Arch Virol DOI 10.1007/s00705-014-1997-3

ORIGINAL ARTICLE

Physical interaction between bovine viral diarrhea virus nonstructural protein 4A and adenosine deaminase acting on RNA (ADAR) Yassir Mahgoub Mohamed • Norasuthi Bangphoomi Daisuke Yamane • Yuto Suda • Kentaro Kato • Taisuke Horimoto • Hiroomi Akashi

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Received: 27 September 2013 / Accepted: 19 January 2014 Ó Springer-Verlag Wien 2014

Abstract Bovine viral diarrhea virus (BVDV) is a positive-sense RNA virus known to produce double-stranded RNA (dsRNA) during its replication in the cytoplasm. Extended dsRNA duplexes can be hyperedited by adenosine deaminase acting on RNA (ADAR), which catalyzes adenosine (A)-to-inosine (I) editing. A-to-I editing has been reported for various viruses. A number of cellular antiviral defense strategies are stimulated by dsRNA, and this may involve hyperediting of dsRNA by ADARs, followed by targeted cleavage by cytoplasmic endonucleases. Here, we identify ADAR as a binding partner of BVDV NS4A in vitro and in vivo and show that the N-terminal domain of NS4A is the ADAR-binding domain. We also show that ADAR has an inhibitory effect on BVDV replication when overexpressed in BVDV-infected bovine cells. Our findings suggest a role of NS4A in the interaction of BVDV with ADAR that favors virus replication.

Introduction Like RNA splicing, RNA editing alters the sequence of an RNA from that encoded in the DNA. Typically, a single

Present Address: Y. M. Mohamed Department of Microbiology, Tropical Medicine Research Institute, National Centre for Research, P.O.Box: 1304, Khartoum 11111, Sudan Y. M. Mohamed  N. Bangphoomi  D. Yamane  Y. Suda  K. Kato  T. Horimoto  H. Akashi (&) Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan e-mail: [email protected]

RNA splicing reaction removes a large block of contiguous sequence, whereas each RNA editing reaction changes only one or two nucleotides. RNA editing can create, delete, or alter the meaning of a codon, create a splice site, or alter RNA structure [8]. Two types of RNA editing have been found in nuclear-encoded mRNAs [7]. One type involves the deamination of cytidine (C) to create uridine (U), and the other, deamination of adenosine (A) to create inosine (I). RNA editing by adenosine deamination is catalyzed by members of an enzyme family known as adenosine deaminase acting on RNA (ADAR) [4]. ADARs act on RNA that is completely, or largely, double-stranded and catalyze the deamination of adenosine to produce inosine, which is translated as a guanine (G) [2], and most enzymes recognize inosine as guanosine. Thus, ADARs change the primary sequence information in an RNA. ADARs from all organisms have a common domain structure that includes a variable number of double-stranded RNA (dsRNA) binding motifs (dsRBMs), followed by a highly conserved C-terminal catalytic domain. Despite their diversity in primary structure, all ADARs deaminate adenosines within completely base-paired dsRNA, regardless of the identity of their natural, endogenous substrates. Many viruses contain RNA genomes or replicate their genomes through an RNA intermediate, which is often double-stranded in form. Such long dsRNAs are rarely produced endogenously by cellular systems. ADARs can carry out hyperediting of such transcripts, potentially triggering their degradation by the specific nuclease [14]. Bovine viral diarrhea virus (BVDV) is an economically important, major reproductive pathogen of cattle with positive-sense, single-stranded RNA and is a member of the genus Pestivirus of the family Flaviviridae. Due to the action of the virus in cell culture, a non-cytopathic (ncp) and a cytopathic (cp) biotype can be distinguished. ncp

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BVDV has the ability to establish persistent infection in its host. An additional difference between ncp BVDV and cp BVDV is in their interactions with the innate immune responses, as cp BVDV has been shown to induce type I IFN and numerous IFN-stimulated genes in cultured cells [1, 12, 20] while ncp BVDV is able to block IFN. For BVDV, like other viruses known to persist in their host, it is necessity to evolve mechanism(s) to counteract the immune system in order to establish persistent infection and survive. BVDV is known to produce dsRNA intermediates during its replication, which makes this virus a possible target for hyperediting by ADARs as part of the cellular innate immune defense. Unlike HCV NS4A, there is no information available on how BVDV NS4A contributes to the interaction between the virus and the host cell. In this report, we identified ADAR as a binding partner of BVDV NS4A using yeast two-hybrid screening. This binding was confirmed by coimmunoprecipitation and GST pull-down assays in vitro and in BVDV infected cells, and the ADAR binding domain on NS4A was identified. Given the potent antiviral activity of ADAR on BVDV replication, BVDV may have evolved to regulate its antiviral function through direct interaction with NS4A,

Materials and methods Antibodies and reagents Anti-Flag-M2 mouse monoclonal antibody and anti-glutathione-S-transferase (GST) rabbit polyclonal antibody were purchased from Sigma (St. Louis, MO, USA). Monoclonal antibody against BVDV NS4A was a kind gift from Dr. Till Ru¨menapf (Justus-Liebig-Universita¨t, Giessen, Germany). Goat anti-mouse IgG (L?H) antibodies conjugated with Alexa 488 and goat anti-rabbit IgG antibodies conjugated with Alexa 546 were from Invitrogen (Carlsbad, CA, USA). Recombinant human interferon alpha 2a (hIFN-a 2a) was purchased from ProSpec (Charlotte, NC, USA). Cells and viruses Primary bovine fetal muscle (BFM) cells and the LB9.K cell line have been described previously [18, 21]. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum (FCS) at 37 °C in a humidified 5 % CO2 atmosphere. LB9.K cells, obtained from the American Type Cell Culture Collection (ATCC), were confirmed to be free of BVDV by reverse transcriptase-polymerase chain reaction (RT-PCR). BVDV strains KS86-1cp, KS86-1ncp and Nose have been described previously [10].

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RNA extraction Total RNA was extracted using an SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Plasmids The bovine ADAR complementary DNA (cDNA; GenBank accession number XM_581374) was generated from mRNA extracted from IFN-a-treated BFM cells by RT-PCR using primers that incorporated XhoI/Flag-tag sequences and NotI sites at the 50 and 30 ends, respectively. Flag-ADAR cDNA was then inserted into the pME18S mammalian expression vector at XhoI/NotI sites to generate an N-terminal Flagtagged bovine ADAR expression vector termed pME.FlagADAR. pCAG vector was described previously [21]. An NS4A sequence derived from the BVDV Nose strain was amplified by RT-PCR using primers that incorporated EcoRI/XhoI sites at the 50 and 30 ends, respectively, and cloned into EcoRI/XhoI sites of pGEX-5X-1 (GE Healthcare, Milwaukee, WI, USA) as a GST-NS4 fusion. The GST tag sequence, with or without the NS4A sequence, was then subcloned into the pCAG vector at XhoI/PstI sites to generate pCAG.GST and pCAG.GST-NS4A. For the yeast twohybrid assay, full-length NS4A derived from the BVDV Nose strain was amplified by RT-PCR using primers that incorporated an EcoRI and a PstI site at the 50 and 30 end, respectively, and cloned into EcoRI/PstI sites of pGBKT7 (Clontech, Palo Alto, CA, USA) in-frame with the GAL4 DNA-binding domain to express N-terminal Myc-tagged NS4A designated pGBKT7-NS4A. Fragments encoding a series of NS4A deletion mutants were amplified by PCR using pCAG.GST-NS4A(wt) as template and primers that incorporated a PstI and a NotI site at the 50 and 30 end, respectively, and cloned into PstI/NotI sites of pCAG.GST in-frame with the GST sequence to express N-terminal GSTtagged NS4A mutants designated as pCAG.GST-NS4A(aa122), pCAG.GST-NS4A(aa21-44), and pCAG.GSTNS4A(aa42-64). A plasmid encoding adenovirus VAI RNA (pVAI HDV4) was kindly provided by Dr. Graeme Conn (Emory University, Atlanta, GA, USA). Yeast two-hybrid screening To identify the potential interacting partners of NS4A, we used a Matchmaker Gal4 two-hybrid system according to the manufacturer’s instructions (Clontech). The bait plasmid (pGBKT7-NS4A) was constructed as described above. NS4A expression was confirmed by western blot analysis using anti-Myc antibody (data not shown). To construct a BFM cDNA library, subconfluent cells were treated with recombinant human IFN-alpha 2a (1000 U/ml) for 6 h

BVDV NS4A binds ADAR

prior to RNA extraction. First-strand cDNA was synthesized using random primers from 0.2 lg mRNA, which was purified from total RNA using an OligotexdT30\Super[ mRNA Purification Kit (TaKaRa, Shiga, Japan). Double-stranded cDNA amplification and Saccharomyces cerevisiae strain AH109 co-transformation were performed as described previously [21], except that NS4A was used as bait. Fifty-three clones were identified. The insert cDNA fragments of isolated clones were amplified by PCR using LD-Insert Screening Amplimer Sets (Clontech) according to the manufacturer’s protocol and then sequenced. Homology searches were carried out using the BLAST algorithm through the NCBI website. Transfection, immunoprecipitation and immunoblotting LB9.K cells were seeded into 6-well plates 24 h before transfection. Cells were then transiently transfected with 4 lg plasmid per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Protein extraction from transfected cells was performed as described previously [21]. The same quantity of precleared cell lysates was immunoprecipitated with anti-GST polyclonal antibody or anti-NS4A monoclonal antibody for 2 h with rotation at 4 °C. Immunocomplexes were precipitated by addition of protein G-Sepharose beads (GE Healthcare) for another 1 h at 4 °C with rotation. The immunoprecipitates were analyzed by SDS-PAGE or Tricine-SDS-PAGE [15], transferred to nitrocellulose membranes, probed with antibodies, and detected by enhanced chemiluminescence (ECL; GE Healthcare). The following antibodies were used: horseradish peroxidase-conjugated monoclonal antibody against Flag (1:1000), rabbit polyclonal antibody against GST (1:3000), and mouse monoclonal antibody against NS4A (1:400). Images were taken with an LAS4000mini image analyzer system (Fujifilm, Tokyo, Japan).

four times with GST-soluble buffer [21]. Bound proteins were analyzed by SDS-PAGE and western blot using antibodies against Flag and GST. Immunofluorescence microscopy LB9.K cells were seeded on four-well chamber slides (Nunc, Roskilde, Denmark) at 4 9 104 cells/well. Twentyfour hours later, cells were transfected with Flag-ADAR plasmid, followed by inoculation with BVDV as described in the respective figure legends. At 18 h postinfection (p.i.), cells were washed twice with phosphate-buffered saline (PBS), fixed with PBS containing 4 % paraformaldehyde for 20 min, permeabilized with 0.5 % Triton X-100 in PBS, and blocked with 10 % BSA in PBS for 15 min at room temperature (RT). Cells were then double-stained with rabbit anti-Flag antibody and mouse anti-NS4A antibody for 2 h at RT. After washing three times with 0.1 % Tween 20 in PBS, cells were incubated with Alexa-546conjugated goat anti-rabbit IgG and Alexa-488-conjugated goat anti-mouse IgG (L?H) for 1 h at RT. Cells were then washed three times with 0.1 % Tween 20 in PBS, mounted in Dako fluorescent medium (Dako Corporation, Carpinteria, CA, USA), and observed under an LSM 510 microscope (Carl Zeiss, Tokyo, Japan). Quantitative real-time RT-PCR RNA was isolated using an SV Total RNA Isolation Kit (Promega) and converted to cDNA using the PrimeScript RT Reagent Kit (TaKaRa) according to the manufacturer’s protocol. GAPDH mRNA and viral RNA were quantified using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) as described previously [19].

GST pull-down assay

Results

The GST-BVDV NS4A plasmid was generated as described above. Recombinant protein was expressed, extracted, and eluted as described previously [21]. Eluted proteins were concentrated using Ultrafree-0.5 Centrifugal Filter Devices (10,000- and 30,000-Da cutoff; Millipore, Billerica, MA, USA). For GST pull-down assays, LB9.K cells were transiently transfected with pME.Flag-ADAR and lysed as described above. ADAR-containing lysates were mixed with equal amounts of either GST or GST-NS4A recombinant protein and incubated with gentle agitation at 4 °C for 2 h. Then, a 50 % slurry of glutathione Sepharose 4B (GSH4B) was added, and the mixture further incubated for 1 h. GSH4B beads (with bound proteins) were sedimented by centrifugation at 50009g for 1 min and washed

Identification of ADAR as a binding partner of NS4A In an attempt to identify interacting partners of NS4A, we performed yeast two-hybrid screening using a cDNA library prepared from IFN-treated BFM cells, such that the library contained abundant IFN-stimulated gene transcripts and full-length NS4A as bait. Sequence analysis of a positive colony identified a 120-amino-acid sequence corresponding to the dsRNA binding motif of an ADAR enzyme (data not shown). ADAR enzymes are family of proteins that catalyze deamination of adenosine (A) to form inosine (I) in a process called ‘‘RNA editing’’. The RNA editing process has been shown to contribute to virus elimination from infected cells through cellular endonucleases.

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Fig. 1 Association between NS4A and ADAR in vitro. (A) GST alone or recombinant GST-NS4A protein was expressed in E. coli, and protein expression was detected by western blot. (B) LB9.K cells were transiently transfected with pME.Flag-ADAR plasmids. Cell lysates containing ADAR were mixed with an equal amount of purified GST alone or GST-NS4A proteins, incubated for 2 h, and pulled down using glutathione Sepharose beads. Bound proteins were then detected by western blot using antibodies against Flag-tag

Association between NS4A and ADAR in vitro To confirm the results obtained from yeast two-hybrid screening, we performed a GST pull-down assay using purified recombinant proteins. Recombinant NS4A protein was expressed in E. coli as a GST fusion protein (GSTNS4A) and checked for expression by western blotting using anti-GST antibodies (Fig. 1A). A mammalian cell lysates containing Flag-ADAR was mixed with GST alone or GST-NS4A. The results showed that ADAR was pulled down with GST-NS4A, but not with GST alone (Fig. 1B), indicating binding between ADAR and NS4A in vitro. NS4A binds ADAR in mammalian cells In order to confirm the binding between NS4A and ADAR in mammalian cells, we performed coimmunoprecipitation and GST pull-down assays. LB9.K cells were transfected with GST alone or GST-NS4A expression vectors together with Flag-tagged ADAR expression vector. The results

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IP: NS4A WB: NS4A

Fig. 2 NS4A binds ADAR in mammalian cells. (A) LB9.K cells were cotransfected with pME.Flag-ADAR and either empty vector or pCAG.GST-NS4A. Proteins immunoprecipitated (IP) with anti-GST were subjected to western blotting using anti-Flag monoclonal antibody (mAb). GST, GST-NS4A, and Flag-ADAR expression was confirmed in untreated lysates using anti-GST and anti-Flag. (B) The lysates from (A) were mixed with glutathione Sepharose beads (GSH4B), and bound proteins were detected by western blotting using anti-Flag. (C) One h after inoculation with either KS86-1cp or KS861ncp at an m.o.i. of 5, LB9.K cells were transfected with Flag-ADAR expression vector. At 24 h p.i., BVDV NS4A was immunoprecipitated with anti-NS4A mAb. Coimmunoprecipitated proteins were subjected to western blotting using anti-Flag mAb

showed that ADAR was immunoprecipitated with GSTNS4A but not GST alone (Fig. 2A). A GST pull-down assay gave the same result, as ADAR was pulled down with GST-NS4A only (Fig. 2B). To determine whether ADAR binds NS4A in the context of BVDV infection, we transfected LB9.K cells infected with either cp or ncp BVDV with Flag-ADAR vector and performed coimmunoprecipitation. The results showed that ADAR was immunoprecipitated with NS4A when anti-NS4A antibodies were used (Fig. 2C). These results indicate that NS4A is able to bind to ADAR in mammalian cells upon infection with the two biotypes of BVDV. NS4A co-localizes with ADAR in the cytoplasm We used confocal immunofluorescence microscopy to verify the binding and cellular localization of NS4A and ADAR. LB9.K cells were transfected with ADAR expression

BVDV NS4A binds ADAR

(A) wt 66aa 1-22 21-44 42-64

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Fig. 3 NS4A co-localizes with ADAR in the cytoplasm. LB9.K cells were transiently transfected with Flag-ADAR expression plasmid. After 6 h, cells were inoculated with cp BVDV (cp) or ncp BVDV (ncp) and incubated for a further 18 h. Cells were immunostained with anti-Flag (red) and anti-NS4A (green) antibodies. Co-localization was observed by the superimposition of red and green images (color figure online)

plasmids and then mock-infected or infected with either cp or ncp BVDV. Staining with antibodies against NS4A and Flag showed that NS4A partially co-localized with ADAR (Fig. 3) in the cytoplasm in cells infected with both BVDV biotypes, consistent with the data obtained from co-immunoprecipitation and GST pull-down assays. These data suggest a possible interaction between ADAR and NS4A in the cytoplasm of BVDV-infected cells. Mapping the ADAR-binding domain on NS4A To identify the ADAR-binding domain on NS4A, GSTtagged NS4A mutants were co-expressed with ADAR in LB9.K cells and immunoprecipitated using anti-GST polyclonal antibody. The results showed that ADAR was coimmunoprecipitated with an NS4A mutant lacking amino acids 23-64 in the N-terminal region (Fig. 4). No precipitation was detected between ADAR and the other two mutants. These results show that the ADAR-binding site of on NS4A is in the be N-terminal domain. ADAR has antiviral activity against BVDV replication ADAR is known to have both positive and negative effects on virus replication. As the influence of ADAR on pestivirus infection has not been reported previously, we first investigated the effect of ADAR on BVDV replication. LB9K cells were transfected with the ADAR expression

IP: GST WB: Flag

Fig. 4 Mapping the ADAR-binding domain on NS4A. (A) Schematic representation of NS4A and its deletion mutants. (B) LB9.K cells were cotransfected with pME.Flag-ADAR and pCAG.GSTNS4A(aa1-22), GST-NS4A(aa21-44), or GST-NS4A(aa42-64). Proteins immunoprecipitated with anti-GST were subjected to western blotting using anti-Flag mAb

plasmid alone or together with plasmids expressing ADAR and adenovirus VAI RNA, and the cells were then infected with either cp or ncp BVDV. The quantity of viral RNA was measured using quantitative real-time PCR (qRTPCR). The results showed that ADAR has an inhibitory effect on both cp BVDV (Fig. 5B) and ncp BVDV (Fig. 5C).

Discussion The occurrence of extended dsRNA in cells is relatively uncommon and is frequently associated with infection by DNA or RNA viruses. Antiviral defense mechanisms such as the protein kinase R (PKR) and 20 -50 -adenylate synthase/ RNase L pathways are stimulated by the presence of dsRNA. An additional mechanism to remove dsRNA from cells may involve hyperediting of dsRNA by ADARs, followed by targeted cleavage by specific cytoplasmic endonuclease [14]. ADARs play important roles during viral infections. They can have either a positive or negative effect on virus replication, depending upon the virus-host combination

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β-actin

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Fig. 5 ADAR inhibits BVDV replication. LB9.K cells were mocktransfected, transfected with ADAR-expressing plasmid, or cotransfected with ADAR- and VA RNAI-expressing plasmids. At 24 h post-transfection, cells were infected with cp BVDV or ncp BVDV. Cells were further incubated for 24 h and then harvested. Expression of ADAR was confirmed by western blot using anti-Flag antibodies (top panel) and b-actin (bottom panel) as internal control (A). The BVDV RNA level from cp-infected (B) and ncp-infected (C) cells was determined by real-time PCR analysis, and data are presented as percentage of control (Mock). *, p \ 0.05 (Student’s t-test)

(reviewed in ref. [13]). The role of RNA editing through ADAR in the innate immune response against viruses has been demonstrated in the case of measles virus infection, as viral RNAs extracted from the brains of measles patients show a large number of U-to-C and A-to-G conversions [5], consistent with hyperediting of both the sense and antisense viral genomes [3, 6]. A study by Taylor et al. investigating replication of subgenomic replicons suggested a role of ADAR1 in antiviral pathways against HCV, a close relative of BVDV. In that study, inhibition or

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knockdown of ADAR1 stimulated viral RNA replication from subgenomic replicons, indicating that ADAR1 has a role in limiting replication of HCV RNA [16]. Using yeast two-hybrid screening, we identified ADAR as a binding partner of BVDV NS4A, and this binding was confirmed in vitro and in vivo. Binding between ADAR and influenza A virus NS1 protein has been demonstrated previously by yeast two-hybrid screening [11]. BVDV is an RNA virus that produces dsRNA intermediates during its replication cycle. The cellular processing on BVDV dsRNA during replication is still not understood. There is a possibility that BVDV dsRNA undergoes hyperediting, which makes the virus subject to elimination from the cell. Here, we showed that ADAR had an antiviral effect against BVDV replication, and this inhibitory effect was partially abolished by using adenovirus VA RNAI, which has been shown to bind and inhibit ADAR1 [9] and PKR [17]. Identification of ADAR as a binding partner of NS4A may suggest a role of NS4A in allowing the virus to evade being eliminated if viral dsRNA is hyperedited by ADARs. The ADAR binding domain on NS4A was determined to be the N-terminal domain. Possibly, binding between NS4A and ADAR may affect the ability of ADAR to bind with dsRNA, since yeast two-hybrid screening identified a dsRNA-binding motif. It will be important to determine whether the BVDV dsRNA intermediates undergo hyperediting during viral replication and to investigate the mechanism by which NS4A interferes with ADAR to clarify the functional significance of this binding. This study may suggest a novel strategy of the virus to evade the host cell defense machinery mediated by ADARs. Acknowledgments We thank Dr. Till Ru¨menapf and Dr. Graeme Conn for providing research materials. This work was supported in part by a Research and Development Project for Application in Promoting New Policies in Agriculture, Forestry and Fisheries grant from the Ministry of Agriculture, Forestry and Fisheries, and by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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