, 1995) and replication (Aizaki et al., 2006; Chang and Luo, 2006). However, controversy exists over the role of PTB in HCV translation and replication has not been reproducibly observed (Brocard et al., 2007; Tischendorf et al., 2004).
HCV is an ideal model to study how the infected cell is altered to allow the establishment of a chronic infection. After infection, the transcriptome of the cell changes in response to the virus or to the antiviral pathways induced by infection. The cell has evolved to sense HCV soon after infection and to activate antiviral pathways. In turn, HCV has evolved to block the antiviral pathways induced by the cell and, at the same time, to use some for its own benefit. In this review, we summarize the proviral and antiviral factors induced in HCV infected cells. These factors can be proteins and microRNAs, but also long noncoding RNAs (lncRNAs) that are induced by infection. Interestingly, several of the lncRNAs upregulated after HCV infection have oncogenic functions, suggesting that upregulation of lncRNAs could explain, at least in part, the increased rate of liver tumors observed in HCV-infected patients. Other lncRNAs induced by HCV infection may regulate the expression of coding genes required for replication or control genes involved in the cellular antiviral response. Given the evolutionary pressure imposed by viral infections and that lncRNAs are specially targeted by evolution, we believe that the study of proviral and antiviral lncRNAs may lead to unexpected discoveries that may have a strong impact on basic science and translational research.
Identification of proteins that mediate the pro-viral functions of the interferon stimulated gene 15 in hepatitis C virus replication
2013, Antiviral Research
Citation Excerpt :
Hnrnps are RNA-binding proteins, that shuttle between the nucleus and the cytoplasm and have been identified to bind the HCV IRES (internal ribosom entry side) to support translation: HnrnpA1 (Kim et al., 2007), HnrnpD (Paek et al., 2008), HnrnpE2/PCBP2 (Fukushi et al., 2001; Wang et al., 2011), HnrnpL (Hwang et al., 2009) and HnrnpM (Yao et al., 2011). Some of these proteins additionally bind to the 3′UTR of HCV: HnrnpA1 (Kim et al., 2007), HnrnpE2/PCBP2 (Wang et al., 2011), HnrnpI/PTB (Chung and Kaplan, 1999), HnrnpL (Hwang et al., 2009) and HnrnpC (Gontarek et al., 1999). Thereby playing an important role in initiation and regulation of HCV RNA replication via the viral RNA-dependent-RNA-polymerase NS5b, which itself is directly interacting with HnrnpA1 as well (Kim et al., 2007).
In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.
Regulation of hepatitis C virus translation initiation by iron: Role of eIF3 and La protein
2012, Virus Research
Citation Excerpt :
HCV IRES can functionally replace the eIF4F protein complex (eIF4E, eIF4G, and eIF4A), and therefore translation initiation of HCV requires just two of the canonical initiation factors, eIF2 and eIF3, for correct positioning of the initiator tRNA during cap-independent initiation (Cai et al., 2010). Several cellular RNA-binding proteins, including La protein (Ali and Siddiqui, 1997), polypyrimidine tract binding protein (PTBP) (Ali and Siddiqui, 1995), heterogeneous nuclear ribonucleoprotein L (RNPL) (Chung and Kaplan, 1999), poly(rC)-binding protein (PCBP)-2 (Spångberg and Schwartz, 1999), and ribosomal protein S9 (Pestova et al., 1998), have been implicated in efficient HCV IRES-dependent translation. Clinical evidences have found that patients with hepatic iron overload have a poor response to treatment with IFN-α (Fujita et al., 2007).
Eukaryotic initiation factors (eIFs) are required for encoding polyprotein of hepatitis C virus (HCV) which is mediated by an internal ribosome-entry site (IRES). Iron overload, a common finding among HCV patients, may be correlated with HCV pathology, but the underlying molecular mechanisms are poorly understood. In this study, we investigated the possible relationship among iron status, eIFs and HCV IRES-mediated translation in vitro. Using bicistronic reporter gene constructs carrying HCV IRES sequence, we found that the levels of intracellular iron were positively associated with the HCV IRES-dependent translation initiation in Huh-7 cells. RT-PCR method showed that iron treatment specifically increased the levels of eIF3A mRNA and La mRNA, whereas iron chelation reduced them. Western blots also confirmed that iron-dependent changes in eIF3A mRNA and La mRNA affected the expression of their proteins. Moreover, antisense phosphorothioate oligodeoxynucleotides to eIF3A and La successfully suppressed the levels of eIF3A and La protein and significantly reduced iron-dependent HCV translation. Taken together, our results suggest that iron promotes the translation initiation of HCV by stimulating the expression of eIF3A and La proteins. Inhibition of eIF3A and La proteins substantially repressed iron-dependent HCV translation, a beneficial effect that may have significant clinical implications.
Inhibition of translation initiation factors might be the potential therapeutic targets for HCV patients with hepatic iron overload
2012, Medical Hypotheses
Citation Excerpt :
Just two canonical initiation factors, eIF2 and eIF3, are required for correct positioning of the initiator tRNA during cap-independent initiation [19]. Many noncanonical translation initiation factors (NCTFs), such as La protein [20], polypyrimidine tract binding protein (PTB) [21], heterogeneous nuclear ribonucleoprotein L (RNPL) [22], poly(rC)-binding protein (PCBP)-2 [23], and ribosomal protein S9 [24], also interact with HCV IRES and might regulate viral translation. In the host, iron overload can lead to a variety of cellular toxicity and tissue damage, but the mechanism remains unclear.
Standard therapy, interferon-alpha (IFN-α) and ribavirin, remains the only available option for treatment of patients with hepatitis C virus (HCV) infection. However, iron overload, a common finding among HCV patients, have a poor response to treatment with current therapy. These data suggest that both host and viral factors are involved in the determination of the outcome of the therapy. Currently, novel antiviral compounds focus on the development of indirect antiviral drugs. The process of the viral translation is considered as the potential therapeutic targets. Coincidentally, study has found that hepatic iron load enhances the levels of eukaryotic initiation factor 3 (eIF3), which is essential for HCV translation. Reversely, iron chelation could reduce eIF3 p170 translation. Our hypothesis is that iron overload may specifically enhance cellular eIFs. As a result, the cellular mechanisms, in patients with iron overload, are utilized for translating viral mRNA into protein. Thus, treatment strategies that target eIFs should be an exceptionally good candidate therapeutic method for HCV patients with hepatic iron overload.
Hepatitis C virus is restricted at both entry and replication in mouse hepatocytes
2009, Biochemical and Biophysical Research Communications
Citation Excerpt :
It is very plausible that host factors and other HCV viral proteins are also required in this RNA replication process. Several cellular proteins such as polypyrimidine track-binding protein (PTB) have been identified to be involved [22–24]. The conserved nature of these known host proteins does not seem to provide any insights into the significant difference of HCV replication that we showed between human and mouse cells in this study.
Human hepatitis C virus (HCV) does not replicate in mouse hepatocytes. The underlying mechanisms are largely unknown. In this study, we took advantage of a series of direct and unique molecular strategies and dissected the HCV life cycle in human hepatoma Huh7.5 cells and mouse hepatoma Hepa1–6 cells. We showed that HCV entry was restricted in Hepa1–6 cells and was not rescued by expression of human HCV receptors. We also showed that HCV RNA replication was impaired in Hepa1–6 cells. In contrast, we showed that the HCV IRES translation activity and HCV production in Hepa1–6 cells were either comparable to, or even slightly higher than those in Huh7.5 cells. Thus, we conclude that entry and RNA replication are the two major HCV restrictions in mouse cells. These studies provide new insights into HCV interaction with mouse cells and new clues for formulating strategies for development of HCV mouse models.
Therapeutic application of RNA interference for hepatitis C virus
2007, Advanced Drug Delivery Reviews
RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing by double-stranded RNA. Because the phenomenon is conserved and ubiquitous in mammalian cells, RNAi has considerable therapeutic potential for human pathogenic gene products. Recent studies have demonstrated the clinical potential of logically designed small interfering RNA (siRNA). However, there are still obstacles in using RNAi as an antiviral therapy, particularly for hepatitis C virus (HCV) that displays a high rate of mutation. Furthermore, delivery is also an important obstacle for siRNA based gene therapy. This paper presents the potential applications and the hurdles facing anti-HCV siRNA drugs. The present review provides insight into the feasible therapeutic strategies of siRNA technology, and its potential for silencing genes associated with HCV disease.

View all citing articles on Scopus

^☆: Fields, B. N.Knipe, D. M.Howley, P. M.

¹: To whom correspondence should be addressed at Gastrointestinal Unit, GRJ-724, Massachusetts General Hospital, Boston, MA 02114-2696. Fax: 617-726-4422. E-mail:[email protected].

View full text

Regular ArticleHeterogeneous Nuclear Ribonucleoprotein I (hnRNP-I/PTB) Selectively Binds the Conserved 3′ Terminus of Hepatitis C Viral RNA☆

Abstract

Biochem. Biophys. Res. Commun.

Virology

Cell

FEBS Lett.

Virology

Virology

J. Biol. Chem.

Fields Virology

Hepatology

J. Virol.

J. Virol.

J. Virol.

EMBO J.

J. Virol.

Jpn. J. Exp. Med.

RNA

J. Virol.

Science

Genes Dev.

Nucleic Acids Res.

Regular Article
Heterogeneous Nuclear Ribonucleoprotein I (hnRNP-I/PTB) Selectively Binds the Conserved 3′ Terminus of Hepatitis C Viral RNA☆