Casaca et al. Virology Journal 2011, 8:358 http://www.virologyj.com/content/8/1/358

RESEARCH Open Access The heterogeneous ribonuclear C interacts with the hepatitis delta virus small antigen Ana Casaca1, Margarida Fardilha2, Edgar da Cruz e Silva2 and Celso Cunha1*

Abstract Background: Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication. Methods: A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression. Results: Thirty known were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens. Conclusions: S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question. Keywords: Hepatitis delta virus, hepatitis D small antigen, yeast two-hybrid, hnRNPC

Background which a single ORF was identified (reviewed in [3]). Hepatitis delta virus (HDV) is a satellite virus of the Transcription from this ORF results in the production hepatitis B virus (HBV) and the only member of the of a 0.8 kb mRNA molecule that codes for a 195 ami- Deltagenus. The association between the two viruses is noacid protein, the small delta antigen (S-HDAg). Dur- due to the fact that the HDV envelope consists of HBV ing transcription, an editing mechanism catalyzed by surface antigens (HBsAg) which are necessary for virus cellular adenosine deaminase (ADAR 1) converts an propagation [1,2]. The HDV genome consists of a 1.7 amber stop codon UAG to a tryptophan codon UGG Kb, circular, ssRNA molecule of negative polarity in extending this ORF by an additional 19 aminoacids resulting in the production of the large delta antigen (L- HDAg) [4]. It is generally accepted that replication of * Correspondence: [email protected] the genome occurs via a rolling-circle mechanism that 1Unidade de Microbiologia Médica, Centro de Malária e outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de involves the participation of host RNA polymerase II Lisboa, Lisboa, Portugal [5]. As a consequence, multimeric antigenomic Full list of author information is available at the end of the article

© 2011 Casaca et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Casaca et al. Virology Journal 2011, 8:358 Page 2 of 13 http://www.virologyj.com/content/8/1/358

molecules are produced which are subsequently self- identify 30 known proteins, including hnRNPC. This cleaved and ligated at precise monomeric intervals. The protein was found to be expressed in 5 positive clones newly produced antigenomes serve as templates for the initially obtained and was also observed in the above synthesis of genomic RNA by a similar mechanism. Sev- mentioned study by Cao et al. [8] to interact with eral functions have been assigned to both forms of the FLAG-S-HDAg. Here, we report that hnRNPC and S- delta antigen and it is consensual that S-HDAg is neces- HDAg interact in vitro and in vivo,andco-localizein sary for RNA accumulation [6], and L-HDAg interacts human liver cells. Using plasmid constructs coding for with HBsAgs playing an important role during virus different aminoacid sequences, in yeast two-hybrid packaging [7]. However, the host factors that participate assays, we could observe that the two proteins interact in the different steps of the HDV replication cycle inter- through their respective RNA binding domains. Finally, acting with both RNA and antigens are largely knockdown of hnRNPC in cells undergoing HDV repli- unknown. Recently, Cao et al reported the use of an cation, leads to a decrease in both S-HDAg and L- immunoprecipitation approach followed by mass spec- HDAg protein expression. Taken together, our data sug- trometry to identify S-HDAg interactors [8]. Over 100 gest that the S-HDAg/hnRNPC interaction here proteins were identified including nine RNA polymerase reported is specific and may be important for modula- II subunits, transcription and splicing factors, RNA heli- tion of HDV replication. cases, and hnRNPs. hnRNPs are abundant nuclear proteins that belong to Methods the large family of RNA-binding proteins containing Construction of plasmids highly conserved amino acid sequences among verte- For yeast two-hybrid assays, recombinant expression brates [9]. These proteins associate with primary tran- plasmids were constructed using pAS2-1 (BD Bios- scripts of RNA polymerase II to form hnRNP nuclear ciences). In this vector, the inserted are expressed complexes. These complexes assist mRNA processing, in-frame with a GAL4 DNA-binding domain (GAL4BD). including the stabilization of pre-mRNAs for nuclear To construct plasmid pAS2-1/S-HDAg, the cod- export and translation [10,11]. The most abundant pro- ing for the S-HDAg was amplified by PCR using plasmid tein in hnRNP nuclear complexes is hnRNPC. Two iso- pSVL-AgS [19] as template and specific primers (Table forms of hnRNPC (C1 and C2) are produced by 1). After digestion with MunIandSalI, the resulting alternative splicing and hnRNPC2 was found to contain fragment was cloned into the pAS2-1 EcoRI/SalIsites. 13 additional amino acids being expressed at about one- Three S-HDAg deletion mutants were also cloned in third the level of hnRNPC1 [12]. The two isoforms form vector pAS2-1. The deletion mutants, encoding S-HDAg stable heterotetramers [(C1)3C2] that bind cooperatively amino acids 1-62, 63-97 or 98-195 were generated by to RNA [13]. PCR amplification, using pSVL-AgS as template and the It has been previously reported that several viruses primers listed in Table 1. These plasmids were named interact with members of the hnRNP family. In particu- pAS2-1/S-HDAg (1-62), pAS2-1/S-HDAg (63-97) and lar, hnRNPs were shown to play important roles during pAS2-1/S-HDAg (98-195). replication of hepatitis C virus (HCV) [14,15], Sindbis For the production of recombinant proteins, the bac- virus [16], and vesicular stomatitis virus (VSV) [17]. terial expression vectors pGEX-6P-2 (GE Healthcare) Recently, hnRNPC was suggested to influence the viral and pET28c (Novagen) were used to produce GST or replication of dengue virus by favoring survival in the His-tagged fusion proteins, respectively. To construct host [18]. pGEX-6P-2/S-HDAg, for expression of GST/S-HDAg, In an attempt to identify cellular factors that interact the S-HDAg cDNA was obtained by digestion of withtheS-HDAgweusedayeasttwo-hybridsystemto pEGFP-C1-HDAg [20] with BglII and SalI, and then screen a human liver cDNA library. We were able to inserted into the XhoI/BamHI cloning sites of pGEX-

Table 1 Oligonucleotides used to generate specific S-HDAg cDNA fragments by PCR Plasmid name Oligonucleotide sequence (5’ ®3’) Fwd Rev pAS2-1/S-HDAg TTATCAATTGATGAGCCGGTCCGAGTCG TTATGTCGACCTATGGAAATCCCTGGTTTCCC pAS2-1/S-HDAg TTATCAATTGATGAGCCGGTCCGAGTCG TTATGTCGACCCATCCTTATCCTTCTTTCCGAG (1-62) pAS2-1/S-HDAg (63-97) TTATCAATTGCTGGATAAGGATGGAGAGGGG TTATGTCGACGTCGGTGAATCCTCCCCTG pAS2-1/S-HDAg (98-165) TTATCAATTGAAGGAGAGGCAGGATCACCG TTATGTCGACCTATGGAAATCCCTGGTTTCCC pET28c/hnRNPC TTATCAATTGGGATGGCCAGCAACGTTACC TTATGTCGACGTGCTTAAGAGTCATCCTCGCC Casaca et al. Virology Journal 2011, 8:358 Page 3 of 13 http://www.virologyj.com/content/8/1/358

6P-2. The plasmid for the expression of His6/hnRNPC, CodonPlus-(DE3)-RP competent cells, respectively. pET28c/hnRNPC, was constructed by amplifying the Expression of GST/S-HDAg and His/hnRNPC proteins hnRNPC cDNA in pOTB7 (Image clone # 3354131) was induced with 1 mM isopropyl-b-D-thiogalactopyra- using the primers pET28c/hnRNPC listed in Table 1. noside (IPTG) for 2 h. After centrifugation, bacterial The resulting fragment was cleaved with MunI and SalI, cells were ressuspended in PBS containing 0.1 mg/mL and cloned into pET28c, in the EcoRI/SalI sites. lysozyme. Three freeze-thaw cycles were performed and then the samples were treated with DNAse I (Roche). Yeast two-hybrid assay Purification of S-HDAg from whole bacterial protein The Matchmaker GAL4 Two-hybrid system (BD Bios- samples was performed using a GSTrap FF 1 mL col- ciences) was used to screen for proteins that interact umn (GE Healthcare), according to the manufacturer’s with S-HDAg from a human liver cDNA library (BD instructions. Briefly extracts were loaded onto a column Biosciences). Yeast two hybrid screening was performed and extensively washed. PreScission protease (GE using materials and protocols provided by the manufac- Healthcare) was then added to remove the GST-tag turer. Briefly, Saccharomyces cerevisiae AH109 strain from GST/S-HDAg. The eluted fraction containg S- pre-transformed with bait plasmid pAS2-1/S-HDAg HDAg protein was dialyzed against PBS and quantified were mated with an Y187 strain pre-transformed with a using a Bradford assay based kit (BioRad). liver cDNA library expressing fusions of GAL4 activa- tion domain. The mating mixture was plated on syn- Blot overlay assay thetic medium lacking tryptophan, leucine, histidine and The blot overlay analysis was performed as previously adenine and then assayed for a-galactosidade activity described [21] using bacterial extracts containing the (SD/-Trp/-Leu/-Hist/-Ade/X-a-gal medium). His6/hnRNPC recombinant protein. Increasing amounts Plasmids isolated from positive colonies were used to of these extracts were separated by SDS-PAGE on a transform E. coli DH5a cells and then sequenced. The 12% gel, and transferred to a nitrocellulose membrane. obtained DNA sequences were then subjected to a The membrane was blocked with PBS containing 5% BLAST search in the GeneBank™ database. low fat milk powder and subsequently probed with 40 To verify the expression of the prey protein identified μg of purified recombinant S-HDAg protein in the same in positive clones, yeast protein extracts were prepared buffer, for 1 h at room temperature. After washing, and analyzed by western blot. Preparation of protein bound S-HDAg was detected by immunoblot using a samples was performed according to the Yeast Protocol rabbit polyclonal anti-HDAg (B3) [22], as Handbook from BD Biosciences. In brief, yeast cultures described below. were grown for 2 days, harvested by centrifugation, and frozen at -80°C. The frozen cell pellets were lysed by Cell culture ressupending in the same volume of warm (60°C) crack- The human hepatocellular carcinoma cell line Huh7 ing buffer [8 M urea, 5% SDS, 40 mM Tris-HCl pH 6.8, (European Collection of Cell Cultures n° 01042712) and 0.1 mM EDTA, 1% b-mercaptoethanol, 0.4 mg/mL bro- the HDV cDNA stably transfected Huh7-D12 cell line mophenol blue] and glass beads (425-600 μm, acid- (Japanese Collection of Research Bioresources JCRB washed, Sigma-Aldrich). The mixture was incubated at 0403) were used in this study. The Huh7-D12 cell line 70°C for 10 min and then vortexed for 1 min at high was obtained by stable transfection of Huh7 cells with a speed. The lysates were then cleared by centrifugation plasmid containing a trimer of full-length genomic HDV for 5 min at 14,000 rpm, and the supernatants were cDNA. The cDNA was initially obtained from a wood- boiled at 100°C for 5 min. Finally, western blot analysis chuck liver infected with human serum containing was performed as described below. HDV. Huh7-D12 cells constitutively express HDV ribo- To confirm the detected interactions and to map the in a pattern consistent with ongoing S-HDAg domains involved in the interaction with HDV replication [23,24]. hnRNPC, yeast co-transformation assays were per- Both cell lines were cultured as monolayers in RPMI formed. Yeast strain AH109 was co-transformed with 1640 medium (Sigma) supplemented with 10% fetal plasmids encoding S-HDAg deletion mutants and the bovine serum (Invitrogen) and grown at 37°C in a humi- prey plasmid. Growth was assayed on high stringency dified atmosphere containing 5% CO2. medium (SD/-Trp/-Leu/-Hist/-Ade/X-a-gal) to evaluate the activation reporter genes. Co-immunoprecipitation For co-immunoprecipitation, protein extracts from Purification of recombinant proteins Huh7-D12 or Huh7 cells were prepared. Cells were first Plasmids pGEX-6P-2/S-HDAg and pET28c/hnRNPC sedimented by centrifugation and resuspended in lysis were used to transform E. coli BL21 and BL21- buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Casaca et al. Virology Journal 2011, 8:358 Page 4 of 13 http://www.virologyj.com/content/8/1/358

Nonidet P-40 and 10% glycerol] supplemented with out overnight at 37°C and the digoxigenin-labeled probe complete protease inhibitor cocktail (Roche). After pre- was detected after incubation with a monoclonal clearing with protein G Dynabeads (Invitrogen), extracts anti-digoxigenin antibody conjugated with FITC (Roche) were incubated with a rabbit polyclonal antibody anti- followed by incubation with a secondary anti-FITC anti- HDAg, at 4°C for 2 h. The resulting immunocomplexes body conjugated with Alexa-488 (Jackson Immuno- were precipitated with Protein G Dynabeads (Invitro- Research Laboratories). gen), extensively washed with PBS, and analyzed by wes- tern blot. Confocal microscopy and image analysis Fluorescent labeled samples were analyzed under a Zeiss Western blot LSM 510 META confocal microscope. Green fluores- Western blot analysis was performed essentially as pre- cence was detected using a 488-nm Argon laser and red viously described [23]. Protein samples were separated fluorescence was detected using a 543-nm HeNe laser. on 12% SDS-PAGE gels and transferred to nitrocellulose The equipment was calibrated with multicolor fluores- membranes (Schleicher & Schuell). The membranes cent beads (Molecular probes, USA) and a dual-band fil- were blocked with PBS containing 5% low fat milk pow- ter allowing simultaneous visualization of green and red der and probed with specific primary , fol- fluorescence. lowed by incubation with appropriate secondary Co-localization coefficients were calculated using Ima- antibodies conjugated with horseradish peroxidase geJ software and the JACoP plugin [25]. The Manders’ (BioRad). Blots were developed with an enhanced che- overlap coefficient (OC) was calculated in order to eval- miluminescence system (ECL, GE Healthcare). When uate co-localization between pairs of images in the red needed, the density of the protein bands was determined and green channels. The values of Mander’s coefficients using the NIH ImageJ software http://rsbweb.nih.gov/ij/. range from 0 to 1, with 1 indicating 100% co-localiza- The following primary antibodies were used: mouse tion and 0 indicating no co-localization [26]. monoclonal antibody against hnRNPC (Santa Cruz Bio- technology), rabbit polyclonal anti-HDAg antibody, siRNA knockdown assays mouse anti-His-tag monoclonal antibody (Sigma- The knockdown of endogenous hnRNPC in Huh7 cells Aldrich), and rabbit polyclonal anti-clathrin antibody was performed using a short hairpin RNA (shRNA) (Santa Cruz Biotechnology). strategy. To construct shRNA-expressing vectors, oligonucleo- Immunofluorescence and in situ hybridization tides targeting the human hnRNPC mRNA and the cor- Immunofluorescence and in situ hybridization were per- responding complementary sequences, were inserted formed on Huh7-D12 cells, as earlier described [23]. into the pSIREN-RetroQ vector (BD Biosciences). The For immunofluorescence cells, grown on coverslips, oligonucleotide sequences were designed according to were fixed with 3.7% formaldehyde and then permeabi- the guidelines provided in http://bioinfo.clontech.com/ lized with 0.5% Triton-X100. Cells were then sequen- rnaidesigner, and were as follows: 5’gatccgcagtagagatgaa- tially incubated with primary antibodies (rabbit gaatgttcaagagacattcttcatctctactgcttttttacgcgtg 3’ (sense) polyclonal anti-HDAg and mouse monoclonal anti- and 5’aattcacgagtaaaaaagcagtagagatgaagaatgtctcttgaa- hnRNPC) for 1 h at room temperature, followed by cattcttcatctctactgcg 3’ (antisense). The underlined incubation with appropriate secondary antibodies conju- sequences denote the hnRNPC shRNA sequence target- gated either with FITC or Texas Red (Jackson Immu- ing the hnRNPC mRNA (nucleotides 673 to 691). The noResearch Laboratories). After washing, the coverslips oligonucleotides were annealed in vitro and subcloned were mounted in Vectashield (Vector Laboratories). into the BamH IandEcoR I sites, downstream of the For fluorescence in situ hybridization a digoxigenin- human U6 promotor thus yielding the pSIREN-RetroQ/ 11-dUTP-labelled probe was used. This probe was pre- hnRNPC vector. A control vector was also generated by pared by nick-translation of plasmid pSVL(D3) [19] as using the negative control siRNA oligonucleotide target- previously described [24]. This plasmid contains a tri- ing the luciferase mRNA supplied by the manufacturer mer of full-length genomic HDV cDNA cloned in pSVL (pSIREN-RetroQ/Luc). (GE Healthcare). Cells, grown on coverslips, were fixed Transfection procedures were performed using the with 3.7% formaldehyde, permeabilized with 0.5% Tri- FuGENE6 transfection reagent (Roche), according to the ton-X100, and treated with 200 U/ml RNase free DNase manufacturer’s instructions. Initially, Huh7 cells were I (Roche) as earlier described [24]. Cells were then transfected with 1 μg pSVL(D3). After 48 h incubation sequentially incubated with 70% formamide and 50% cells were transfected again with pSIREN-RetroQ/ formamide at 73°C, for 3 min. Hybridization was carried hnRNPC or pSIREN-RetroQ/Luc. 24 h post-transfection, Casaca et al. Virology Journal 2011, 8:358 Page 5 of 13 http://www.virologyj.com/content/8/1/358

puromycin was added at a final concentration of 2 μg/ strain Y189. Yeast colonies were first selected on med- mL. Finally, cells were harvested and analyzed by wes- ium lacking leucine, tryptophan, histidine and adenine tern blot as described above. and subsequently assayed for a-galactosidase activity. Plasmids were isolated from 112 positive clones and res- Results cued by transformation into E. coli. The identity of the Identification of S-HDAg interacting proteins in a yeast obtained plasmids was established by sequencing allow- two hybrid screening ing the identification of 30 known proteins in the Gen- In order to identify proteins interacting with S-HDAg, a Bank database (Table 2). These proteins were found to yeast two hybrid screening was performed. The entire S- be involved in different metabolic pathways and func- HDAg cDNA was fused in-frame to the Gal4 DNA tions, namely cell communication and signal transduc- binding domain and the resulting construct was trans- tion, transport, protein metabolism, immune response, formed into the yeast strain AH109. Using this bait, we and regulation of nucleic acid metabolism (Figure 1). In screened approximately 108 clones from a human liver a previous study aimed to detect S-HDAg interactors, cDNA library, previously pre-transformed into the yeast Cao et al. [8] used a combined immunoprecipitation

Table 2 Proteins that interact with S-HDAg in the yeast two-hybrid screening Protein Number of Biological function clones Heterogeneous nuclear ribonucleoprotein C isoform 1 (HNRNPC) [NP_004491.2] 5 Raly RNA binding protein-like (RALY) [NP_057951.1] 1 Chromodomain helicase DNA binding protein 7 (CHD7) [NP_060250.2] 1 ELAV-like protein 1 (ELAVL1) [NP_001410.2] 1 Heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB) [NP_112556.2] 1 Regulation of nucleic acid metabolism Ankyrin repeat domain 11 (ANKRD11) [NP_037407.4] 1 SWI/SNF related. matrix associated actin dependent regulator of chromatin subfamily a member 2 1 (SMARCA2) [NP_003061.3] Zinc finger proteín 533 (ZNF533) isoform 2 [NP_001106868.1] 1 Ribosomal protein S15 (RPS15) [NP_001009.1] 1 TRAF and TNF receptor associated protein [EAW55458.1] 1 Cell communication, signal transduction Ribosome receptor P180 [BAF73807.1] 1 EBNA1 binding protein 2 (EBP2) [NP_006815.2] 1 Unknown Ribosomal L1 domain containing 1 (RSL1D1) [CAA07491.1] 1 Apolipoprotein E (APOE) [NP_000032.1] 5 Transport Proteasome activator complex subunit 2 (PSME2) [NP_002809.2] 1 BRCA1 associated protein (BAP1) [NP_004647] 1 Antithrombin III (SERPINC1) [NP_000479.1] 1 Protein metabolism Ribosomal protein L36A like (RPL36AL) [NP_000992.1] 1 Ribosomal protein L13 (RPL13) [NP_000968.2] 2 a-1 acid glycoprotein (ORM1) [NP_000598.2] 5 Complement component C3 (C3) [NP_000055.2] 1 Immune response Natural killer tumor recognition sequence (NKTR) [NP_005376] 1 17b hidroxyesteroid dehydrogenase 4 (HSD17B4) [NP_000405.1] 2 17b hidroxyesteroid dehydrogenase 6 (HSD17B6) [NP_003716.2] 1 ATP synthase subunit 8 [ACQ75157.1] 2 Cytochrome oxidase subunit 3 (MT-COIII) [ACT53100.1] 1 Metabolism, energy pathways NADH dehydrogenase subunit 2 (MT-ND2) [ACT53095.1] 1 NADH dehydrogenase subunit 4 (MT-ND4) [ACQ76111.1] 1 NADH dehydrogenase (ubiquinone) 1b subcomplex subunit 7 (NDUFB7) [NP_004137.2] 1 Cytochrome oxidase subunit 1 (MT-COI) [ACI04331.1] 1 Casaca et al. Virology Journal 2011, 8:358 Page 6 of 13 http://www.virologyj.com/content/8/1/358

Figure 2 Identification of hnRNPC as an S-HDAg interactor in a yeast two hybrid screening. (A) Schematic representation of hnRNPC and the GAL4AD fusion proteins identified in yeast positive

clones (GAL4AD/hnRNPC and GAL4AD/hnRNPC1-108). GAL4AD/ hnRNPC and GAL4AD/hnRNPC1-108 were identified in four and one positive clones, respectively. GAL4AD/hnRNPC1-108 codes for a truncated form of hnRNPC, that contains only the first 108 aminoacids. Both proteins contain a 5’untranslated region of hnRNPC mRNA (black bar). GAL4AD represents the activation domain of the GAL4 transcription factor and RRM and CC represent Figure 1 S-HDAg interacting partners in a yeast two-hybrid the RNA recognition or coiled-coil motifs, respectively, present in screen. The identified proteins were grouped according to their hnRNPC. (B) Western blot analysis of hnRNPC expression in yeast respective biological functions using the human protein reference positive clones. Yeast extracts were prepared, separated on 12% database http://www.hprd.org/. SDS/PAGE gels, and immunoblotted with an anti-hnRNPC antibody.

The positive clone expressing GAL4AD/hnRNPC1-108 was used in this analysis and AH109 yeast extracts were used as negative control. and mass spectrometry approach. The authors reported the identification of over 100 known proteins able to western blot analysis. Yeast protein extracts from a posi- interact with FLAG-S-HDAg. Among the identified pro- tive clone were prepared and the presence of protein teins three were also detected as S-HDag interactors in GAL4AD/hnRNPC1-108 (37 KDa) was investigated using this study: hnRNPC, hnRNP A/B, and the RNA binding an antibody specific for hnRNPC protein. As shown in protein RALY. Interestingly, these proteins are involved Figure 2B, a band of the expected molecular weight in the regulation of RNA metabolism and belong to the could be detected in the positive clone extracts but not same functional group allowing to speculate about a in yeast AH109 extracts (negative control), confirming possible involvement in HDV replication. Taking into the expression of GAL4AD/hnRNPC1-108 protein. consideration that hnRNPC was previously shown to modulate the replication of a number of RNA viruses hnRNPC and S-HDAg interact in vitro and in vivo including HCV, VSV, and influenza A [14-17], Having established that S-HDAg and hnRNPC interact we decided to further explore the possible interaction in the yeast two-hybrid assay, we decided to investigate between S-HDAg and hnRNPC. Four of the prey plas- if the two proteins also interact in vitro and in vivo in mids from the positive clones initially obtained in the human hepatoma cells. To test for possible in vitro yeast two-hybrid screening contained the entire interactions we performed a blot overlay assay. To do hnRNPC ORF sequence while a fifth plasmid expressed this, both S-HDAg and hnRNPC were expressed in E. a truncated form of the hnRNPC protein (GAL4AD/ coli, as GST or His-tag fusion proteins, respectively. hnRNPC1-108), as shown in Figure 2A. Increasing amounts of E. coli protein samples containing Our first approach was designed in order to confirm His6/hnRNPC were then separated by electrophoresis, the expression of a GAL4AD/hnRNPC fusion protein by and transferred to a nitrocellulose membrane. The Casaca et al. Virology Journal 2011, 8:358 Page 7 of 13 http://www.virologyj.com/content/8/1/358

membrane was then overlaid with recombinant S-HDAg assays using lysates from either Huh7-D12 or the paren- and the bound protein was detected with a rabbit anti- tal, non-transfected Huh7 cell lines. A rabbit polyclonal S-HDAg antibody. The obtained results are displayed on anti-HDAg antibody was used in the assays and inter- Figure 3A, and show that increasing amounts of His6- acting proteins were detected by western blot analysis hnRNPC correspond to a proportional increase in the with a mouse monoclonal antibody against hnRNPC. As amounts of bound S-HDAg, suggesting a specific inter- negative controls we used both Huh7-D12 lysates in the action between the two proteins. absence of antibody and Huh7 lysates immunoprecipi- Having established that S-HDAg and hnRNPC interact tated with anti-HDAg antibody. As shown in the Figure in vitro, we next decided to investigate if both proteins 3B, hnRNPC and HDAg can be co-immunoprecipitated also interact in vivo in the human hepatoma cell line in Huh7-D12 cells, but not in the negative controls, pro- Huh7-D12 which is stably transfected with HDV cDNA viding evidence that the two proteins are probably pre- and constitutively expresses virus RNAs and antigens. sentinthesamecomplex.Itisthuspossiblethatthe This was performed using co-immunoprecipitation two proteins also interact in vivo in human liver cells.

A.

1 OLIGNLS RNA-BD 195 S-HDAg S-HDAg(1-62) S-HDAg(63-97) S-HDAg(98-195)

B.

2 1- pAS2-1/S-HDAg + pGADT7-Rec/hnRNPC1-108 132- pAS2-1/S-HDAg 3-pGADT7-Rec/hnRNPC1-108 4- pAS2-1/S-HDAg (1-62) + pGADT7-Rec/hnRNPC 4 6 1-108 5 5- pAS2-1/S-HDAg (63-97) + pGADT7-Rec/hnRNPC1-108 6- pAS2-1/S-HDAg (98-195)+ pGADT7-Rec/hnRNPC1-108

SD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/- Hist/-Ade/X-α-Gal

Figure 3 In vitro and in vivo interaction between hnRNPC and S-HDAg. (A) Overlay of bacterially expressed His6/hnRNPC with recombinant S-HDAg. E. coli protein extracts were prepared after induction of recombinant protein expression with IPTG. Increasing amounts of His6/hnRNPC- containing extracts (lanes 2 to 6), were separated by SDS/PAGE, blotted onto nitrocellulose membranes and overlaid with the same amount of purified recombinant S-HDAg. Detection of S-HDAg was performed with a specific rabbit polyclonal antibody. E. coli protein extracts lacking

His6/hnRNPC (lane 1) were used as a negative control (upper panel). The presence of His6/hnRNPC in the extracts was confirmed using a mouse anti-His-tag monoclonal antibody (middle panel). In the lower panel, the assay was performed in the absence of purified S-HDAg showing that the anti-HDAg polyclonal antibody does not recognize hnRNPC. (B) Co-immunoprecipitation of hnRNPC with S-HDAg. Huh7-D12 cell lysates were immunoprecipitated with an anti-HDAg antibody bound to protein G beads. The immunoprecipitates were separated on 12% SDS/PAGE gels and immunoblotted with an anti-hnRNPC antibody. The negative controls were performed by incubation of Huh7-D12 lysates with beads protein G beads and by immunoprecipitating Huh7 lysates with an anti-HDAg antibody. Casaca et al. Virology Journal 2011, 8:358 Page 8 of 13 http://www.virologyj.com/content/8/1/358

Subcellular localization of hnRNPC, HDAg, and HDV RNA by double immunofluorescence staining and confocal hnRNPC proteins are predominantly localized in the microscopy. Huh7-D12 cells were dual labeled using nucleus and bind to nascent RNA polymerase II tran- FITC or Texas Red-conjugated antibodies to detect scripts, playing important roles in mRNA biogenesis HDAg and hnRNPC respectively. The distribution of S- [27]. Since HDV viral transcription takes place in the HDAg is consistent with previous findings [24], showing nucleus and the involvement of RNA polymerase II in a nuclear localization, with preferential accumulation in this process has been proposed [5], it is plausible to nuclear foci (Figure 4A). hnRNPC is widely distributed consider that hnRNPC might influence HDV replication. throughout the nucleus and apparently overlaps with To learn more about the interaction between S-HDAg HDAg (Figure 4B). The degree of co-localization was and hnRNPC at the cellular level, the sub-cellular distri- quantified by determining Mander’s overlap coefficient bution of the two interacting proteins was determined (OC). The mean OC (n = 10) determined for HDAg

Figure 4 Co-localization of hnRNPC with HDV antigens and RNA in human liver cells. (A-C) Double indirect immunofluorescence was used to detect HDAg and hnRNPC proteins in cultured Huh7-D12 cells. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X- 100, and incubated with an anti-HDAg antibody (A, green staining) and an anti-hnRNPC antibody (B, red staining). Combined in situ hybridization and immunofluorescence was performed to detect HDV RNA and the hnRNPC protein. After fixation and permeabilization, cells were hybridized with a digoxigenin-labeled probe to detect HDV RNA (D, green staining) and with an anti-hnRNPC antibody (E, red staining). Overlaps of images are shown in panels C and F. (G) 10 individual cells from each experiment were analyzed using the ImageJ software and the JaCoP plugin to determine the Mander’s overlap coefficient. Casaca et al. Virology Journal 2011, 8:358 Page 9 of 13 http://www.virologyj.com/content/8/1/358

and hnRNPC was 0.823 (Std. deviation = 0.007) which inconsistent with a possible interaction during virus indicate that the two proteins significantly co-localize. replication. However, this hypothesis, as well as an even- Since the diffused nucleoplasmic distribution with addi- tual role of the virus RNA in mediating and promoting tional accumulation in foci of HDAg, is consistent with this process, needs to be further investigated. ongoing HDV RNA replication, the co-localization with hnRNPCmayrepresentabiologicallyrelevant Analysis of interacting domains of S-HDAg observation. In the yeast two-hybrid screening, we found that the N After obtaining evidences on HDAg and hnRNPC co- terminal aminoacid sequence of hnRNPC (amino acids localization in HDV expressing cells, we combined in 1 to 108), which includes the RNA recognition motif, situ hybridization and immunofluorescence experiments was able to interact with the S-HDAg. In order to iden- to analyze the co-localization between HDV RNAs and tify the regions of the S-HDAg required for interaction hnRNPC protein. Indeed, because HDAgs and HDV with this region of hnRNPC, three truncated mutants of RNAs exhibit a similar sub-cellular distribution in cells S-HDAg were cloned fused to GAL4-BD and their inter- undergoing HDV replication [24] and hnRNPC is also action with hnRNPC1-108 was assessed using yeast two an RNA binding protein, it is likely that HDV RNAs hybrid assays. Since three functional domains were pre- and hnRNPC cellular distributions are also related. To viously identified in the S-HDAg, a nuclear localization exclude a possible contribution of hybridization signals signal (aa 66-75) [28], an oligomerization domain (aa corresponding to the HDV cDNA integrated in the 12-60) [29], and two bipartite RNA-binding domains (aa , Huh7-D12 cells were treated with DNase 97-143) [30], the three deletion constructs were gener- prior to hybridization as earlier described [24]. Figure 4 ated in order to independently include one of the three (D-F) shows examples of the obtained results and the S-HDAg functional domains. Accordingly, these respective OC. The mean OC value was found to be constructs included the coding sequence for aminoacids high (0.801; std. deviation = 0.066; n = 10), indicating 1-62, 63-97, and 98-195, respectively, in the S-HDAg that both HDV RNAs and antigens significantly coloca- (Figure 5A). lize with hnRNPC in human cells. The observed coloca- Reporter expression was reproduced using intact S- lization of hnRNPC with HDV RNA and HDAgs is not HDAg and hnRNPC1-108, confirming the interaction

A B C

G

S-HDAg hnRNPC Merge D E F

HDV RNA hnRNPC Merge Figure 5 Identification of S-HDAg domains involved in the S-HDAg/hnRNPC interaction, by yeast two-hybrid analyses. (A) Schematic representation of the deletion constructs of S-HDAg cloned in the bait vector pAS2-1. OLIG, NLS and RNA-BD represent the oligomerization, nuclear localization signal or RNA binding domains, respectively, identified in S-HDAg. (B) AH109 yeast cells were (co)transformed with the indicated plasmids. Interactions were tested by growth selection on dropout media lacking the amino acids leucine or tryptophan (SD/-Leu or SD/-Trp) to select for pGADT7/Rec or/and pAS2-1 plasmid constructs, respectively. Activation of reporter genes was tested on plates lacking leucine, tryptophan, histidine and adenine, and containing X-a-Gal (SD/-Trp/-Leu/-Hist/-Ade/X-a-Gal). Casaca et al. Virology Journal 2011, 8:358 Page 10 of 13 http://www.virologyj.com/content/8/1/358

previously detected in the yeast two hybrid screening. A Additionally, the C-terminal deletion mutant, containing N iR residues 98-195, could activate the expression of repor- A. s ol ter genes. In contrast, the two remaining constructs cor- tr on responding to aa 1-62 and 63-97, were not able to C interact with hnRNPC1-108 and thus the activation of reporter genes could not be detected (Figure 5B). This hnRNP C result suggests that the S-HDAg C-terminal region con- taining the RNA-binding domain mediates the interac- L-HDAg tion with hnRNPC. S-HDAg hnRNPC knockdown reduces the HDAg expression levels Clathrin Several members of the hnRNP family of proteins have been recently shown to positively regulate the replica- B. tion of hepatitis C and influenza A viruses [15,30]. Similarly to these two RNA viruses it would be possi- ble that hnRNPs are also involved in the modulation of HDV replication. We thus decided to investigate the ability of hnRNPC to influence HDV replication, using a shRNA-mediated knockdown technology. Plasmid pSVL-D3, which contains three copies in tandem of the HDV genome and was previously shown to be able to initiate HDV replication [19], was used to trans- fected Huh7 cells. After 48 hrs incubation, the cells were transfected again with the pSIREN-RetroQ/ hnRNPC vector, encoding a shRNA specific for hnRNPC, or the pSIREN-RetroQ/Luc, targeting the luciferase protein, here used as a negative control. The subsequent addition of puromycin allowed the selec- tion of pSIREN-RetroQ expressing cells. The efficiency of the pSIREN-RetroQ/hnRNPC construct in silencing hnRNPC was analysed by western blot. Figure 6 dis- plays the obtained results showing that in the presence of the pSIREN-RetroQ/hnRNPC plasmid, the endogen- Figure 6 Silencing of endogenous hnRNPC reduces expression ous hnRNPC levels were reduced to approximately of HDAgs. (A) Western blot analysis of Huh7 cells transfected with 45% when compared with pSIREN-RetroQ/Luc trans- plasmid pSVD3, and pSIREN-RetroQ constructs. Knockdown of the hnRNPC protein was performed using a specific siRNA in Huh7 cells. fected cells. Although the obtained reduction in intra- 48 hrs after addition of puromycin, total protein extracts were cellular hnRNPC levels was about 55%, we believe it prepared, separated by SDS-PAGE, and transferred to a nitrocellulose represented an acceptable compromise in order to membrane. Blots were incubated with anti-hnRNPC, anti-clathrin or reduce of possible side effects in cellular metabolism. anti-HDAg antibodies. The displayed images are representative of In fact, although longer incubation times resulted in a three independent experiments. (B) Images were digitalized and band intensity was determined using the ImageJ program. The more marked inhibition of hnRNPC expression (up to relative amounts of protein were obtained after normalization with 80%), cell viability was also dramatically reduced (often the housekeeping protein clathrin. Inhibition of HDAg expression is less than 10%) limiting the significance of the obtained presented as a percentage after comparison with the values results (data not shown). obtained with the negative control vector pSIREN-RetroQ/Luc After confirming that pSIREN-RetroQ/hnRNPC provided by the manufacturer. transfection results in a reduction of endogenous expression of hnRNPC, we decided to use the same approach to investigate the influence of hnRNPC HDAg expression levels, respectively, when compared silencing on HDAgs expression. Figure 6A shows the with the control pSIREN-RetroQ/Luc transfected results obtained after western blot analysis of the cells (Figure 6B). These results suggest that hnRNPC relative expression of HDAgs in these experiments. may be involved in the modulation of HDV RNA As can be observed, knockdown of hnRNPC resulted transcription or stabilization of HDAgs coding in a decrease of 66% and 92% of S-HDAg and L- mRNAs. Casaca et al. Virology Journal 2011, 8:358 Page 11 of 13 http://www.virologyj.com/content/8/1/358

Discussion able to find hnRNPC1 among the putative interacting Despite being a small and simple virus the host factors clones in the yeast two hybrid screen. This result may involved in HDV replication remain largely unknown. be explained by the fact that hnRNPC2 is expressed at Nevertheless, using different molecular biology and bio- approximately one-third of the level of hnRNPC1 [12], chemical approaches a number of host proteins that thus being less abundant in the human liver cDNA interact with the virus RNA and antigens were already library. However, the fact that we also identified a trun- identified [31]. In this work we made use of a yeast two- cated form of hnRNPC comprising the first 108 amino hybrid approach to screen 108 clones from a human acids which are common to both isoforms, may suggest liver cDNA library and identify possible S-HDAg inter- that S-HDAg shall be able to interact with hnRNPC2 as acting proteins. The use of a similar approach has been well. A detailed analysis of the sequences obtained from previously reported by Wang et al allowing identifying all the five prey plasmids coding for hnRNPC showed NES1 as a L-HDAg interactor [32]. Interestingly, we an in-frame stop codon between the GAL4-AD and the found in our system that when using L-HDAg as bait hnRNPC ORF. Accordingly, the GAL4AD/hnRNPC this protein could self activate transcription of HIS3, fusion protein could only be produced through a stop ADE2, and MEL-1 reporter genes (data not shown). codon bypass event. However it has been proposed that Accordingly, this observation did not allow us to further S. cerevisiae enablesthesynthesisofpolypeptidesvia use L-HDAg in the yeast two-hybrid screening. How- stop-codon readthrough [34]. Furthermore, in previous ever, using S-HDAg as bait, we initially obtained 112 yeast two hybrid screens this mechanism was also clones that after subsequent sequence analysis and data- observed, namely in the cDNA library screen with the base search allowed identifying 30 known proteins Lyssavirus phosphoprotein as bait, where the protein (Table 2). These proteins were reported to be involved dynein LC8 was identified as an interactor and the cor- in different cellular processes including nucleic acid responding sequence also exhibited an in-frame stop metabolism, cell communication, transport, protein codon between the GAL4-AD and the dynein LC8 cod- metabolism, immune response, and energy pathways. ing region [35]. Two of the identified proteins were found to belong to Having identified hnRNPC as an S-HDAg interactor the hnRNP family of proteins: hnRNP A/B and in the yeast two-hybrid system, we decided to confirm hnRNPC. These two proteins together with the RNA this interaction in vitro using a blot overlay assay with binding protein RALY, also identified in this yeast two- purified S-HDAg and increasing amounts of bacterially hybrid screen, were also previously identified by Cao et expressedhnRNPC.Theresultsindicatedthatthe al. [8] as S-HDAg interactors using an immunoprecipita- interaction of the two proteins is specific since the tion followed by mass spectrometry approach. These obtained band intensity was proportional to the proteins are involved in the regulation of RNA metabo- amount of hnRNPC present in the bacterial protein lism and may thus be involved in the modulation of samples. Next, we decided to investigate if S-HDAg HDV RNA transcription and replication. This observa- and hnRNPC interact in vivo in human liver cells. This tion, and the fact that five of the positive clones was performed using a co-immunoprecipitation assay detected in the yeast two-hybrid screening were found and a polyclonal antibody against HDAg allowing con- to code for hnRNPC, prompted us to further investigate firming the previously detected interaction between the a possible functional significance of the interaction two proteins. between this protein and S-HDAg. HDV replication occurs in the nucleus of infected liver The family of hnRNP proteins has been associated cells. It was previously demonstrated that both virus with various cellular functions namely mRNA biogen- RNA and antigens co-localize in the nucleus displaying esis, transport and translation [33]. Since HDV is an a diffuse pattern with additional accumulation in foci. RNA virus highly dependent on host proteins, the iden- These foci, however, do not correspond to preferential tification of hnRNPC as an interactor for S-HDAg may sites of RNA synthesis or processing [23]. If hnRNPC is suggest a possible role for this protein in the HDV repli- involved in HDV replication it can be expected to co- cation cycle. Thus we decided to explore this interaction localize with HDV components in the nucleus, namely in order to elucidate its biological relevance. in the nucleoplasm. Immunofluoresecence and in situ Two isoforms of hnRNPC were previously identified: hybridization analysis of a human liver cell line that the more abundant hnRNPC1, and a splicing variant constitutively expresses HDV RNAs and antigens, with a 13 aminoacid insertion, known as hnRNPC2 [12]. allowed calculating the Mander’s overlap coefficient Both proteins form stable heterotetramers able to bind between hnRNPC, HDV RNA, and HDAgs. The to mRNA cooperatively [13]. Although the two isoforms obtained values, 0.823 for hnRNPC and HDV RNA and share most of the aminoacid sequence we were only 0.801 for hnRNPC and HDAgs, revealed a significant Casaca et al. Virology Journal 2011, 8:358 Page 12 of 13 http://www.virologyj.com/content/8/1/358

degree of co-localization, supporting an eventual role of shRNA experiments to knock down several of the iden- hnRNPC in virus replication. tified proteins and determined the effect on the accumu- In an attempt to map the domains of the S-HDAg that lation of HDV RNA. However, hnRNPC was not among interact with hnRNPC we made three plasmid constructs the investigated proteins. coding for aminoacids 12-60, 66-75, and 98-195. These regions in the S-HDAg include the oligomerization Conclusions domain, the nuclear localization signal, and the RNA In conclusion, a yeast two-hybrid screening of a human binding domain, respectively. The ability to interact with liver cDNA library allowed the identification of 30 S- hnRNPC was tested in the yeast two-hybrid assay show- HDAg interacting proteins. Among the proteins ing that only the polypeptide comprising aminoacids 98- involved in nucleic acid metabolism hnRNPC was found 195 could activate transcription of reporter genes. Since to bind in vitro and in vivo to S-HDAg. Although in the yeast two-hybrid screen, we found that the N shRNA-mediated knockdown of hnRNPC resulted in a terminal aminoacid sequence of hnRNPC (amino acids 1 marked decrease of HDAg accumulation, further studies to 108), which comprises the RNA recognition motif [13] are mandatory in order to clarify the precise role of this interacts with the S-HDAg it is plausible that the interac- protein in the HDV replication cycle. tion between the two proteins is mediated by their respective RNA binding domains. However, our results Acknowledgements seem to indicate that the interaction between the two We acknowledge Dr. José Rino and Dr. António Temudo (Institute of proteins is not dependent on the presence of HDV RNA. Molecular Medicine, Lisbon) for help with the confocal microscope. This In fact, the interaction between the two proteins can be work was supported by Fundação para a Ciência e Tecnologia (PTDC/SAU- MII/098314/2008). AC is a recipient of a FCT PhD grant. detected in the absence of HDV RNA. Since hnRNPC is the most abundant protein in the hnRNP complexes it Author details 1 would be possible that the interaction with HDAg repre- Unidade de Microbiologia Médica, Centro de Malária e outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de sents an artificial observation mediated by this abun- Lisboa, Lisboa, Portugal. 2Laboratório de Transdução de Sinais, Centro de dance. However, taken together, our data, including blot Biologia Celular, Universidade de Aveiro, Aveiro, Portugal. overlay, coimmunoprecipitation and experiments aimed Authors’ contributions at mapping the interaction domains, show that the two AC carried out the experiments and helped draft the manuscript. MF proteins interact specifically. participated in the yeast two-hybrid screening, coordination of the study hnRNPC has been shown to play important roles in the and helped to draft the manuscript; ECS participated in the design and coordination of the study. CC conceived the study, participated in its design replication cycle of several RNA viruses including influ- and coordination, and drafted the manuscript. All authors read and enza A, hepatitis C, and polioviruses. Recently, Brunner approved the final manuscript. et al, showed that the rate of poliovirus RNA synthesis is Competing interests slower in a cell line expressing reduced levels of hnRNPC The authors declare that they have no competing interests. [36]. Furthermore, knockdown of hnRNPC using shRNAs resulted in a decrease of positive strand polio- Received: 23 May 2011 Accepted: 20 July 2011 Published: 20 July 2011 virus RNA accumulation [37]. 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