Bovine viral diarrhea virus non-structural protein 5A interacts with NIK-and IKK {beta}-binding protein

Bovine viral diarrhea virus non-structural protein 5A interacts with NIK-and IKK {beta}-binding protein
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  See discussions, stats, and author profiles for this publication at: Bovine viral diarrhea virus non-structuralprotein 5A interacts with NIK- and IKK -binding protein  Article   in  Journal of General Virology · May 2010 DOI: 10.1099/vir.0.020990-0 · Source: PubMed CITATIONS 15 READS 91 8 authors , including:Muhammad Atif ZahoorMcMaster University 24   PUBLICATIONS   142   CITATIONS   SEE PROFILE  Yassir M MohamedTropical Medicine Research Institute of Sudan 10   PUBLICATIONS   84   CITATIONS   SEE PROFILE Kyousuke KobayashiThe University of Tokyo 26   PUBLICATIONS   219   CITATIONS   SEE PROFILE  Yukinobu TohyaNihon University 213   PUBLICATIONS   2,767   CITATIONS   SEE PROFILE All content following this page was uploaded by Muhammad Atif Zahoor on 14 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Bovine viral diarrhea virus non-structural protein 5Ainteracts with NIK- and IKK b -binding protein Muhammad Atif Zahoor, Daisuke Yamane, Yassir Mahgoub Mohamed,Yuto Suda, Kyousuke Kobayashi, Kentaro Kato, Yukinobu Tohyaand Hiroomi Akashi Correspondence Hiroomi 15 February 2010Accepted 3 May 2010 Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences,University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes aneconomically important livestock disease worldwide. Previous studies have suggested that non-structural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role duringvirus infection. Extensive reports are available on HCV NS5A and its interactions with the hostcellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. Toidentify the cellular proteins that interact with the N terminus of NS5A and could be involved in itsfunction, we conducted a yeast two-hybrid screening. As a result, we identified a cellular proteintermed bovine NIK- and IKK b -binding protein (NIBP), which is involved in protein trafficking andnuclear factor kappa B (NF- k B) signalling in cells. The interaction of NS5A with NIBP wasconfirmed both  in vitro  and  in vivo . Complementing our glutathione  S  -transferase pull-down andimmunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5Acolocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells.Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597–623. Inaddition, overexpression of NS5A inhibited NF- k B activation in HEK293 and LB9.K cells asdetermined by luciferase reporter-gene assay. We further showed that inhibition of endogenousNIBP by small interfering RNA molecules enhanced virus replication, indicating the importance ofNIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and aviral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cellmachinery for mediating trafficking as well as NF- k B signalling. INTRODUCTION Pestiviruses are important livestock pathogens that belongto the family   Flaviviridae  , which also includes the closely related genera  Hepacivirus   ( Hepatitis C virus  ; HCV) and Flavivirus   ( Yellow fever virus   and  West Nile virus  ). Thegenus  Pestivirus   includes the species  Bovine viral diarrhea virus 1  (BVDV-1), BVDV-2,  Border disease virus   and Classical swine fever virus  . As a well-characterized prototypemember of the genus  Pestivirus   within the family  Flaviviridae  , BVDV sometimes serves as a surrogate modelfor HCV life-cycle studies. BVDV is an enveloped viruscontaining a single, positive-sense RNA of approximately 12.3 kb. The genomic RNA consists of one long openreading frame (ORF), which is flanked by untranslatedregions at both ends. The ORF encodes a single polyproteinof about 4000 aa, which is processed co- and post-translationally by both viral and cellular proteases into atleast 11 mature viral proteins (N pro , C, E rns , E1, E2, p7,NS2–3, NS4A, NS4B, NS5A and NS5B) (Lindenbach  et al. ,2007).BVDV non-structural protein 5A (NS5A) is a phospho-protein of 56–58 kDa that plays an essential role in BVDVreplication (Tellinghuisen  et al. , 2006) and shares many features with its counterpart HCV NS5A, e.g. both proteinsare phosphorylated by the same or similar serine/threonineprotein kinase(s) (Reed  et al. , 1998), both are the only non-structural (NS) proteins that are complemented  in trans  (Appel  et al. , 2005; Grassmann  et al. , 2001), both share anN-terminal membrane-anchor  a -helix  (Brass  et al. , 2007)and, finally, both contain zinc-binding cysteine residues attheir N terminus (Tellinghuisen  et al. , 2005, 2006). Although HCV NS5A has been shown to be associatedwith a range of cellular proteins involved in cellularsignalling pathways (Macdonald & Harris, 2004), such asinterferon-induced kinase PKR (Gale  et al. , 1998), p53 (Lan et al. , 2002) and TRAF2 (Park   et al. , 2002), and withproteins involved in protein trafficking and membranemorphology, such as apolipoprotein A1 (Shi  et al. , 2002),amphiphysin II (Zech  et al. , 2003), soluble  N  -ethylmalei-mide-sensitive-factor attachment protein receptor (SNARE)protein (Tu  et al. , 1999), human vesicle-associated Journal of General Virology   (2010),  91,  1939–1948  DOI  10.1099/vir.0.020990-0020990 G 2010 SGM  Printed in Great Britain  1939  membrane protein-associated protein subtype B (hVAP-B)(Hamamoto  et al. , 2005) and Rab-GTPase (Sklan  et al. ,2007), little is known about the role of NS5A in BVDV RNAreplication.The nuclear factor kappa B (NF- k B) family of transcriptionfactors comprises homodimers and heterodimers of fiverelated proteins  [  NF- k B1 (p105/p50), NF- k B2 (p100/p52),c-Rel, RelA (p65) and RelB ] . NF- k B activation is inducedby a variety of stimuli including tumour necrosis factoralpha (TNF- a ), interleukin-1 (IL-1), lipopolysaccharideand double-stranded RNA (dsRNA) (Hatada  et al. , 2000;Tato & Hunter, 2002). In resting cells, they are held asinactive complexes in the cytoplasm by inhibitor of   k B(I k B a ) proteins. Activation of NF- k B occurs via I k B kinase(IKK) complex-mediated Ser 32,36 phosphorylation of I k B a ,followed by ubiquitin–proteasome-dependent degradation.Thereby, released NF- k B is translocated to the nucleus,where it regulates expression of numerous genes that play important roles in inflammation, innate immune responsesand cell survival (Hacker & Karin, 2006; Hatada  et al. ,2000).The members of the IKK complex (IKK a  and  b ) arethemselves phosphorylated and activated by one or moreupstream activating kinases, which are likely to bemembers of the mitogen-activated protein (MAP) kinasekinase kinase (MAPKKK) family of enzymes (Hirano  et al. ,1996). One such upstream kinase, NIK (NF- k B-inducingkinase), was identified by its ability to bind directly withTRAF2, an adaptor protein thought to couple both TNF- a and IL-1 receptors to NF- k B activation. The IKK complex is critical for the activation of NF- k B (Hatada  et al. , 2000;Hiscott  et al. , 2006) and is a logical target for modulation by viruses (Hiscott  et al. , 2006). Earlier studies have shown thatNIK and IKK a  and/or IKK b , along with some otherunidentified cellular proteins, are involved in Epstein–Barrvirus (Luftig  et al. , 2004), human cytomegalovirus (Jarvis et al. , 2006), human T-cell leukemia virus type 1 (Geleziunas et al. , 1998) and vaccinia virus (Chen  et al. , 2008) infections.In a search for potential binding partners of BVDV NS5A,we performed a yeast two-hybrid screening of a bovinecDNA library using BVDV NS5A as bait and identified acellular protein named NIK- and IKK b -binding protein(NIBP), which plays a dual role as an activator of NF- k Bthrough its direct interactions with NIK and IKK b  (Hu et al. , 2005) and in protein trafficking through its directinteraction with Bet3 (Cox   et al. , 2007; Kummel  et al. ,2008). The Bet3 protein is an essential component of thetrafficking protein particle (TRAPP) complex (Menon  et al. , 2006), which, together with SNAREs and Rab-GTPases(Kim  et al. , 2005; Loh  et al. , 2005; Rossi  et al. , 1995; Sacher et al. , 2008), is involved in vesicular transport in eukaryoticcells. We report here that NS5A from BVDV interactsphysically with NIBP and inhibits NF- k B activation, whichmay play a role in BVDV pathogenesis. Being the firstdemonstration of an interaction between NIBP and a viralprotein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediatingtrafficking as well as NF- k B signalling. RESULTS Identification of bovine NIBP as an interactingpartner of BVDV NS5A To investigate the interacting partners of BVDV NS5A, weperformed a yeast two-hybrid screening of a bovineMadin–Darby bovine kidney (MDBK) cDNA library usingthe N terminus of NS5A as bait and identified a cloneencoding the cDNA sequence for 110 aa of the middleregion of a novel protein termed NIBP. Database analysisshowed that NIBP has other aliases, i.e. TRAPP complex 9(TRAPPC9), KIAA1882 ( TULARIC GENE 1 ), IKK b -binding protein or TRS 120 kDa subunit. Bovine NIBP, a1138 aa peptide (126.9 kDa), is found on chromosome 14and shares a high degree of amino acid sequence similarity ( . 90%) with human, rat and mouse sequences. Direct association of NIBP with recombinantNS5A One approach to determine the significance of theinteraction between NS5A and NIBP identified in the yeasttwo-hybrid assay was to study the interaction  in vitro  .Thus, we performed a pull-down assay using a glutathione S  -transferase (GST)–NS5A fusion protein and an epitope-tagged interaction partner. Full-length NS5A was expressedas a GST fusion protein in  Escherichia coli   as describedpreviously (Zahoor  et al. , 2009). Mammalian cell lysatecontaining FLAG–NIBP was incubated with GST–NS5A orGST alone. The immune complex was pulled down usingSepharose 4B beads as described by  Hamamoto  et al. (2005) and subjected to Western blot analyses using anti-FLAG, anti-NS5A monoclonal antibodies (mAbs) or rabbitanti-GST polyclonal antibody. The results showed thatNS5A interacted specifically with NIBP, whereas no bandwas observed in the case of GST alone, which was used as anegative control (Fig. 1). NIBP interacts with BVDV NS5A in mammaliancells To examine whether bovine NIBP interacts with NS5A inmammalian cells, c-Myc-tagged NS5A (c-Myc–NS5A) wascoexpressed withFLAG-tagged NIBP (FLAG–NIBP) in LB9.Kcells and immunoprecipitated with an antibody to the c-Mycor the FLAG tag. The results indicated that FLAG–NIBP andc-Myc–NS5A were coimmunoprecipitated by either antibody (Fig. 2a). To further confirm the interaction between NIBPand NS5A in BVDV-infected cells, lysates of LB9.K cellsinfected with cp (Nose) and ncp (KS86-1ncp) strains of BVDV were subjected to immunoprecipitation analysiswith a rabbit polyclonal antibody raised against NIBP. Theresults showed that NS5A was coimmunoprecipitated with M. A. Zahoor and others1940  Journal of General Virology   91  endogenous NIBP in the cp and ncp BVDV-infected cells(Fig. 2b), whereas NS5A did not show immunoprecipitationwith rabbit IgG1 alone, which was used as a negative control.Collectively, these results indicated that NIBP interacts withNS5A in mammalian cells. NIBP interacts with NS5A through aa 597–623 As NS5A interacts specifically with NIBP  in vitro   and  in vivo  (Figs 1 and 2a, b), we tried to determine the region of NIBPresponsible for binding with NS5A. NIBP is composed of three domains separated by two low-complexity sequences(LCS) at 511–524 and 993–1004 aa (Fig. 3a). Variousdeletion mutants of FLAG-tagged NIBP were constructed asshown in Fig. 3(b). Each deletion mutant was coexpressedwith c-Myc–NS5A in LB9.K cells and immunoprecipitatedwith an anti-c-Myc antibody. An empty vector was used as anegative control in the immunoprecipitation analyses. Theresults showed that c-Myc–NS5A was immunoprecipitatedwith FLAG–NIBP spanning the region corresponding toaa 597–623 (Fig. 3c). These results indicated that NIBPinteracts with NS5A through aa 597–623. NS5A colocalizes with NIBP in the endoplasmicreticulum (ER) membrane We reported previously that BVDV NS5A proteins from thecp and ncp biotypes are localized predominantly in the ER incultured cells (Zahoor  et al. , 2009). To determine thesubcellular localization of NS5A and NIBP in BVDV-infectedLB9.K cells, we transfected LB9.K cells with an expressionvector encoding FLAG–NIBP and performed immunostain-ing using anti-NIBP rabbit polyclonal and anti-NS5A Fig. 1.  Direct association of NIBP with NS5A by GST pull-downassay. LB9.K cells were transiently transfected with FLAG–NIBP.Either GST alone or GST–NS5A purified from  E. coli   wasincubated with cell lysate containing NIBP. Bound proteins wereprecipitated by glutathione beads as described in Methods anddetected by immunoblotting. Both NIBP and NS5A were verifiedby anti-FLAG (top panel) and anti-NS5A (middle panel) mAbs,showing that GST–NS5A, but not GST alone, associated withFLAG–NIBP. As a control, cell lysates were immunoblotted usinganti-GST rabbit polyclonal antibody (bottom panel). IB,Immunoblot. Arrows indicate GST–NS5A and GST alone atapproximately 82 and 26 kDa, respectively. Fig. 2.  NIBP interacts with BVDV NS5A in mammalian cells. (a) c-Myc–NS5A of strain Nose from BVDV and FLAG-taggedNIBP were expressed in LB9.K cells and immunoprecipitated (IP) with anti-c-Myc or anti-FLAG antibody. Immunoprecipitateswere subjected to immunoblotting (IB) to detect coprecipitated counterparts. As a negative control, an empty plasmid was usedinstead of the plasmid encoding FLAG–NIBP or c-Myc–NS5A. Anti-FLAG or anti-c-Myc did not recognize c-Myc-taggedNS5A and FLAG-tagged NIBP, respectively. (b) Endogenous NIBP in LB9.K cells infected with cp and ncp BVDV wasimmunoprecipitated with normal control rabbit IgG1 (lane 1) or anti-NIBP rabbit IgG (lane 2), and immunoprecipitates wereanalysed by immunoblotting with specific antibodies. BVDV NS5A interacts with NIBP 1941  monoclonal antibodies. Results showed thatNS5Afromboththe cp and ncp biotypes of BVDV colocalized with NIBP inthe cytoplasm (Fig. 4a). To further demonstrate thesubcellular localization of NIBP in the cells, the ER wasimmunostained with antibody against Grp78/Bip. Theresults showed that NIBP colocalized with Grp78/Bip (Fig.4b), which is involved in the ER and post-ER secretory pathway. Collectively, the colocalization of NS5A with NIBPin the ER suggested that NS5A interacts specifically withNIBP under intracellular conditions in BVDV-infected cells. NS5A inhibits TNF- a - and dsRNA-induced NF- k  Bactivation To investigate the role of NS5A on TNF- a - or polyriboinos-inic:polyribocytidylic acid  [  poly(I:C) ]  (dsRNA)-inducedNF- k B activity, we performed a luciferase reporter-geneassay. Plasmids encoding NS5A and NIBP were cotrans-fected into human embryonic kidney (HEK) 293 (Fig. 5a)and LB9.K cells (Fig. 5b, c) along with a NF- k B luciferasereporter plasmid. Later, the cells were induced with TNF- a for 6 h (HEK293 and LB9.K cells) or with poly(I:C) for12 h (LB9.K cells). The results showed that luciferaseactivities were lower when NS5A was expressed, indicatingthat NS5A expression causes the suppression of TNF- a -induced NF- k B activation compared with the control inboth HEK293 and LB9.K cell lines, regardless of their srcin(Fig. 5a, b). Moreover, NS5A also inhibited poly(I:C)-induced NF- k B activity in LB9.K cells (Fig. 5c). In contrast,NIBP only caused TNF- a -induced NF- k B activation, whichis consistent with a previously described report (Hu  et al. ,2005), whereas it did not induce poly(I:C)-mediated NF- k Bactivation, further highlighting that NIBP is necessary forTNF- a -induced NF- k B activation through the classicalsignalling cascade (Hu  et al. , 2005). Inhibition of NIBP by small interfering RNA(siRNA)-enhanced virus replication In order to determine the possible implication of NIBP inBVDV replication, we examined the effect of knockdown of endogenous NIBP by siRNA and measured the intracellular Fig. 3.  Determination of the NS5A-binding region in NIBP. (a) Structure and functional domains of NIBP. (b) Full-length ordeletion mutants of NIBP used in the study and the results of binding to NS5A. N-terminally FLAG-tagged NIBP mutantsencoding the regions indicated were designated 1–1138, 1–786, 624–1138, 191–596 or 528–864, respectively. A summaryof immunoprecipitation results is given on the right. (c) Each mutant or full-length NIBP was coexpressed with c-Myc–NS5A inLB9.K cells, immunoprecipitated with an anti-c-Myc antibody and analysed by immunoblotting with an anti-FLAG antibody. As anegative control, an empty plasmid was used instead of the plasmid encoding c-Myc–NS5A. The anti-c-Myc antibody did notrecognize FLAG-tagged NIBP or its mutants. The arrow indicates the full-length NIBP from aa 1 to 1138 on the immunoblot. M. A. Zahoor and others1942  Journal of General Virology   91
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