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Journal of Virology, April 2007, p. 3852-3865, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.01311-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

An RNA-Binding Protein, hnRNP A1, and a Scaffold Protein, Septin 6, Facilitate Hepatitis C Virus Replication{triangledown}

Chon Saeng Kim,1 Su Kyoung Seol,1 Ok-Kyu Song,2 Ji Hoon Park,1 and Sung Key Jang1*

PBC, Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Republic of Korea,1 Panbionet Corp., Pohang, Kyungbuk, Republic of Korea2

Received 22 June 2006/ Accepted 7 January 2007


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. NS5b is an RNA-dependent RNA polymerase that polymerizes the newly synthesized RNA. HCV likely uses host proteins for its replication, similar to other RNA viruses. To identify the cellular factors involved in HCV replication, we searched for cellular proteins that interact with the NS5b protein. HnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, septin 6 protein also interacts with hnRNP A1. Moreover, hnRNP A1 interacts with the 5'-nontranslated region (5' NTR) and the 3' NTR of HCV RNA containing the cis-acting elements required for replication. Knockdown of hnRNP A1 and overexpression of C-terminally truncated hnRNP A1 reduced HCV replication. In addition, knockdown of septin 6 and overexpression of N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV through RNA-protein and protein-protein interactions.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Approximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV), and these individuals account for most cases of chronic liver disease, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (45, 70).

HCV has a single-stranded RNA genome of positive polarity that contains a single long open reading frame flanked by nontranslated regions (NTRs) at its 5' and 3' ends. Nearly the entire 5' NTR is needed for efficient RNA amplification, although the minimal replication element is within the first 120 nucleotides (20, 35, 58). In addition, the 5' NTR has been found to contain an internal ribosomal entry site (IRES), which is required for the translation of polyprotein (29, 67, 75). The 3' NTR is composed of a nonessential variable region, a poly(U)/poly(U·C) tract, and a highly conserved and essential 3' X domain (19, 40, 74). The viral proteins are translated as a single large polyprotein of 3,010 to 3,040 amino acids, which is cotranslationally and/or posttranslationally processed by cellular and viral proteases into mature structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) viral proteins (57). NS5b is an RNA-dependent RNA polymerase that produces complementary RNAs from template RNAs (3, 66).

Subgenomic HCV RNA replicons have been developed to mimic the replication of a viral RNA infecting a host cell. These replicons are composed of the HCV 5' NTR; a selection marker, such as neomycin phosphotransferase; the IRES of encephalomyocarditis virus; HCV nonstructural proteins NS3, -4a, -4b, -5a, and -5b; and the HCV 3' NTR. When introduced into human hepatoma (Huh 7) cells, the HCV replicon RNA replicates autonomously (47). Using this replicon system, several groups have reported that HCV replication occurs in a distinct replication complex, which comprises viral RNA and HCV proteins (1, 12, 23, 54). The replication complex is formed on intracellular membranes with vesicular structures termed the "membranous web." Recently, several cellular proteins, such as FBL2, hVAP-33, and cyclophilin B, were suggested to function in HCV replication (22, 69, 71). The geranylgeranylated FBL2, which interacts with NS5a, augments HCV RNA replication (69). hVAP-33, which interacts with both NS5a and NS5b, mediates formation of the HCV RNA replication complex on the lipid raft (22). Furthermore, phosphorylation of HCV NS5a modulates its interaction with hVAP-33 and, therefore, viral RNA replication (14). Cyclophilin B, which interacts with NS5b, stimulates HCV RNA replication by increasing the affinity with which NS5b binds to the viral RNA (71).

HnRNP A1 is an RNA-binding protein that shuttles between the nucleus and the cytoplasm. It is involved in several RNA metabolic processes, such as pre-mRNA splicing and transport of cellular RNAs (11, 73). The hnRNP A1 protein is composed of 320 amino acids and contains two RNA-binding domains and a glycine-rich domain that is responsible for protein-protein interactions. The signal that mediates shuttling between the nucleus and cytoplasm has been identified as the C-terminal 38 amino acids, termed M9 (62, 72). Cytoplasmic and nuclear hnRNP A1 have different RNA-binding profiles. The cytoplasmic hnRNP A1 has high affinity with AU-rich elements (26, 27), whereas the nuclear hnRNP A1 has high affinity with a polypyrimidine stretch bordered by AG at the 3' ends of introns (52, 64). Interestingly, hnRNP A1 has been shown to be involved in the replication of an RNA virus (mouse hepatitis virus [MHV]) through an interaction with the transcription-regulatory region of MHV RNA (44, 60).

Septins belong to a family of GTP-binding proteins that function as dynamic, regulatable scaffolds recruiting other proteins (16, 24, 25, 36). Proteins of the septin family, originally identified in Saccharomyces cerevisiae as proteins required for normal cytokinesis, are proposed to be involved in many processes, including membrane dynamics, vesicle trafficking, apoptosis, and cytoskeletal remodeling (25, 31, 36). Septins are expressed throughout the animal kingdom but seem to be absent from plants (49). To date, at least 12 distinct septins with multiple splice variants have been identified in mammals (50). The central GTP-binding domain (GBD) is highly conserved in all human septins. The N-terminal parts of the GBD are composed of basic residues, and the C termini are predicted to form alpha-helical coiled-coil (CC) regions that are likely to participate in oligomerization of proteins (25). Septins are known to polymerize, forming homo- and hetero-oligomeric structures as functional units that can in turn form higher-order filaments in vitro and in vivo (6, 15, 16, 18, 39, 53). The detailed mechanism of this oligomerization remains to be elucidated, although some reports have suggested that the CC domain (59) and the GBD (6) have roles in the oligomerization.

As described above, several host proteins have been proposed to facilitate the replication of HCV; however, the molecular details of the replication complex largely remain to be elucidated. In the present work, we sought to identify cellular factors involved in HCV replication using both coimmunoprecipitation and the yeast two-hybrid screening system to identify cellular proteins that interact with the NS5b protein. The hnRNP A1 and septin 6 proteins were identified by coimmunoprecipitation and yeast two-hybrid screening, respectively. Interestingly, the septin 6 protein also directly interacts with hnRNP A1, suggesting that NS5b, septin 6, and hnRNP A1 form a complex. We also show that hnRNP A1 interacts with the 5' NTR and the 3' NTR of HCV RNA, which contain the cis-acting elements required for viral replication. Knockdown of hnRNP A1 and overexpression of a C-terminally truncated hnRNP A1 reduced HCV replication. Moreover, knockdown of septin 6 and overexpression of an N-terminally truncated septin 6 inhibited HCV replication. These results indicate that the host proteins hnRNP A1 and septin 6 play important roles in the replication of HCV by forming a complex with NS5b and viral RNA.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructions. For the generation of pcDNA Flag-NS5b{Delta}21, the NS5b sequence in the replicon construct, I389neo/NS3-3' (47) was PCR amplified with the following oligomers: 5'-ACT GCG GCC GCG TCG ATG TCC TAC ACA TGG AC-3' and 5'-ATC AAG CTT TCA GCG GGG TCG GGC ACG AG-3 '. The amplified DNA was digested with NotI and HindIII and then inserted into the pcDNA-Flag vector.

Plasmid pBSK9286-9604/7 was a gift from R. Bartenschlager at the University of Heidelberg (46). pSK-3'NTR was obtained by insertion of a PCR fragment corresponding to the variable region, poly(U)/poly(U·C) track, and X tail into the KpnI-BamHI site of pBSK9286-9604/7. pSK-Xtail was similarly constructed by insertion of a PCR fragment corresponding to the X tail into the KpnI-BamHI site of pBSK9286-9604/7.

The hnRNP A1 cDNA was amplified by PCR using a pGBT9-hnRNP A1 vector (32) and a set of primers representing the 5' and 3' ends of the hnRNP A1 coding region and cloned into the pcDNA 3.1(+)-hygro vector (Invitrogen). The 9-amino-acid hemagglutinin (HA) tag was attached to the N terminus of hnRNP A1 by including the HA tag in the forward PCR primer. The truncated HA-hnRNP A1{Delta}C was similarly constructed, using a PCR-amplified fragment corresponding to hnRNP A1 (amino acids 1 to 245).

To construct the plasmid pCT1-NS5b{Delta}21, which was used in the yeast two-hybrid screening, the NS5b sequence corresponding to amino acids 1 to 570 was PCR amplified from I389neo/NS3-3'. The amplified DNA fragment and pCT1 vector (Panbionet Corp.) were digested with NotI and SalI and then ligated together.

We obtained the specific cDNA clone of septin 6 transcript variant II (GenBank accession no. NM_015129) from the 21C Frontier Human Gene Bank (http://genbank.kribb.re.kr). To construct the plasmid pACT2-septin 6, which was used as prey for the yeast two-hybrid assay, septin 6 cDNA encoding amino acids 1 to 434 was amplified by PCR. The amplified cDNA fragment was digested with BamHI and SalI, the pACT2 vector (Clontech) was digested with BamHI and XhoI, and the two were ligated together. The truncated septin 6 derivatives (pACT2-septin 6{Delta}N, pACT2-septin 6{Delta}C, pACT2-septin 6{Delta}NC, pACT2-septin 6PB, and pACT2-septin 6CC) were similarly constructed using PCR-amplified fragments that represented septin 6{Delta}N (amino acids 62 to 434), septin 6{Delta}C (amino acids 1 to 317), septin 6{Delta}NC (amino acids 62 to 317), septin 6PB (amino acids 1 to 61), and septin 6CC (amino acids 318 to 434).

To construct pEGFP-septin 6, the amplified cDNA fragment and the pEGFP-C1 vector (Clontech) were digested with HindIII and BamHI and then ligated together.

To construct pcDNA HA-septin 6, an amplified septin 6 cDNA fragment was cloned into the pcDNA 3.1(+)-hygro vector. The 9-amino-acid HA tag was attached to the N terminus of septin 6 by including the HA tag in the forward PCR primer. The truncated HA-septin 6{Delta}N, HA-septin 6{Delta}C, and HA-septin 6{Delta}NC were similarly constructed using PCR-amplified fragments that represented septin 6{Delta}N (amino acids 62 to 434), septin 6{Delta}C (amino acids 1 to 317), and septin 6{Delta}NC (amino acids 62 to 317). The sequences of all constructed plasmids were confirmed by DNA sequencing.

Antibodies. Antibodies against hnRNP A1 (4B10) and against hnRNP C (4F4) were gifts from G. Dreyfuss of Howard Hughes Medical Institute at the University of Pennsylvania, and a rabbit polyclonal antibody against hnRNP A1/A2 was a gift from B. Chabot of Telogene Inc. The monoclonal antibody 5B-12B7 against NS5b was a gift from D. Moradpour at the University of Freiburg, and the antibodies against septin 2 and septin 6 were gifts from I. G. Macara at the University of Virginia School of Medicine. Anti-PTB antibody was described elsewhere (10). Flag and {gamma}-tubulin antibodies were purchased from Sigma, and actin antibody was purchased from ICN. Antibodies against HA, NS5b, and green fluorescent protein (GFP) were purchased from Santa Cruz.

Cell culture and transient transfection. Huh 7 cells and Huh 5-15 cells (containing the subgenomic replicon I389neo/NS3-3') and Huh-luc/neo-ET cells (containing the subgenomic replicon carrying both the neomycin phosphotransferase and the firefly luciferase genes) were provided by R. Bartenschlager at the University of Heidelberg (47, 68). 293T cells and Huh 7 cells were grown at 37°C in Dulbecco's modified Eagle's medium (Gibco) supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 10 µg/ml) and 10% fetal bovine serum (HyClone) in the presence of 6.0% CO2. Huh 5-15 and Huh-luc/neo-ET cells were grown under the same conditions but with the addition of the antibiotic G418 (600 µg/ml; Calbiochem). 293T cells were electroporated as previously described (35).

Knockdown of hnRNP A1 and septin 6 using siRNAs. Duplex small interfering RNAs (siRNAs) targeted to hnRNP A1, hnRNP A2, and septin 6 and control siRNA were purchased from Bioneer Inc. (Korea). The siRNA sequences targeting septin 6 (septin 6 siRNA 1 and septin 6 siRNA 2) were 5'-GAG ACA UAU GAG GCC AAA AdTdT-3' and 5'-GAA AGA AGC GGA GCU CAA AdTdT-3', respectively. The control siRNA sequence was 5'-CCU ACG CCA CCA AUU UCG UdTdT-3'. The siRNA sequences targeting hnRNP A1 (hnRNP A1 siRNA) and hnRNP A2 (hnRNP A2 siRNA) were 5'-CUA CAA UGA UUU UGG GAA UdTdT-3' and 5'-GCU UUG AAA CCA CAG AAG AdTdT-3', respectively. In order to transfect siRNA into Huh 7 cells containing an assayable replicon, 100 nM of siRNA mixed with 3 µl of Lipofectamine 2000 (Invitrogen) was added to each well of a six-well plate. Two consecutive transfections were performed with a 2-day interval (55). The cells were harvested for other experiments 5 days after the first transfection. In the cases of hnRNP A1 and hnRNP A2 siRNA transfections, the cells were harvested for other experiments 3 days after a single transfection.

Mass spectrometry. To identify the coprecipitated proteins, we used peptide fingerprinting by matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide sequencing by MALDI-TOF/TOF. The peptides were investigated with MALDI-TOF and MALDI-TOF/TOF at the Biomolecular Diversity core facility in the Postech Biotech Center (Pohang, Korea). Database searches were performed with the Mascot program at the same facility.

Immunoprecipitation. Immunoprecipitation was performed as described previously (34).

Fluorescence microscopy. Fluorescence microscopy was performed as described previously (34). Primary rabbit antibody against hnRNP A1/A2 and mouse antibody against NS5b (12B7) were used to detect colocalization of the two proteins.

RNA affinity chromatography. RNA affinity chromatography was performed as previously described (33). Huh 7 cytoplasmic lysate was prepared by the method used by El-Hage et al. (13). To generate biotinylated 3'-NTR RNA of the negative strand, the 5'-NTR sequence in I389neo/NS3-3' was amplified with reverse primers containing the T7 RNA polymerase promoter. The gel-purified PCR-amplified DNA templates were used directly for in vitro transcription. To generate the biotinylated positive-strand 1-to-389 RNA, we linearized the I389neo/NS3-3' plasmid with AscI enzyme. Biotinylated HCV 3'-NTR RNAs were produced from pSK-3'NTR and pSK-Xtail that had been linearized with BamHI. RNA transcripts were produced by T7 RNA polymerase (Stratagene) with NTPs containing Bio-16-UTP (Roche).

Yeast two-hybrid screening. Yeast two-hybrid screening was performed by Panbionet Corp.

Luciferase assays. Luciferase assays were performed with a firefly luciferase assay kit (Promega) according to the manufacturer's instructions. The luciferase activity in the cells was normalized to the protein concentration, as determined using the Bradford assay.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hnRNP A1 is coimmunoprecipitated with HCV NS5b. Many RNA viruses exploit cellular proteins for their replication. We investigated cellular proteins that interact with NS5b because HCV may also rely on cellular machineries for its replication. A recombinant NS5b protein with a Flag tag at the N terminus and a 21-amino-acid truncation at the C terminus, which has been shown to be required for association with the membrane, was overexpressed in 293T cells. NS5b was immunoprecipitated using an anti-Flag monoclonal antibody, and the coprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1A). With Coomassie blue staining, several proteins were observed to be associated with NS5b (compare lanes F and V in Fig. 1A). Prominent bands were excised from the gel, and proteins were identified by peptide mass fingerprinting. Trypsinized peptide mixtures were subjected to MALDI-TOF MS or MALDI-TOF/TOF. A 65-kDa protein band (Fig. 1A) was identified as the NS5b protein. Several protein bands were identified as large ribosomal subunit proteins (Fig. 1A). Two representative ribosomal proteins, L6 and L7, are indicated in Fig. 1A. These results are consistent with a previous report (65) that HCV NS5b RNA replicase specifically binds to ribosomes. A protein band (Fig. 1A) was identified as hnRNP A1 protein by MALDI-TOF/TOF analysis. The amino acid sequences of three polypeptides were determined by the analysis, and the sequences of these polypeptides perfectly matched that of the hnRNP A1 protein (Fig. 1C). To further confirm the identities of these proteins, the presence of the hnRNP A1 protein in the NS5b precipitate was confirmed with anti-NS5b and anti-hnRNP A1 antibodies (4B10). As shown in Fig. 1B, hnRNP A1 was detected in the Flag-NS5b precipitate (lane F), but not in the control precipitate (lane V).


Figure 1
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  		FIG. 1. hnRNP A1 is coimmunoprecipitated with HCV NS5b. (A) Proteins coimmunoprecipitated with HCV NS5b. A recombinant NS5b protein with a Flag tag at the N terminus and a 21-amino-acid truncation at the C terminus was overexpressed in 293T cells (lane F). Using an anti-Flag antibody, NS5b was immunoprecipitated, and coprecipitated proteins were then resolved by SDS-PAGE. A similar experiment was performed with 293T cells transfected with control vector (lane V). (B) Equal amounts of the coimmunoprecipitates used in panel A were separated by SDS-PAGE and analyzed by Western blotting using antibodies against NS5b and hnRNP A1 (4B10). (C) Peptide mixtures obtained by in-gel digestion of the protein in band i in panel A with trypsin were analyzed by MALDI-TOF MS and MALDI-TOF/TOF. The peptide sizes matched those predicted for hnRNP A1. The matching peptides are shown in boldface and underlined. (D) Immunoprecipitations (IPs) using an anti-Flag antibody were carried out with 293T cell lysates transfected with empty vector (Vec) or Flag-NS5b expression vector (Flag-NS5b). The cell lysates were preincubated either with (+) (lanes 3 and 6) or without (–) (lanes 1, 2, 4, and 5) RNase A (10 µg/ml). The precipitates were resolved by SDS-PAGE and then analyzed by Western blotting with antibodies against hnRNP A1 (4B10) and NS5b. (E) Immunoprecipitation was carried out using anti-hnRNP A1 antibody with Huh 7 (lanes 1, 2, 5, and 6) and Huh 5-15 (lanes 3, 4, 7, and 8) cell lysates that had been preincubated either with (+) (lanes 2, 4, 6 and 8) or without (–) (lanes 1, 3, 5, and 7) RNase A.

 
NS5b and hnRNP A1 are RNA-binding proteins, so it is possible that the NS5b-hnRNP A1 interaction was mediated by an RNA. To investigate the effect of RNA on the interaction, immunoprecipitation was carried out after treatment of the lysates with RNase A. The RNase treatment dissociates RNA-hnRNP complexes (63). Indeed, there was a marked reduction in the intensity of the hnRNP A1 band after the RNase treatment (Fig. 1D, compare lane 5 and lane 6). However, some hnRNP A1 proteins were still associated with NS5b after RNase treatment (Fig. 1D, lane 6). The interaction between NS5b and hnRNP A1 was again confirmed by immunoprecipitation of hnRNP A1 with Huh 5-15 cells containing a subgenomic RNA replicon, I389neo/NS3-3' (47). These cells contain a subgenomic HCV replicon that expresses nonstructural proteins that direct replication of the replicon RNA. As shown in Fig. 1E, lanes 7 and 8, NS5b proteins in the replicon-containing cells coprecipitated with endogenous hnRNP A1, and RNase treatment reduced this association, similar to the results shown in Fig. 1D. On the basis of these data, we concluded that hnRNP A1 interacts with NS5b and that the complex formation is augmented by the presence of RNA.

HnRNP A1 partially colocalizes with NS5b. We then examined the subcellular localization of hnRNP A1 in Huh 5-15 cells containing the HCV replicon. Immunocytochemical analyses of NS5b (Fig. 2A, a, d, and g) and hnRNP A1 and A2 (Fig. 2A, b, e, and h) were performed using a monoclonal antibody, 12B7 ({alpha}-NS5b), and a polyclonal antibody recognizing both hnRNP A1 and hnRNP A2 proteins (55). In naïve Huh 7 cells, hnRNP A1 was found mainly in the nucleus (Fig. 2A, b). In Huh 5-15 cells, a redistribution of hnRNP A1 into the cytoplasm was detected in many cells expressing NS5b, although hnRNP A1 was found in both the nucleus and cytoplasm in some NS5b-positive cells (Fig. 2A, e). We detected a similar cytoplasmic localization for hnRNP A1 in Huh 5-15 cells, using a monoclonal antibody (4B10) recognizing hnRNP A1 (data not shown). At x1,000 magnification, NS5b and hnRNP A1 proteins were partially colocalized at speckles in the cytoplasm (Fig. 2A, i). By contrast, other hnRNP proteins, such as PTB and hnRNP C1, mainly remained in the nucleus in Huh 5-15 cells (Fig. 2B). In Huh 7 cells transiently expressing NS5b without HCV replication, no marked relocalization of hnRNP A1 was detected (data not shown). Thus, the relocalization of hnRNP A1 from the nucleus to the cytoplasm seems to be specific to HCV replication. Interestingly, relocalization of hnRNP A1 was also observed in cells infected with MHV, which requires hnRNP A1 for replication (60).


Figure 2
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  		FIG. 2. hnRNP A1 partially colocalizes with NS5b. (A) Huh 7 and Huh 5-15 cells were grown on coverslips. The cells were fixed, permeabilized, and treated with primary antibodies (anti-hnRNP A1/A2 polyclonal antibody and anti-NS5b monoclonal antibody) and secondary antibodies (tetramethyl rhodamine isocyanate [TRITC]-conjugated goat anti-rabbit immunoglobulin G and fluorescein-5-isothiocyanate [FITC]-conjugated goat anti-mouse immunoglobulin G). The localization patterns of hnRNP A1 and NS5b are shown in red and green, respectively. The merged images are shown in the Merged column. The yellow dots in panel i show the partial colocalization of NS5b and hnRNP A1 proteins. (B) The immunofluorescence experiments were performed with Huh 5-15 cells and primary anti-hnRNP C and anti-PTB antibodies. The localization of hnRNP C and PTB is shown in red.

 
Septin 6 interacts with NS5b and hnRNP A1. We investigated the interaction between NS5b and hnRNP A1 using the yeast two-hybrid system (17). A direct protein-protein interaction between NS5b and hnRNP A1 was not observed by the yeast two-hybrid analysis (data not shown). The yeast two-hybrid system was further used to identify cellular proteins interacting with NS5b, using HCV NS5b with a 21-amino-acid C-terminal deletion as bait and a human thymus cDNA library (Clontech) as prey. Seven positive clones were obtained from the screening. DNA sequencing showed that one of the seven positive clones encoded the middle portion of eukaryotic translation initiation factor 4A, isoform 2 (eIF4AII), from amino acid 144 (data not shown). This protein has been reported to interact with NS5b in vitro (42). The other six positive clones matched the sequence of the septin 6 gene (NM_015129). Our research was focused on the newly identified protein septin 6. A full-length cDNA clone of septin 6 was obtained as described in Materials and Methods. To determine the region of septin 6 required for interaction with NS5b, two-hybrid analyses were carried out using the bait vector containing the NS5b sequence spanning amino acids 1 to 570 (BD-NS5b) and prey vectors encoding full-length septin 6 (AD-septin 6) or mutant derivatives (AD-septin 6{Delta}N, AD-septin 6{Delta}C, AD-septin 6{Delta}NC, AD-septin 6PB, and AD-septin 6CC) (Fig. 3A). The mutants were generated according to the prediction that the structure of septin 6 consists of the polybasic (PB) domain, GBD, and the CC domain consecutively. AD-septin 6{Delta}N, AD-septin 6{Delta}C, AD-septin 6{Delta}NC, AD-septin 6PB, and AD-septin 6CC contain the GAL4 activation domain (AD) fused with the GBD plus CC domain, PB domain plus GBD, GBD, PB domain, and CC domain of septin 6, respectively (Fig. 3A). Full-length septin 6 and the C-terminal-truncation mutant of septin 6 (AD-septin 6{Delta}C) supported the growth of yeasts on the selection media deficient in leucine, tryptophan, and adenine (Fig. 3B, i, spots 1 and 3). The remaining constructs did not support the growth of yeast on the selection media (Fig. 3B, i, spots 2, 4, 5, and 6). These results indicate that both the PB domain and GBD are required for interaction with NS5b.


Figure 3
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  		FIG. 3. Septin 6 interacts with NS5b and hnRNP A1. (A) Schematic diagram of full-length septin 6 and its derivatives fused with the GAL4 AD. (B) (i) Protein-protein interactions between septin 6 and NS5b were monitored with the yeast two-hybrid system. Prey vectors expressed septin 6 (AD-septin 6) or its derivatives (AD-septin 6{Delta}N, AD-septin 6{Delta}C, AD-septin 6 {Delta}NC, AD-septin 6PB, and AD-septin 6CC), and the bait vector expressed NS5b (amino acids 1 to 570). Yeast cells transfected with both bait and prey vectors were cultivated on a synthetic dextrose medium deficient in leucine, tryptophan, and adenine. Spots 1 to 6, +, and – represent yeasts containing prey vectors AD-septin 6, AD-septin 6{Delta}N, AD-septin 6{Delta}C, AD-septin 6{Delta}NC, AD-septin 6PB, and AD-septin 6CC; a positive control for protein-protein interaction; and a negative control, respectively. Yeasts containing bait and prey vectors expressing proteins that interact grew on this medium (spots 1, 3, and +). (ii) Coimmunoprecipitations were performed using 293T cells expressing both eGFP-tagged septin 6 (eGFP-septin 6) and Flag-tagged NS5b (Flag-NS5b). Cell extracts (input) were prepared, and immunoprecipitations (IPs) were then performed using anti-Flag antibody (lanes 4 and 5) or control anti-HA antibody (lane 6). The precipitates were subjected to Western blotting with an anti-NS5b (Flag-NS5b) or anti-GFP (eGFP-septin 6) antibody. (C) (i) Protein-protein interactions between septin 6 and hnRNP A1 were monitored using the yeast two-hybrid system with a prey vector expressing septin 6 (AD-septin 6) and a bait vector expressing amino acids 1 to 320 of hnRNP A1 (BD-hnRNP A1). Yeast cells transfected with both bait and prey vectors were cultivated on synthetic dextrose medium deficient in leucine, tryptophan, and adenine. Yeast containing bait and prey vectors producing septin 6 and hnRNP A1 (spot 4) and the positive control (spot +) grew on this medium, but the bait or prey with negative control did not grow (spots 1, 2, and 3). (ii) 293T cells were transfected with plasmids expressing eGFP-tagged septin 6. Forty-eight hours after transfection, 293T cells were lysed and subjected to immunoprecipitation using anti-GFP antibody (lane 3) and control anti-HA antibody (lane 4). The precipitated proteins were analyzed by Western blotting using anti-GFP (eGFP-septin 6) and anti-hnRNP A1 (hnRNP A1) antibodies. (D) Immunoprecipitation with anti-septin 6 antibody was performed with cell lysates from Huh 7 and Huh 5-15 cells containing a subgenomic replicon. The precipitated proteins were analyzed by Western blotting using anti-NS5b (NS5b), anti-septin 2 (septin 2), and anti-hnRNP A1 (hnRNP A1) antibodies.

 
To confirm the protein-protein interaction between NS5b and septin 6 by a biochemical method, a coimmunoprecipitation experiment was performed using 293T cells ectopically expressing both enhanced GFP (eGFP)-tagged septin 6 (eGFP-septin 6) and Flag-tagged NS5b (Flag-NS5b). The cell lysates were then subjected to immunoprecipitation using an anti-Flag antibody. The immunoprecipitates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting using the anti-NS5b and anti-eGFP antibodies (Fig. 3B, ii). Flag-NS5b and eGFP-septin 6 were expressed at similar levels (Fig. 3B, ii). eGFP-septin 6 was coprecipitated with Flag-NS5b only when Flag-NS5b was precipitated with an anti-Flag antibody (Fig. 3B, ii, IP, lane 5). NS5b precipitation by the negative-control anti-HA antibody was not observed (Fig. 3B, ii, IP, lane 6). These results indicate that NS5b interacts with septin 6 in vivo.

Two-hybrid analysis was also performed to investigate the protein-protein interaction between septin 6 and hnRNP A1 using hnRNP A1 in the bait vector and AD-septin 6 as prey, because hnRNP A1 exists in a complex with NS5b (see above). This bait-and-prey combination supported the growth of yeast (Fig. 3C, i, spot 4), which indicates that septin 6 directly interacts with hnRNP A1. We then tried to confirm the protein-protein interaction between septin 6 and hnRNP A1 by a biochemical method. Immunoprecipitation was performed with an anti-GFP antibody using 293T cells ectopically expressing eGFP-septin 6. Endogenous hnRNP A1 was coprecipitated with eGFP-septin 6 by the anti-GFP antibody (Fig. 3C, ii, IP, lane 3). By contrast, control antibody (anti-HA antibody) did not precipitate with either eGFP-septin 6 or hnRNP A1 (Fig. 3C, ii, IP, lane 4). The yeast two-hybrid analysis and immunoprecipitation data indicate that septin 6 interacts with hnRNP A1.

From the coimmunoprecipitation data and two-hybrid analyses, we concluded that NS5b, septin 6, and hnRNP A1 can form a protein complex through NS5b-septin 6 and septin 6-hnRNP A1 interactions. Formation of this protein complex is also indicated by the coimmunoprecipitation of hnRNP A1 with NS5b (Fig. 1). To confirm the formation of this protein complex during HCV replication, we sought to detect NS5b-septin 6 and septin 6-hnRNP A1 interactions using a monoclonal antibody against septin 6 (41) and the Huh 5-15 cell line containing the HCV replicon I389neo/NS3-3' (Fig. 3D). Coprecipitation of NS5b and hnRNP A1 was shown by Western blotting using antibodies against NS5b and hnRNP A1 (Fig. 3D, NS5b and hnRNP A1). The endogenous hnRNP A1 was coprecipitated with the septin 6 antibody from both Huh 7 and Huh 5-15 cell extracts irrespective of the presence of the HCV replicon (Fig. 3D, lanes 4 and 5). NS5b was coprecipitated with the septin 6 antibody from Huh 5-15 cell extracts (Fig. 3D, lane 5). Precipitation of septin 6 by the septin 6 antibody was indirectly monitored by the presence of septin 2, which is known to interact with septin 6 (38), because septin 6 is the same mass (50 kDa) as the immunoglobulin heavy chain, which can be detected as a thick, nonspecific band in the Western blot. Curiously, the amount of septin 2 precipitated by septin 6 antibody was greatly increased in the presence of the HCV replicon, even though the total amount of septin 2 remained the same. This may indicate a change in the configuration of septin 6 in HCV-replicating cells, possibly through the interaction with NS5b (Fig. 3D, septin 2, compare lanes 4 and 5). One plausible explanation is that there is a change in the polymerization statuses of septin 2 and septin 6 because of the replication of HCV (53). However, the molecular basis of this phenomenon remains to be elucidated. These data indicate that endogenous hnRNP A1 and septin 6 form a complex with NS5b.

hnRNP A1 binds to the NTRs of HCV RNA. We sought to identify cellular proteins that interact with the 3' NTR of negative-strand RNA, because the 3' end of negative-strand RNA is used as the template for initiation of replication. Therefore, a cellular protein with a role in HCV replication is likely to interact with this region. To isolate cellular proteins interacting with this RNA, a biotinylated RNA corresponding to the 389 nucleotides at the 3' end (–1 to 389 RNA) of negative-strand RNA was generated by in vitro transcription, and RNA affinity chromatography was performed with the biotinylated RNA and a cytoplasmic extract of Huh 7 cells. After precipitation and washing of the RNA-protein complex, RNA-bound proteins were resolved by SDS-PAGE (Fig. 4A). Many RNA-binding proteins were revealed by the RNA affinity chromatography, as shown by comparison with the control lane loaded with proteins bound to the resin without RNA (Fig. 4A, lane +). Proteins with apparent molecular masses of 32 kDa and 35 kDa were excised from the gel and analyzed by MALDI-TOF peptide mass fingerprinting. The 35-kDa protein band (Fig. 4A, i) was identified as GADPH (glyceraldehyde-3-phosphate dehydrogenase) (Fig. 4B, i). The mass values of 11 peptides were well matched to theoretically predicted tryptic peptides of GAPDH by the Mascot program, and sequence coverage was 45%. The 32-kDa protein band (Fig. 4A, ii) was identified as hnRNP A1 (Fig. 4B, ii). The mass values of nine peptides were well matched to theoretically predicted tryptic peptides of hnRNP A1 by the Mascot Program, and sequence coverage was 26%.


Figure 4
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  		FIG. 4. Interaction of hnRNP A1 with the HCV RNA. (A) To isolate the cellular proteins interacting with the HCV 3' NTR of negative-strand RNA, RNA affinity chromatography was performed with a biotinylated RNA corresponding to the 389 nucleotides at the 3' terminus of the negative-sense RNA (lane +). The resin-bound proteins were resolved by SDS-PAGE and visualized by Coomassie staining. A similar experiment was performed without RNA (lane –). (B) MALDI-TOF analysis was performed as described in the legend to Fig. 1C, and the peptides that match GAPDH and hnRNP A1 are shown in boldface and underlined. (C) RNA pull-down experiments were performed using biotinylated RNAs corresponding to various regions of the HCV RNA (lanes 1 to 3 represent 3'-end region nucleotides 1 to 389, 1 to 104, and 119 to 389 of negative-sense RNA, respectively; lanes 4 and 5 represent the 3' NTR and the X tail of positive-sense RNA; lane 6 represents the 5' NTR [nucleotides 1 to 389] of positive-sense RNA). (D) RNA pull-down experiments were performed using biotinylated RNAs corresponding to two regions of the HCV RNA (lane 1 represents the 5' NTR [nucleotides 1 to 389] of HCV RNA; lane 2 represents the NS3 RNA [nucleotides 24 to 428 from the first nucleotide of NS3]). Western blotting analyses were performed with an antibody recognizing hnRNP A1 (top) and an antibody recognizing both hnRNP A1 and hnRNP A2 (bottom).

 
Binding of hnRNP A1 to HCV RNA was further analyzed using various regions of HCV RNAs that may have important roles in replication. Biotinylated RNAs corresponding to parts of the 5' NTR and 3' NTR of HCV in both polarities were generated by in vitro transcription and then incubated with a cytoplasmic extract of Huh 7 cells. After pulling down of RNA-bound proteins with streptavidin agarose resin and washing of the precipitate, an immunoblot analysis using an hnRNP A1 antibody was performed. Consistent with the data shown in Fig. 4A, the 3'-terminal 389 nucleotides of negative-sense RNA bind strongly with hnRNP A1 (Fig. 4C, lane 1). The 3'-terminal nucleotides 119 to 389 of negative-sense RNA seem to be responsible for this binding (Fig. 4C, lane 3), because very weak, if any, binding of hnRNP A1 was observed from the RNA corresponding to the 3'-terminal nucleotides 1 to 104 (Fig. 4C, lane 2). The 3'-terminal part of positive-sense RNA that corresponds to the 3' NTR of viral RNA, which has an important role in negative-sense RNA synthesis, also interacts with hnRNP A1 (Fig. 4C, lane 4). The X tail at the very 3' end of positive-sense RNA binds weakly to hnRNP A1 (Fig. 4C, lane 5). This indicates that the variable region and/or the poly(U)/poly(U·C) tract are responsible for the interaction with hnRNP A1. Interestingly, strong binding of hnRNP A1 was observed when the 5' NTR with a part of the core-coding RNA was used as a probe (Fig. 4C, lane 6). To further demonstrate the specificity of hnRNP A1 for different regions of HCV RNA, pull-down experiments were performed with biotinylated RNAs corresponding to the 5' NTR and to a part of the NS3-coding sequence (Fig. 4D). No binding of hnRNP A1 or hnRNP A2 was detected from the RNA corresponding to the NS3-coding sequence (Fig. 4D, top, lane 2). Intriguingly, three bands were detected by antibody recognizing both hnRNP A1 and hnRNP A2 from the protein pool precipitated with the RNA corresponding to the 5' NTR and a part of the core-coding region (Fig. 4D, bottom, lane 1). The bottom two bands are hnRNP A1 and hnRNP A2, as indicated (Fig. 4D, bottom). The identity of the top band recognized by the antibody is not known. These data indicate that the 5' and 3' ends of HCV RNA in both polarities interact with hnRNP A1.

Knockdown of hnRNP A1 and hnRNP A2 by siRNAs reduces HCV replication. To analyze the role of hnRNP A1 in HCV replication, we used RNA interference to knock down hnRNP A1 mRNA. An siRNA against hnRNP A1 and a control siRNA were transfected into Huh-luc/neo-ET cells containing an assayable replicon (Fig. 5A). This assayable replicon expresses a firefly luciferase-ubiquitin-neomycin phosphotransferase fusion protein under the control of the HCV IRES and the HCV polyprotein (comprising NS3, NS4a, NS4b, NS5a, and NS5b) under the control of the encephalomyocarditis virus IRES (Fig. 5A). The effects of the siRNAs on hnRNP A1 levels were observed by Western blotting using anti-hnRNP A1/A2 antibody, which recognizes both hnRNP A1 and hnRNP A2 (Fig. 5B, top). Compared with cells that had been transfected without siRNA and with control siRNA, cells transfected with hnRNP A1 siRNA showed marked reduction in the level of hnRNP A1 (Fig. 5B, top, lane 3). The effect of hnRNP A1 knockdown on HCV replication was monitored by luciferase activity in the Huh luc/neo-ET cells containing an assayable replicon. Treatment of cells with siRNA against hnRNP A1 showed a modest effect on the replication of HCV, indicated by the luciferase activity in the cells containing assayable replicon (Fig. 5C, lane 3), even though the level of hnRNP A1 was highly reduced in the cells treated with hnRNP A1 siRNA. We speculated that the moderate reduction of HCV replication by the knockdown of hnRNP A1 was attributed to putative functional substitutions of hnRNP A1-related proteins for the activity of hnRNP A1 in HCV replication. Functional substitutions of hnRNP A1-related proteins for hnRNP A1 have been reported in several biological processes. For instance, hnRNP A/B, hnRNP A2/B1, and hnRNP A3, which are highly homologous to hnRNP A1, can substitute for hnRNP A1 in the regulation of alternative splicing of cellular pre-mRNA (51) and that of human immunodeficiency virus pre-mRNA (4). It was also suggested that the role of hnRNP A1 in the replication of MHV RNA can be substituted for by hnRNP A1-related proteins (61). To confirm this possibility, we tested the effect of knockdown of hnRNP A2 mRNA on HCV replication. Knocking down of hnRNP A2 showed a similar effect to knocking down of hnRNP A1 on the replication of HCV RNA (Fig. 5C, lane 4). Moreover, cells transfected with both hnRNP A1 and hnRNP A2 siRNAs showed dramatic reduction (50%) in the replication of HCV RNA compared with the cells transfected without siRNA and with control siRNA (Fig. 5C, lane 5). There was also a reduction in the expression levels of virus-encoded proteins in the cells treated with both hnRNP A1 and hnRNP A2, as shown by Western blotting of NS5b (Fig. 5B, NS5b, lane 5). This indicates that hnRNP A2, and possibly other hnRNP A1-related proteins, can substitute for the activity of hnRNP A1 in HCV replication. By contrast, the levels of actin and {gamma}-tubulin (Fig. 5B, actin and {gamma}-tubulin) and the amount of total cellular protein measured by Bradford assay (data not shown) were not affected under the same conditions, indicating that suppression of hnRNP A1 and hnRNP A2 did not have detrimental side effects on the transfected cells. These data indicate that both hnRNP A1 and hnRNP A2 may play important roles in the replication of HCV RNA.


Figure 5
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  		FIG. 5. hnRNP A1 and hnRNP A2 are required for HCV replication. The effects of hnRNP A1 on HCV replication were investigated by knockdown of hnRNP A1 (A, B, and C) or overexpression of hnRNP A1 and its truncated mutant (D, E, and F). (A) Schematic diagram of an assayable subgenomic replicon, I389luc-ubi-neo/NS3-3'/ET. The Huh-luc/neo-ET cell line contains a bicistronic selectable HCV replicon containing both the neomycin phosphotransferase (Neo) gene and the firefly luciferase (FLuc) gene. (B) Western blot analysis of Huh-luc/neo-ET cells transfected with siRNAs. Protein levels were analyzed by Western blotting with anti-hnRNP A1/A2, which recognizes both hnRNP A1 and hnRNP A2 (top); anti-NS5b (NS5b); anti-actin (actin); and anti-{gamma}-tubulin ({gamma}-tubulin) antibodies. The relative amounts of hnRNP A1 and hnRNP A2, which were normalized to those in control siRNA-transfected cells, are depicted below the hnRNP A2/hnRNP A1 gel. (C) The effects of siRNAs on HCV replication were monitored indirectly by measuring luciferase activities in cells. The luciferase activities were normalized to that in mock-transfected cells, which was set to 1. Experiments were performed three times, and mean and standard deviation values of the experiments are depicted as bars and lines, respectively. (D) Schematic diagram of the wild-type and a mutant hnRNP A1 with a C-terminal truncation. RBD, Gly, and M9 denote the RNA-binding domain, the glycine-rich region, and the M9 sequence responsible for shuttling between the nucleus and cytoplasm, respectively. (E) Effect of a truncated hnRNP A1 on HCV replication. The control vector and plasmids expressing HA-hnRNP A1 and HA-hnRNP A1{Delta}C were transiently transfected into Huh-luc/neo-ET cells. Protein levels were analyzed by Western blotting with anti-HA (HA), anti-NS5b (NS5b), and anti-actin (actin) antibodies. (F) Effects of wild-type and mutant hnRNP A1 expression on HCV replication were monitored by measuring the luciferase activities in the replicon-containing cells. The luciferase activities were normalized to that obtained from cells transfected with the control vector, which was set to 1. Experiments were performed three times, and the results are depicted as in panel C.

 
Overexpression of hnRNP A1 with a C-terminal truncation reduces HCV replication. To further explore the potential role of hnRNP A1 in HCV RNA replication, we constructed plasmids expressing the HA-tagged wild-type hnRNP A1 (HA-hnRNP A1) or a mutant hnRNP A1 with a 75-amino-acid C-terminal deletion (HA-hnRNP A1{Delta}C) (Fig. 5D). This mutant lacks a part of the glycine-rich domain and the M9 sequence responsible for shuttling hnRNP A1 between the nucleus and the cytoplasm. This truncation mutant had previously been shown to inhibit the RNA synthesis of MHV through dominant-negative effects (60). We transfected plasmids expressing the wild-type and mutant hnRNP A1 into Huh-luc/neo-ET cells containing the assayable HCV replicon. The HA-tagged full-length hnRNP A1 and the HA-tagged truncated hnRNP A1 were well expressed, as shown in Fig. 5E (HA, lanes HA-hnRNP A1 and HA-hnRNP A1{Delta}C, respectively). Similar levels of NS5b were observed with the cells transfected with the control plasmid and the plasmid expressing the full-length hnRNP A1 (Fig. 5E, NS5b,lanes control and HA-hnRNP A1, respectively). On the other hand, the level of NS5b was reduced in cells expressing the truncated hnRNP A1 (Fig. 5E, NS5b, lane HA-hnRNP A1{Delta}C). This effect can be attributed, at least in part, to the reduced level of HCV RNA that was indirectly measured by luciferase activities in the cell extracts (Fig. 5F). These results indicate that the C-terminal domain of hnRNP A1, including the M9 sequence and the glycine-rich region, is important for HCV replication. Together with the results from siRNA against hnRNP A1, these data strongly indicate that hnRNP A1 plays an important role in HCV RNA replication.

Knockdown of septin 6 by siRNA reduces HCV replication. The role of septin 6 in HCV replication was investigated by knockdown of septin 6 expression by siRNA against septin 6. Two siRNAs against septin 6 (septin 6 siRNA 1 and septin 6 siRNA 2) reduced the expression of septin 6 (Fig. 6A, . The effect of the septin 6 knockdown on HCV replication was monitored by luciferase activity in the Huh-luc/neo-ET cells containing an assayable replicon. Treatment with the siRNAs against septin 6 reduced the level of luciferase activity in the cells by about 70% compared with the activity in the cells transfected without siRNA and with the control siRNA (Fig. 6B). There was also a reduction in the expression levels of virus-encoded proteins in the septin 6-siRNA-treated cells, as shown by Western blotting of NS5b (Fig. 6A, NS5b). By contrast, the levels of actin and {gamma}-tubulin were not affected (Fig. 6A, actin and {gamma}-tubulin). Transfection of each siRNA did not reduce the amount of total cellular protein measured by Bradford assay (data not shown), indicating that suppression of septin 6 and transfection of siRNA did not have detrimental side effects on transfected cells. These data indicate that septin 6 may play an important role in HCV RNA replication.


Figure 6
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  		FIG. 6. Effects of an siRNA against septin 6 and a septin 6 mutant on HCV replication. Whether septin 6 is required for HCV replication was investigated by monitoring the effects of knockdown of septin 6 (A and B) or overexpression of full-length septin 6 and its deletion mutants (C, D, and E). (A) Levels of septin 6, NS5b, actin, and {gamma}-tubulin in cells treated with siRNAs against septin 6 were examined by Western blotting using anti-septin 6 (septin 6), anti-NS5b (NS5b), anti-actin (actin), and anti-{gamma}-tubulin ({gamma}-tubulin) antibodies. The relative amounts of septin 6, which were normalized to those in control siRNA-transfected cells, are depicted below the septin 6 gel. (B) The effects of siRNAs against septin 6 on HCV replication were investigated by measuring luciferase activity in Huh-luc/neo-ET cells. The luciferase activity in cells transfected with siRNAs against septin 6 was normalized to that in mock-transfected cells, which was set to 1. Experiments were performed three times, and the results are depicted as in Fig. 5C. (C) Schematic diagram of full-length septin 6 and its deletion mutants. (D) Plasmids expressing full-length septin 6 and its deletion derivatives were transiently transfected into Huh-luc/neo-ET cells. Protein levels were analyzed by Western blotting with anti-HA (HA), anti-NS5b (NS5b) and anti-actin (actin) antibodies. (E) The effects of overexpression of full-length septin 6 and its derivatives on HCV replication were monitored by measuring the luciferase activities in the cells. The luciferase activities were normalized to that obtained by transfection of a control vector, which was set to 1. Experiments were performed three times, and the results are depicted as in Fig. 5C.

 
Overexpression of septin 6 with an N-terminal truncation reduces HCV replication. To further explore the potential role of septin 6 in HCV replication, we constructed plasmids expressing the HA-tagged full-length septin 6 (HA-septin 6) or mutants of septin 6 with various truncations or deletions based on the predicted structure of septin 6 (Fig. 6C) (25). Septin 6 variants with the predicted molecular mass were expressed by cells transfected with the vectors shown in Fig. 6C (Fig. 6D, HA). Reduced levels of luciferase activities, which reflect viral RNA levels, were seen in cells expressing septin 6 with an N-terminal truncation (Fig. 6E, lanes septin 6{Delta}N and septin 6{Delta}NC). Reduced levels of NS5b were also seen in cells expressing septin 6 with an N-terminal truncation (Fig. 6D, lanes 3 and 5). On the other hand, cells expressing full-length septin 6 or septin 6 with a C-terminal truncation did not show significant changes in luciferase activity (Fig. 6E, lanes septin 6 and septin 6{Delta}C) and NS5b protein levels (Fig. 6D, NS5b, lanes 2 and 4). Together with the siRNA data, these data indicate that septin 6 plays an important role in HCV replication.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Host proteins are involved in various processes of the viral proliferation cycle (43). In the present work, we investigated cellular proteins with important roles in viral RNA replication by identifying proteins that form a complex with the viral RNA-dependent RNA polymerase NS5b. Two independent approaches were used for this purpose. The first was a coimmunoprecipitation method that is suitable for identifying whole proteins in the replication complex irrespective of whether they have direct interaction with NS5b. Using this method, hnRNP A1, which interacts with the 5' NTR and the 3' NTR of HCV (Fig. 4), was shown to exist in the replication complex (Fig. 1). Interestingly, in HCV-replicating cells, hnRNP A1 is relocalized from the nucleus to the cytoplasm (Fig. 2). The second method was the yeast two-hybrid assay, which is suitable for identifying proteins that directly interact with NS5b. Septin 6 was shown to interact with NS5b by this method (Fig. 3). Intriguingly, as shown by this assay, hnRNP A1 in the replication complex interacts with septin 6 (Fig. 3C, i). These data indicate that NS5b, septin 6, and hnRNP A1 form a complex.

To investigate whether hnRNP A1 and septin 6 have positive roles in HCV replication, the effects of protein knockdown (using siRNAs against these proteins) and overexpression of truncated mutants of these proteins on replication of an HCV replicon in a Huh 7 cell were assessed. Knockdown of both proteins inhibited the replication of viral RNA. In addition, expression of a C-terminally truncated hnRNP A1 and an N-terminally truncated septin 6 inhibited the replication of viral RNA (Fig. 5E and F and 6D and E, respectively). These data indicate that both hnRNP A1 and septin 6 are involved in HCV replication.

How do these proteins contribute to the replication of HCV RNA? As hnRNP A1 is an RNA-binding protein, it may direct the HCV RNA replication complex containing NS5b, septin 6, and hnRNP A1 to the proper region(s) of the genomic RNA. Interaction of hnRNP A1 with the 3'-end regions of both positive-sense and negative-sense viral RNAs (Fig. 4C) may direct the replication complex to the sites where replication of the negative- and positive-sense RNAs is initiated. Septin 6 may function as a scaffolding molecule, connecting NS5b and hnRNP A1, which in turn is associated with the viral RNA. Several reports have suggested that septins form a scaffold by which they direct the localization of other proteins involved in diverse processes (7-9, 16, 37, 48). The scaffolding functions of septins have been observed in cytokinesis, bud site selection, and chitin deposition in Saccharomyces cerevisiae. Considering the reported physiological roles of septins, we speculate that septin 6 may be involved in localization of the replication complex to a membranous compartment in the cell and may also have a role in recruiting cellular proteins, which might facilitate the replication of HCV RNA, to the replication complex.

On the basis of the data presented here, we propose a hypothetical model of the HCV replication complex involved in the synthesis of a negative-sense RNA from the positive-sense template RNA. The RNA-dependent RNA polymerase is recruited to the 3' end of the positive-sense RNA due to protein-protein interactions between NS5b and septin 6 and between septin 6 and hnRNP A1 and an RNA-protein interaction between the 3' NTR and hnRNP A1. Specific RNA-protein interaction between HCV RNA and NS5b may also participate in the augmentation of NS5b-HCV RNA interaction and the precise localization of NS5b to the very 3' end of viral RNA. Circularization of viral RNA, which often occurs in replication complexes of positive-sense RNA viruses (2, 21, 28, 30, 56), might occur due to an interaction between HCV RNA and hnRNP A1 composed of the 5' NTR-hnRNP A1-hnRNP A1-3' NTR RNA. In this respect, it is important to note that the truncation of the glycine-rich C-terminal region of hnRNP A1, which mediates homomeric interaction of the protein (5, 32), resulted in inhibition of HCV replication. One plausible explanation for this is that the truncated hnRNP A1 may compete with the endogenous hnRNP A1 for binding to the HCV RNA and inhibit RNA circularization, thereby resulting in inhibition of viral RNA replication. Other viral proteins (such as NS3 and NS5a) and cellular proteins (such as hVAP-33, FBL2, and cyclophilin B) (22, 69, 71), which are known to play important roles in HCV replication, may also participate in the replication of HCV RNA in concert with hnRNP A1 and septin 6.

To our knowledge, this is the first report that describes an RNA-binding protein (hnRNP A1) actively participating in HCV replication in molecular detail (indirect protein-protein interaction). These studies of the HCV replication complex not only improve our understanding of the replication of HCV, but also have the potential for use as the basis for developing a drug against HCV that acts by inhibiting HCV replication. Moreover, investigations into the cellular proteins involved in the proliferation of HCV will improve our understanding of the molecular basis of pathological phenomena of virus infection. For instance, we found that hnRNP A1 is relocalized in HCV replicon-containing cells. Therefore, it is likely that the normal function of hnRNP A1 is prevented by HCV infection. In the same way, the normal function of septin 6, which plays a key role in cytokinesis, is likely to be disrupted by the interaction with NS5b. Therefore, NS5b of HCV has the potential to affect the proliferation cycle of host cells. The effect of NS5b on the physiology of host cells remains to be investigated.


   ACKNOWLEDGMENTS
 
We are grateful to G. Dreyfuss of the Howard Hughes Medical Institute at the University of Pennsylvania for the hnRNP A1 antibody, B. Chabot of Telogene Inc. for the hnRNP A1/A2 antibody, D. Moradpour at the University of Freiburg for the NS5b antibody, and I. G. Macara at the University of Virginia for the septin 2 and septin 6 antibodies.

The present study was supported in part by the Program for the Training of Graduate Students in Regional Innovation, which was conducted by the Ministry of Commerce Industry and Energy of the Korean Government, and grant MCBRG (M10501000022-05-N0100-02200) and grant SBD-NCRC (R15-2004-033-01001-0) from MOST, grants 02-PJ2-PG1-CH16-0002 and A050291 from KHIDI, KRF-2003-005-C0001, grant FPR05B 1-310 of the 21C Frontier Functional Proteomics Project from KMST, and a grant from POSCO.


   FOOTNOTES
 
* Corresponding author. Mailing address: PBC, Department of Life Science, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, Republic of Korea. Phone: 82-54-279-2298. Fax: 82-54-279-8009. E-mail: sungkey{at}postech.ac.kr Back

{triangledown} Published ahead of print on 17 January 2007. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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