1 Department of Microbiology, School of Medicine, University of Washington, Seattle, WA, USA
2 Regional Primate Research Center, University of Washington, Seattle, WA, USA
3 Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA
Correspondence
Michael Katze (at address 1)
honey{at}u.washington.edu
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ABSTRACT |
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Present address: Antiviral Research, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, 60064 IL, USA
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INTRODUCTION |
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A Flaviviridae family member, HCV possesses a positive-sense, ssRNA genome of about 9600 nucleotides (Reed & Rice, 2000). The HCV genome consists of highly conserved 5'- and 3'-noncoding regions (NCR), and a large open reading frame (ORF) that encodes a polyprotein of approximately 3010 amino acids. The polyprotein is processed co- and post-translationally by both host and viral proteases into at least 10 structural (core, E1, E2 and p7) and nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Recent studies also show the existence of an alternative ORF within the core-coding region that encodes a novel HCV protein of unknown function (Varaklioti et al., 2002
; Walewski et al., 2001
; Xu et al., 2001
). The 5' terminus of the HCV genome possesses a complex secondary structure that functions as an internal ribosome-entry site (IRES) to mediate viral protein translation in a cap-independent manner (Hellen & Pestova, 1999
; Rijnbrand & Lemon, 2000
). The HCV IRES was mapped between about 40 and 370 nucleotides from the 5'-end of the genome, including most of the 5'-NCR and a small beginning portion of the core-coding region (Honda et al., 1996
; Reynolds et al., 1995
; Rijnbrand et al., 1995
). Three distinct elements have been shown to be involved in HCV IRES-mediated translation: (1) integrity of the global structure of HCV IRES (Hellen & Pestova, 1999
); (2) the 3'-terminal region of the HCV genome; (3) trans-acting cellular factors that interact with the HCV IRES element and assist in translation initiation. Furthermore, the HCV core protein-coding sequence, but not the core protein, has been suggested to modulate HCV IRES-directed translation efficiency, possibly through long-range RNARNA interaction (Honda et al., 1999
; Wang et al., 2000
). However, another recent study suggested that core protein caused autogenous inhibition of HCV IRES translation, which may imply a mechanism for switching from translation to RNA replication and/or encapsidation during the virus life-cycle (Zhang et al., 2002
). Interestingly, HCV IRES-mediated translation seems to be regulated by cell cycle-associated mechanisms, and it is known that the HCV IRES activity varies with the cell cycle (Honda et al., 2000
; Pietschmann et al., 2001
).
Being a highly conserved, virus-specific mechanism, the IRES-mediated translation of HCV constitutes an excellent target for development of the next generation of antiviral drugs for HCV (Jubin, 2001). However, because of the lack of an efficient in vitro infection system, the regulatory mechanisms of HCV IRES-mediated translation still remain poorly characterized. Previous studies focused on the roles of HCV genomic RNA sequences and structures, and in trans cellular proteins, in regulation of HCV IRES-directed translation. Few studies have characterized the effects of HCV-encoded proteins on HCV translation, and it remains largely unknown whether any HCV-encoded nonstructural protein(s) can regulate IRES-directed translation, as part of the virus's strategies to control its life-cycle. In this study, using both the HCV replicon cells and human liver cells transiently expressing HCV nonstructural proteins, we investigated the function of HCV nonstructural proteins in regulating HCV IRES-mediated translation. We also characterized the possible molecular mechanisms underlying the phenotype and suggest a novel mechanism by which HCV modulates its cap-independent translation and facilitates virus persistence.
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METHODS |
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Interferon and ribavirin treatment.
Human type I IFN was purchased from Access. Ribavirin was purchased from Sigma. For IFN treatment, Huh7 and HCV replicon cells were incubated with media containing type I IFN (5 and 50 IU ml-1) for 24 h at 37 °C in the presence of 5 % CO2. For ribavirin treatment, Huh7 and HCV replicon cells were incubated with media containing ribavirin (400 and 1000 µM) for 24 h at 37 °C in the presence of 5 % CO2.
Antibodies and immunoblot assays.
Antibodies specific to HCV NS3, NS4A and NS5B were described previously (Tomei et al., 1993). The antibody specific to HCV NS5A was purchased from ID Labs. Antibodies specific to phosphorylated eIF2
(Ser51), total eIF2
, phosphorylated eIF4E (Ser209), total eIF4E, phosphorylated Erk1/2 (Thr180/Tyr182) and total Erk1/2 were previously described (He et al., 2001
). For immunoblot analysis, cell lysates were collected and protein concentrations were determined as described previously (He et al., 2001
). Equal amounts of cell lysates were resolved on SDS-PAGE (12 %), followed by electroblotting to nitrocellulose membrane (Schleicher and Schuell). The immunoblot analysis was performed as previously described (He et al., 2001
). The relative levels of protein phosphorylation were determined by quantifying the immunoblots with ImageQuant (version 5.1). The signals from the phospho-specific immunoblots were normalized against their individual control signals and the ratio of phospho-specific signal to control signal was calculated as previously described (He et al., 2001
).
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RESULTS |
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We examined the effect of HCV replicon on HCV IRES-mediated translation using an established dual-luciferase reporter gene construct containing a genotype 1b HCV IRES element (Fig. 1B). In this bicistronic reporter construct, translation of the upstream Renilla luciferase gene is initiated in a cap-dependent mechanism, while translation of the downstream firefly luciferase gene is mediated by an HCV IRES element contained within the HCV 5'NTR in a cap-independent manner. This construct allows internally controlled quantification of the IRES-directed translational level since the two luciferase reporters are apparently translated from the same transcripts. The HCV-IRES dual-luciferase reporter construct was transiently transfected into either the HCV replicon cells (clone 10A) (Blight et al., 2000
) or Huh7 cells, the parental control cell line. Cell lysates were collected at different time-points post-transfection and dual-luciferase assays were performed on the lysates (Fig. 2
A). The relative IRES-activity levels were expressed as the ratio of the activity of firefly luciferase (IRES-dependent) over that of Renilla luciferase (cap-dependent). The activity of the HCV IRES in Huh7 cells was arbitrarily taken as 100 % with the activity of the IRES in replicon cells expressed relative to this. As shown in Fig. 2(A)
, at 12 h post-transfection, there was no significant difference in HCV IRES-directed translational levels between the replicon and Huh7 cells. However, at 24 h post-transfection, an almost 2-fold increase in relative HCV IRES activity was observed in the replicon cells over that of Huh7 cells. In our experiments the effects of NS proteins on IRES were not observed at early time-points after transfection, which is probably due to the recovery phase of cells following the rather cytotoxic transfection procedure. Examination of the absolute Renilla and firefly luciferase activity levels showed that HCV replicon caused an increase in the IRES-dependent firefly luciferase activity, but not in cap-directed Renilla luciferase reporter activity (data not shown). In addition, no significant difference in the global cellular protein synthesis rates was observed between Huh7 and replicon cells by pulse-labelling analysis (data not shown). This suggests that the HCV replicon cells specifically stimulate HCV IRES-directed translation, but not cap-dependent global mRNA translation. Similar, but more dramatic, results (a 5·5-fold increase) were acquired with another HCV replicon cell line clone (clone Huh-9-13) (Lohmann et al., 1999
), although slightly different kinetics was observed in this cell line (Fig. 2B
). The difference in relative HCV IRES activity levels and kinetics may be caused by variations in either experimental conditions and/or replicon clones. Collectively, these results suggest that the stimulatory effect of HCV replicon on HCV IRES activity is not clone-specific, and is thus likely due to HCV-encoded function(s).
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The effect of HCV replicon on HCV IRES-mediated translation is sensitive to both IFN and ribavirin treatment
However, it was unclear whether the enhancement of HCV IRES activity by replicon cells was caused by HCV-encoded function(s) or by adaptive cellular mutations that occurred during the replicon cell line construction process. We next attempted to address this question by inhibiting replication of the HCV replicon in clone 10A replicon cells with IFN and ribavirin treatment. Both IFN and ribavirin are used in the current therapies for HCV infection (Hoofnagle, 1999), and have been shown to perturb HCV genome replication in different cell culture systems, including the replicon system (Blight et al., 2000
; Chung et al., 2001
; Frese et al., 2001
; Maag et al., 2001
). If the HCV IRES-stimulatory effect of the replicon cells were due to HCV-encoded protein function(s), we would speculate that anti-HCV drug treatment, which inhibits HCV genome replication and protein expression, would therefore inhibit the IRES-stimulatory effect of HCV replicon cells.
In the experiment shown in Fig. 3(A), both Huh7 and clone 10A replicon cells were transiently transfected with the HCV IRES-containing dual-luciferase reporter construct, immediately followed by treatment either with different concentrations of type I IFN (lanes 36) or with ribavirin (lanes 710). Untreated cells were used as controls (lanes 1 and 2). At 24 h post-transfection and drug treatment, cell lysates were collected and dual-luciferase assays were performed to determine relative IRES activity levels as described above. The activity of the HCV IRES in untreated Huh7 cells was arbitrarily taken as 100 % with the activities of the IRES under other conditions expressed relative to this. As shown in Fig. 3(A)
, the drug treatment did not have a significant effect on relative HCV IRES activity in the parental Huh7 cells (lanes 3, 5, 7 and 9), while in replicon cells 2- to 3-fold inhibitions of HCV IRES activity were observed (lanes 4, 6, 8 and 10). The inhibition of relative HCV IRES activity in replicon cells was primarily caused by a decrease in IRES-directed firefly luciferase activity level, while the cap-dependent Renilla luciferase activity level remained almost unchanged (Fig. 3B
). These results suggest that the mechanism(s) responsible for the enhancement of HCV IRES activity by replicon cells is sensitive to anti-HCV drug treatment, and is thus likely to be an HCV-encoded function(s), rather than adaptive cellular mutations in the replicon cells. Inhibition of HCV replicon RNA levels by IFN treatment in our experiments was observed by RT-PCR analysis (data not shown), which is consistent with the results of previous studies (Blight et al., 2000
; Chung et al., 2001
). IFN treatment was also found to reduce HCV protein levels in the replicon cells (data not shown). All these results argue forcefully in supporting the notion that HCV upregulates its own cap-independent translation process.
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HCV replicon cells show decreased levels of eIF2 and eIF4E phosphorylation
Our previous study (He et al., 2001) showed that NS5A was able to decrease the phosphorylation levels of both eIF2
and eIF4E, suggesting a possible mechanism by which HCV may differentially regulate cap-dependent and -independent translation initiation. So we next examined the phosphorylation status of eIF2
and eIF4E in Huh7 and clone 10A replicon cells, in order to probe the possible molecular mechanisms by which HCV replicon stimulates HCV IRES activity. By performing immunoblot analysis with antibodies that specifically recognize the phosphorylated forms of eIF2
and eIF4E (Fig. 5
A, B), lower phosphorylation levels of both eIF2
and eIF4E were detected in replicon cells than in Huh7 parental cells, which is consistent with our previous results. Importantly, HCV replicon did not seem to alter the total protein levels of eIF2
and eIF4E, as shown by immunoblot analysis with antibodies that recognize total eIF2
and eIF4E (Fig. 5A, B
). However, it remains uncertain whether in replicon cells the modulation of eIF2
and eIF4E phosphorylation levels is due to NS5A action. Since it is known that the phosphorylation of eIF4E is regulated by the mitogen-activated protein kinase (MAPK) pathway, the activation level of the Erk1/2 MAPK was also compared in Huh7 and replicon cells, by performing immunoblot analysis with antibody specific for the phosphorylated, activated forms of Erk1/2 MAPKs. Consistent with the reduced level of eIF4E phosphorylation in replicon cells, the level of Erk1/2 MAPK phosphorylation observed in replicon cells was lower than in Huh7 cells (Fig. 5C
) (He et al., 2002
). These results suggest that HCV replicon caused reduced phosphorylation levels of both eIF2
and eIF4E, which may play a role in the enhancement of HCV IRES activity in these cells. However, considering the fact that HCV replicon stimulated HCV IRES function specifically, and had no significant effect of other viral IRES elements, it is likely that an additional HCV-specific mechanism(s) is responsible for the HCV IRES-specific phenotype of the replicon cells.
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DISCUSSION |
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In persistent viral infections, such as those by HCV or the DNA tumour viruses, constitutive modulation of host translational control pathways and release of translational suppression may make important contributions to viral pathogenesis/oncogenesis. However, little is known of the nature of viral translational programming as it pertains to persistent infection, although it clearly requires that the host mRNA translation remain sufficient to sustain the host cell and support virus persistence. Analyses of the mechanisms by which viruses may mediate persistence and latency suggest that host cell integrity and translational competence are maintained through (i) viral modulation of specific cellular mRNA translation and (ii) viral modification of host signalling and translational regulatory pathways (Gale et al., 2000). In the case of HCV, our understanding of viral translational control mechanisms is further limited by the lack of efficient virus infection systems, and the current working models are based on results from in vitro and surrogate systems. Both NS5A and E2 proteins of HCV have been shown to interact with and inhibit PKR (Gale et al., 1997
, 1998
; Taylor et al., 1999
). Inhibition of PKR-dependent eIF2
phosphorylation can be seen as a mechanism to ensure overall translational competence during virus infection. Our previous study also showed that NS5A protein inhibits eIF4E (the mRNA cap-binding protein) phosphorylation, through both Grb2- and PKR-dependent pathways (He et al., 2001
). The downregulation of eIF4E phosphorylation and activation may negatively affect the translation of at least a subset of cellular mRNAs, especially those that are more sensitive to the phosphorylation status of eIF4E, such as some genes regulating cell growth and stress response (Gingras et al., 1999
; Sonenberg & Gingras, 1998
). So in addition to favouring viral protein synthesis, the modulation of eIF4E by NS5A may contribute to regulation of host cell growth and stress response, suggesting a new mechanism of viral pathogenesis.
The most important implication of this study is that HCV encodes nonstructural proteins, possibly NS4B and NS5A, that specifically enhance HCV IRES-directed translation. This is the first indication that an HCV nonstructural protein upregulates the activity of its own IRES, probably in order to facilitate viral protein synthesis and virus replication. Although the underlying molecular mechanism remains basically unclear, it is possible that HCV nonstructural proteins modulate viral IRES activity through interaction with host cell proteins involved in the translation machinery or translational control pathways. It is also possible that different HCV NS proteins may collaborate in this process. It would be interesting to test the effect of different combinations of HCV NS proteins on HCV IRES activity in future studies. (This would be technically challenging if we consider the number of NS proteins involved and the different possible combinations.) Interestingly, it is known that picornavirus leader and 2A proteinases enhance picornavirus IRES activity either indirectly, by cleaving eIF4G, as well as possibly directly, by an unknown mechanism that does not involve eIF4G cleavage (Hambidge & Sarnow, 1992; Macadam et al., 1994
; Ventoso & Carrasco, 1995
). It seems that these different groups of viruses employ diversified mechanisms to achieve a common goal: to boost viral translation and replication. It is noteworthy that NS5A-1b1 and -1b5, two isolates related to different responses to IFN treatment in patients (Gale et al., 1997
), show different abilities to enhance HCV IRES activity. Thus the biological differences between HCV genotypes/isolates, such as IFN-resistance, may be due, in part, to the variations in IRES-dependent translation efficiency, which in turn influence virus replication. Eventually, the biological relevance of our results awaits careful examination in a biologically relevant virus infection system, which is still not available due to technical obstacles.
The IRES is the most conserved part of the HCV genome, and may play multifunctional roles in translation, replication or packaging of the viral genome (Hellen & Sarnow, 2001). Interestingly, the translation initiation process on the HCV IRES has simpler factor requirements than many other translation initiation mechanisms (only the cricket paralysis virus IRES has even simpler factor requirements that the HCV IRES) (Hellen & Pestova, 1999
; Wilson et al., 2000
), indicating that HCV employs a very unique translation control mechanism, even among the various IRES-utilizing viruses. IRES-mediated translation is not a common feature among cellular mRNAs, and thus may represent a valid and new target for therapeutic intervention in viral mRNA translation (Jubin, 2001
), and remains a promising new area for the development of effective antiviral compounds, which mostly has been limited to inhibitors of viral enzymes.
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ACKNOWLEDGEMENTS |
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Received 13 June 2002;
accepted 24 November 2002.