Interaction Networks of Hepatitis C Virus NS4B: Implications for Antiviral Therapy

Cellular Microbiology (2012) 14(7), 994–1002 doi:10.1111/j.1462-5822.2012.01773.x First published online 5 March 2012 Microreview

Interaction networks of hepatitis C virus NS4B: implications for antiviral therapy

Shanshan Li,1 Xilan Yu,1 Yunli Guo2 and structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A Lingbao Kong2* and NS5B) (Moradpour et al., 2007). HCV non-structural 1Department of Plant Pathology and Microbiology, Iowa proteins (NS3-5B) along with viral RNA comprise the high- State University, Ames, IA, USA. order replication complex associated with endoplasmic 2College of Bioscience and Engineering, Jiangxi reticulum (ER) membrane (Waris et al., 2004). Agricultural University, Nanchang, Jiangxi, China. HCV NS4B is a 27 kDa ER membrane-associated protein that mediates virus–host interactions (Hugle et al., 2001). It is conserved in Flaviviridae family and is able to Summary induce alteration of ER membrane and formation of a Hepatitis C virus (HCV) is an important human ‘membranous web’ structure, which provides a platform pathogen infecting more than 170 million people for the HCV replication complex (Egger et al., 2002; worldwide with approximately three million new Lundin et al., 2003; Welsch et al., 2007). In addition, cases each year. HCV depends heavily on interac- NS4B has been reported to possess some enzymatic tions between viral proteins and host factors for its activities that hydrolyse GTP and ATP and catalyse the survival and propagation. Among HCV viral pro- synthesis of ATP and AMP from two ADP molecules teins, the HCV non-structural protein 4B (NS4B) (Thompson et al., 2009). Inhibition of these functions has been shown to mediate virus–host interactions makes NS4B the subject of antiviral pharmacological that are essential for HCV replication and patho- research and some promising anti-HCV agents have genesis and emerged as the target for anti-HCV been identiﬁed, such as pyrazolopyrimidines, amiloride therapy. Here, we reviewed recent knowledge analogue, clemizole hydrochloride and clemizole-related about the NS4B interaction networks with host indazole series (Dvory-Sobol et al., 2010). In this review, factors and its possible regulatory mechanisms, we focused on recent advances about the interactions of which will both advance our understanding of the NS4B with host cellular proteins as well as their possible role of NS4B in HCV life cycle and illuminate poten- regulatory mechanisms. tial viral and host therapeutic targets.

Structure features of HCV NS4B Introduction Topology structure of NS4B Hepatitis C virus (HCV) is the major causative agent of NS4B is an integral membrane protein consisting of an non-A, non-B (NANB) hepatitis and its infection often N-terminal portion (aa 1–69), a central transmembrane leads to chronic hepatitis, liver cirrhosis and hepatocellu- part (aa 70–190) and a C-terminal portion (aa 191–261) lar carcinoma (HCC) (Hoofnagle, 1997). However, there (Fig. 1A). The N-terminal domain comprises two amphip- are no efficacious vaccines and therapies against HCV athic a-helices, designated as AH1 and AH2. AH2 is con- infection and development of effective antivirals remains a served in all HCV genotypes and has been shown to great challenge. HCV is an enveloped virus that belongs undergo oligomerization and stimulate aggregation of lipid to the genus Hepacivirus of the Flaviviridae family and its vesicles, which is critical for HCV replication (Gouttenoire genome consists of a 9.6 kb positive-stranded linear RNA et al., 2009; 2010a; Cho et al., 2010). The NS4B N-termini that comprises a single large open reading frame encod- is predicted to lie in the cytosolic face. However, by arti- ing three structural (core, E1 and E2) and seven non- ﬁcially inserting glycosylation sites into NS4B, Lundin et al. postulated that NS4B AH2 translocates across the Received 6 December, 2011; revised 26 January, 2012; accepted 1 February, 2012. *For correspondence. E-mail [email protected], membrane into the ER lumen leading to the formation of Tel. (+86) 791 83813459; Fax (+86) 791 83828080. the ﬁfth transmembrane domain called TMX (Lundin et al.,

cellular microbiology HCV NS4B interaction networks 995

Fig. 1. Structural characteristics of HCV NS4B. A. Schematic representation of NS4B structure and topology on ER membrane. Shown are its N-terminal amphipathic helices (AH1 and AH2), four predicted transmembrane domains (TM1–TM4) and C-terminal helices (H1 and H2) (labelled as red). NS4B N-terminus is postulated to translocate across the membrane bilayer into ER, creating a ﬁfth transmembrane domain (TMX) (labelled as light red). All the reported protein domains and motifs are indicated by black arrows according to Dvory-Sobol et al. (2010) and Gouttenoire et al. (2010b). B. Predicted protein–protein binding domains within NS4B by basicELM software. Note that the sequence of NS4B was derived from HCV genotype 1a (GenBank Accession No. AF009606.1). The conserved motifs between HCV NS4B 1a and 1b (GenBank Accession No. AB109543) are indicated by black asterisk. The conserved motifs between HCV NS4B 1a and 2a (GenBank Accession No. AY746460.1) are indicated by yellow asterisk. NTD, N-terminal domain; CTD, C-terminal domain.

2006). Dual topologies of NS4B are proposed to stand for tions and the high hydrophobic nature of NS4B, the only two distinct functions of NS4B during the virus life cycle, reported interaction motif is the basic leucine zipper where N-termini translocation marks the transition (bZIP) (aa 20–55) (Fig. 1A), which mediates the physical between these two states. Additionally, the replicon assay interaction between NS4B N-termini and the central part showed that NS4B N-termini is implicated in membrane of the human protein CREB-RP/ATF6b, an ER stress association and retention, which are required for NS4B- response element (Tong et al., 2002; Welsch et al., 2007). mediated formation of HCV replication platform (Elazar To provide new insights into the NS4B functions as well as et al., 2004). new avenues for future research, we predicted possible The NS4B central domain harbours four predicted interaction motifs within NS4B using basicELM software transmembrane domains (TMs) and a nucleotide-binding (http://elm.eu.org/), which has been widely applied to Walker A motif (129GSIGLK135) (Einav et al., 2004). In predict virus–host protein interactions (Evans et al., 2009; addition, Han et al. identified two conserved dimerization Davey et al., 2011). We identified 22 hypothetical protein– motifs (GXXXG and S/T cluster) within NS4B transmem- protein interaction motifs within NS4B, some of which are brane domains and the mutations within these motifs led consistent with the reported functions of NS4B (Fig. 1B to less efficient HCV replication and reduced replication and Table S1). foci formation (Han et al., 2011). The first class of predicted motifs interacts with proteins The NS4B C-terminal portion is composed of two pre- regulating cell growth and apoptosis. The NS4B first four dicted transmembrane helices (H1 and H2) with H2 residues form a binding site for inhibitors of apoptosis involved in the formation of functional HCV replication proteins (IAPs), which is consistent with previous report complexes (Dvory-Sobol et al., 2010). The NS4B about NS4B’s anti-apoptosis effects (Rai and Deval, C-terminal domain also contains an arginine-rich motif 2011). Additionally, NS4B contains four cyclin binding (192RR193) to bind viral RNA and a nucleotide-binding sites (Table S1), suggesting its role in cell cycle control. In Walker B motif (228DAAA231), both of which are impor- fact, NS4B has been reported to regulate expression of tant for HCV replication in the replicon assay (Einav et al., cell cycle related genes such as p21 and Waf1 (Shimoike 2008). et al., 1999; Florese et al., 2002). The second class of predicted motifs is involved in phosphorylation/dephosphorylation signalling pathways: Protein–protein interaction domains one protein phosphatase 1 catalytic subunit (PP1c) inter- NS4B contains important motifs that enable NS4B to acting motif, seven forkhead-associated domain (FHA)- interact with other proteins. Due to the technical limita- binding motifs, one STAT3 SH2-binding domain, three

STAT5 SH2-binding motifs, and four SH3-binding motifs. Interactions between NS4B and As PP1c interacting motif targets PP1 to its substrate membrane-associated proteins for dephosphorylation (Cohen, 2002), we hypothesized NS4A/4B/5A precursor has been reported to physically that NS4B might modulate phosphorylation status of other interact with a cellular vesicle membrane transport protein proteins. FHA and SH2 domains act as phosphorylation- hVAP-33 (Gao et al., 2004). The replicon assay showed dependent protein–protein interaction modules that that NS4B acts as anchor protein to tether NS4A/4B/5A preferentially bind to phosphor-threonine and phospho- to lipid raft, which then recruit hVAP-33 and NS5B to tyrosine in their targets, respectively (Hammet et al., the membrane foci (Gao et al., 2004). Yeast two-hybrid 2003). The presence of these two modules implies that assay showed that NS4B interacts with a large number NS4B could be phosphorylated and subsequently recruit of membrane-associated proteins: integrins (ITGA2B, FHA- and SH2-containing molecules to the virus replica- ITGAL, ITGB1, ITGB3), transporters (ABCA1, ABCA6, tion foci, thereby providing a link between HCV replication ATP5G2) and transmembrane proteins (TMX2, TMCO1) and cellular processes. Moreover, our predicted data (Tripathi et al., 2010). The membrane association of suggest that NS4B could interact with STAT3 and STAT5 NS4B might facilitate anchoring of NS4B to membrane and participate in STAT signalling pathways, which regu- (Boleti et al., 2010), and has multiple implications in HCV late cell proliferation, survival, carcinogenesis and cytok- life cycle, such as inducing formation of the replication ine production (Jove, 2000). SH3 domain is a conserved complex and activating cellular stress. sequence identiﬁed in a number of signalling adapter molecules, such as Src kinases, Crk adapter protein and phospholipase C-g (Mayer, 2001). Altogether, these data Interactions between NS4B and proteins in suggest that NS4B activates several cellular signalling cellular stress pathways in a phosphorylation-dependent manner, which is consistent with previously reported experimental data NS4B is able to trigger cellular stress signalling pathways (Waris et al., 2007; Li et al., 2009; Park et al., 2009). such as ER stress, which is caused by abnormal accu- Finally, NS4B contains a class 1 PDZ-binding motif, mulation of unfolded or misfolded proteins in the ER and which is one of the most abundant domains encoded in one outcome of ER stress is activating unfolded protein the human genome and its malfunction leads to over 20 response (UPR). UPR is an adaptive strategy employed heritable human diseases (te Velthuis et al., 2011). by cells to ameliorate this stress via activating three dis- All these motifs except SH3-binding motif are con- tinct membrane-associated transducers: (i) ATF6, whose served among HCV NS4B genotype 1a, 1b and 2a function is to activate transcription of chaperone proteins (Fig. 1B), implying their evolutional importance in HCV facilitating viral protein folding, (ii) XBP1, which stimulates pathology. After mapping these motifs to NS4B degradation of misfolded proteins, and (iii) PERK, whose sequence, we found that these motifs are primarily function is to attenuate translation (Schroder and located in the N-terminus and C-terminus (Fig. 1B). Spe- Kaufman, 2005). NS4B has been shown to physically ciﬁcally, the majority of N-terminus motifs were found interact with ATF6a/b (Tong et al., 2002) and our previous within a-helices AH1 and AH2 and most C-terminus work demonstrated that NS4B activates UPR through motifs within helices H1 and H2 (Table S2), which inducing both XBP1 mRNA splicing and ATF6 cleavage emphasize the importance of these domains in mediating pathways in human hepatic cells (Li et al., 2009). In addi- NS4B interactions (Gouttenoire et al., 2009; 2010a; Lief- tion, NS4B might take part in protein degradation as it hebber et al., 2009; Cho et al., 2010). In the central interacts with proteasome subunit, PSMB5 (Florese et al., domain, three FHA-binding motifs and two SH3-binding 2002; Tripathi et al., 2010). Furthermore, there are also motifs were found within transmembrane domains some clues suggesting the participation of NS4B in the (Table S2). Nonetheless, the presence of these hypoth- PERK pathway to suppress protein translation. Kato et al. esized motifs and their relevance to HCV life cycle await found that NS4B inhibited cap-dependent translation of further experimental validation. cellular proteins and IRES-mediated translation of viral proteins (Kato et al., 2002). Additionally, NS4B has been shown to interact with cellular translational factor, RPL22 HCV NS4B interaction networks and downregulate expression of translation factor, eIF3 Based on current reports about NS4B and host proteins (Zheng et al., 2005; Tripathi et al., 2010). However, the interactions, we constructed the NS4B interaction net- direct participation of NS4B in PERK pathway requires works from the following seven major categories: mem- further investigation. brane association, cellular stress, autophagy, immune In addition to ER stress, we also found that NS4B response, lipid metabolism, transformation and tumori- activates oxidative stress by stimulating the production of genesis, and epigenetic regulation (Fig. 2). reactive oxygen species (ROS) (Li et al., 2009). The ROS

Fig. 2. Interaction networks of NS4B. Direct or secondary interactions between NS4B and cellular proteins were labelled in black colour. Red colour indicates genes whose expression has been affected by NS4B (Zheng et al., 2005). The hypothesized NS4B interaction proteins are labelled in yellow boxes and the experimentally validated interaction proteins were labelled in light blue boxes. is produced as a consequence of altered intracellular Ca2+ Interactions between NS4B and proteins in host homeostasis by NS4B. Moreover, NS4B can interact with immune response proteins associated with oxidative stress, such as mito- chondrial proteins COX2 (cytochrome c oxidase II), ND4 Several lines of evidence showed that NS4B inﬂuences (mitochondrially encoded NADH dehydrogenase 4) and the host immune response, which contributes to evasion ATP5G2 (ATP synthase subunit C2) (Tripathi et al., 2010). of HCV from host surveillance and persistent infection (Moriyama et al., 2007; Tasaka et al., 2007; Tripathi et al., 2010). NS4B can suppress NS5B-activated IFN-b pre- Interactions between NS4B and proteins in sumably by inhibiting NS5B activity or recognition of autophagy machinery dsRNA by TLR3 (Moriyama et al., 2007). Tasaka and Another outcome of ER stress is the activation of autoph- colleagues found that NS4B blocked the retinoic acid- agy, which is a cellular response to viral infection in inducible gene I (RIG-I) mediated activation of IFN eukaryotic cells (He and Klionsky, 2009). HCV infection probably by targeting adaptor molecule, Cardif (Tasaka stimulates autophagy pathway, which is required for virus et al., 2007). Yeast two-hybrid assay indicate that NS4B replication and HCV particle assembly probably by regu- interacts with components of antigen presentation, i.e. lating host immune response and providing a scaffold for CD63, CD82 and APOA1 (Tripathi et al., 2010). Addition- HCV replication (Marlène Dreux, 2011). As an ER stress- ally, genome proﬁling data showed that NS4B downregu- inducing protein, NS4B has been shown in the replicon lated transcription of genes in immune response, such as assay to interact with proteins in autophagy machinery, CD81, IL7R (chemokine 7 receptor) and killer cell lectin- such as small GTPase Rab5, which activates autophagy like receptor subfamily C (Zheng et al., 2005). by forming the complex with Beclin 1 and Class III PI-3- Yeast two-hybrid assay also showed that NS4B inter- Kinase Vps34 (Stone et al., 2007; Manna et al., 2010; Su acts with proteins belonging to complement and coagula- et al., 2011). tion process (Tripathi et al., 2010), which play critical

© 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 994–1002 998 S. Li, X. Yu, Y. Guo and L. Kong functions in the host defence against pathogen invasion Epigenetic regulation by NS4B and virus clearance. These proteins are thrombin (F2), Epigenetic regulation refers to heritable changes in gene serine protease inhibitor SERPINA1 and FGG (fibrinogen transcription without any alteration in DNA sequence gamma chain), which are involved in chronic liver injury (Esteller, 2008). Hepatocellular carcinoma has been (Gaca et al., 2002; Kok et al., 2010). Consistent with shown to be associated with aberrant epigenetic regula- these data, NS4B was found to downregulate the tran- tion such as mis-regulated DNA methylation and altered scription of proteins in complement pathway such as C3 histone modifications (Herceg and Paliwal, 2011). In HCV (Complement component 3), HFL3 (H factor-like 3) and replicon-harbouring cells, the IFN-stimulated genes are tachykinin precursor 1 (substance K) (Zheng et al., 2005). silenced by DNA methylation (Naka et al., 2006). Yeast Further efforts are required to explore the outcome of two-hybrid assay indicated that NS4B interacts with these interactions and implications in HCV propagation. enzymes involved in DNA and protein covalent modifications, i.e. METTL7B (methyltransferase like 7B) and AANAT (arylalkylamine N-acetyltransferase) (Tripathi Interactions between NS4B and proteins in et al., 2010). In addition, NS4B has been shown to regu- lipid metabolism late transcription of genes associated with host epigenetic HCV infection leads to altered lipid metabolism with high control such as SWI/SNF, MBD4 (methyl CpG-binding content of cholesterol (Syed et al., 2010). Recent studies domain protein 4) and DNA methyltransferase (AJ223333) found that cholesterol/fatty acid synthesis pathways are (Zheng et al., 2005). Collectively, these data suggest that required for HCV replication (Su et al., 2002; Syed et al., NS4B plays a yet undefined function in the regulation of 2010). The important transcription factors governing these host epigenetic programme. pathways are SREBPs (Horton et al., 2002), which can be activated by HCV infection via Ca2+ signalling and oxidative stress (Waris et al., 2007). In the context of HCV Others replicon or during HCV infection, NS4B has been shown NS4B has been reported to interact with heat shock pro- to stimulate expression of SREBPs and fatty acid synthe- teins such as Hsp70, HSPA8 and HSP90B1, suggesting sis through activating the PI3-K-AKT pathway (Waris that NS4B could improve the folding and/or stability of et al., 2007; Park et al., 2009), suggesting a potential role viral proteins either to reduce the ER stress or to facilitate of NS4B in regulation membrane constituents. In addition, virus replication (Chen et al., 2010). Furthermore, NS4B NS4B interacts with factors participating in lipoprotein might participate in DNA repair as it downregulates DNA metabolism (APOA1BP, APOA1, APOB, APOC1, APOC3, repair proteins: RAD23 and XRCC5 (Zheng et al., 2005). APOF,APOL1 and LRP1), which regulate cholesterol syn- Nonetheless, their relevance to HCV replication awaits thesis and are linked to viral assembly and release (Andre further study. et al., 2005; Popescu and Dubuisson, 2010; Benedicto Notably, we should keep in mind that some of these et al., 2011). These findings suggest that NS4B induces reported data are based on either NS4B alone or overex- membranous web formation, in part, by altering mem- pression studies. We may overestimate or underestimate brane constituents (Waris et al., 2007). NS4B interaction networks given that NS4B could behave properly in the context of the HCV replication and infection. Therefore, future work should be focused on identi- Interactions between NS4B and proteins in cellular fication and confirmation of NS4B interaction networks in transformation and tumorigenesis either replicon or HCV infection assays to recapitulate its NS4B is able to transform NIH3T3 cells and its NBM motif authentic functions in HCV life cycle. mediates cellular transformation with exciting implications for novel therapies (Park et al., 2000; Einav et al., 2004). Regulation of interaction network by covalent Moreover, NS4B has been shown to interact with tumour modifications of NS4B genes, such as MCC (mutated in colorectal cancers) and BRCA1 (breast cancer 1) (Tripathi et al., 2010). Genome Covalent modifications represent an important and profiling data showed that NS4B regulated transcription economical way for virus to modulate protein–protein of tumour-related genes, RAP1, FYN oncogene, DKK1, interactions in a post-translational manner. The best SLIT2 (slit homologue 2), RRM1 (ribonucleotide reduc- experimental evidence for NS4B modifications is cysteine tase M1 polypeptide), p53, p53BP (p53-binding protein) palmitoylation and this modification is important for NS4B and DCC (deleted in colorectal carcinoma) (Zheng et al., to interact with itself and other viral proteins (Yu et al., 2005). Nonetheless, whether these interactions lead to 2006). By virtue of powerful bioinformatics tools, we pre- liver carcinogenesis remains to be elucidated. dicted seven major types of covalent modifications within

Fig. 3. Covalent modiﬁcations of NS4B. Covalent modiﬁcations of NS4B were predicted by software as described in Supporting information. The reported domains within NS4B have been highlighted in colours with yellow stands for N-terminal AH1 and AH2, red stands for four transmembrane domains (TM1–4), and blue stands for C-terminal H1 and H2. Three functional motifs have been labelled by coloured boxes: red box represents bZIP motif, green box represents S/T cluster-like dimerization motif, blue box represents Walker A motif and black box represents Walker B motif. The critical residues required for HCV replication are highlighted as red colour (Blight, 2011). All domains were labelled according to Dvory-Sobol et al. (2010) and Gouttenoire et al. (2009). The sequence of NS4B used for prediction was retrieved from NCBI database (GenBank Accession No. AF009606.1).

NS4B, some of which are consistent with experimental modifications may induce ‘loss-of-function’ mechanism by data (see Supporting information for details). As shown in disrupting original interactions such as cysteine palmitoy- Fig. 3, the majority of these modifications are located in lation (Yu et al., 2006). However, these predicted NS4B the N- and C-terminal domains, especially in domains covalent modifications and their functions are required to critical for HCV replication such as AH1, AH2, TM1, TM4 be verified by biochemical and biophysical tools, i.e. mass and H2. Furthermore, most modifications were found spectrometry. within regions highly enriched in protein interaction motifs (Table S2), suggesting that these modifications may regu- Implications for antiviral therapy late NS4B interaction networks. Intriguingly, most of these modifications occur on residues that are critical for HCV Current therapeutics for HCV involves a combination RNA replication, such as K18, K20, K135, R214 and Y223 of subcutaneous pegylated IFN-a and oral ribavirin. (Blight, 2011). Although the precise mechanisms by which However, this treatment is limited by its low efficiency and these residues are involved in HCV replication are serious side-effects (Dvory-Sobol et al., 2010). Recently, unclear, our predicted data indicated that these residues there are significant advances towards HCV-specific anti- could modulate NS4B interaction networks via their cova- virals development by pharmacologically inhibiting NS4B lent modifications. In this context, these modifications interactions. Glenn’s lab identified a class of small mol- may constitute a ‘gain-of-function’ mechanism by forming ecules that specifically prevent HCV replication (Cho et al., binding sites for proteins containing domains such as 2010). These molecules are inhibitors of NS4B AH2, which FHA, SH2, class 1 PDZ, etc. On the other hand, these disrupt either AH2 oligomerization or its membrane asso-

© 2012 Blackwell Publishing Ltd, Cellular Microbiology, 14, 994–1002 1000 S. Li, X. Yu, Y. Guo and L. Kong ciation. This work provides compelling evidence that NS4B Benedicto, I., Molina-Jimenez, F., Moreno-Otero, R., interaction networks can be promising targets for antiviral Lopez-Cabrera, M., and Majano, P.L. (2011) Interplay treatment (Cho et al., 2010). Like NS4B AH2 domain, AH1, among cellular polarization, lipoprotein metabolism and hepatitis C virus entry. World J Gastroenterol 17: 2683– H1 and H2 domains also contain NS4B interaction 2690. modules that mediate interactions between NS4B and Blight, K.J. (2011) Charged residues in hepatitis C virus human proteins, suggesting that these specific domains NS4B are critical for multiple NS4B functions in RNA rep- and motifs can be the potential targets. In addition to NS4B lication. J Virol 85: 8158–8171. targets, some host factors that interact with NS4B could Boleti, H., Smirlis, D., Dalagiorgou, G., Meurs, E.F., become potential targets such as CD81 and lipoproteins, Christoforidis, S., and Mavromara, P. (2010) ER targeting which have become the targets for antiviral drug develop- and retention of the HCV NS4B protein relies on the con- certed action of multiple structural features including its ment (Rice, 2011). The host targets could also be proteins transmembrane domains. Mol Membr Biol 27: 50–74. that mediate NS4B covalent modifications. For instance, Chen, Y.J., Chen, Y.H., Chow, L.P., Tsai, Y.H., Chen, P.H., inhibitors for histone deacetylases (valproic acid, butyric Huang, C.Y., et al. (2010) Heat shock protein 72 is associ- acid and tributyrin) have been reported to induce apoptosis ated with the hepatitis C virus replicase complex and and cell cycle arrest in hepatocellular carcinoma cells enhances viral RNA replication. J Biol Chem 285: 28183– (Kuroiwa-Trzmielina et al., 2009; Tatebe et al., 2009). 28190. Although host targets are inherently less virus-specific Cho, N.J., Dvory-Sobol, H., Lee, C., Cho, S.J., Bryson, P., Masek, M., et al. (2010) Identification of a class of HCV than NS4B-targeted therapy and could lead to side-effects, inhibitors directed against the nonstructural protein NS4B. therapeutic drugs targeting host factors can overcome the Sci Transl Med 2: 15ra16. genetic variability of NS4B targets. Therefore, it is conceiv- Cohen, P.T. (2002) Protein phosphatase 1 – targeted in many able that a combination of inhibitors specific for both NS4B directions. J Cell Sci 115: 241–256. and host factors will constitute a more efficient regimen for Davey, N.E., Trave, G., and Gibson, T.J. (2011) How viruses HCV-infected patients. hijack cell regulation. Trends Biochem Sci 36: 159–169. Dvory-Sobol, H., Pang, P.S., and Glenn, J.S. (2010) The future of HCV therapy: NS4B as an antiviral target. Viruses Conclusions 2: 2481–2492. Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H.E., The HCV NS4B is a multifunctional protein essential in Moradpour, D., and Bienz, K. (2002) Expression of hepa- HCV life cycle and most of its functions are performed by titis C virus proteins induces distinct membrane alterations interacting with cellular proteins involved in membrane including a candidate viral replication complex. J Virol 76: association, cellular stress, autophagy, immune response, 5974–5984. Einav, S., Elazar, M., Danieli, T., and Glenn, J.S. (2004) A lipid metabolism, transformation and tumorigenesis, and nucleotide binding motif in hepatitis C virus (HCV) NS4B epigenetic regulation. These interactions are mediated by mediates HCV RNA replication. J Virol 78: 11288–11295. protein domains and motifs within NS4B and can be regu- Einav, S., Gerber, D., Bryson, P.D., Sklan, E.H., Elazar, M., lated by its covalent modifications. Therefore, elucidating Maerkl, S.J., et al. (2008) Discovery of a hepatitis C target the sophisticated interaction networks can, to some and its pharmacological inhibitors by microfluidic affinity extent, explain how HCV NS4B contributes to HCV repli- analysis. Nat Biotechnol 26: 1019–1027. cation and pathology. On the other hand, we anticipated Elazar, M., Liu, P., Rice, C.M., and Glenn, J.S. (2004) An N-terminal amphipathic helix in hepatitis C virus (HCV) that unravelling these interactions would shed light on NS4B mediates membrane association, correct localiza- chemoprevention and chemotherapy of HCV as most tion of replication complex proteins, and HCV RNA replica- promising antivirals target host–virus interactions. tion. J Virol 78: 11393–11400. Esteller, M. (2008) Epigenetics in cancer. N Engl J Med 358: 1148–1159. Acknowledgements Evans, P., Dampier, W., Ungar, L., and Tozeren, A. (2009) We apologize to our colleagues whose work was not discussed Prediction of HIV-1 virus – host protein interactions using here due to space limitations. This work was funded by National virus and host sequence motifs. BMC Med Genomics 2: Nature Science Foundation of China (No. 31160034) to L. Kong 27. and Nature Science Foundation of Jiangxi Province (No. Florese, R.H., Nagano-Fujii, M., Iwanaga, Y., Hidajat, R., and 2010GZY0059) to L. Kong. Hotta, H. (2002) Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus. Virus Res 90: 119–131. References Gaca, M.D., Zhou, X., and Benyon, R.C. (2002) Regulation of hepatic stellate cell proliferation and collagen synthesis by Andre, P., Perlemuter, G., Budkowska, A., Brechot, C., and proteinase-activated receptors. J Hepatol 36: 362–369. Lotteau, V. (2005) Hepatitis C virus particles and lipopro- Gao, L., Aizaki, H., He, J.W., and Lai, M.M. (2004) Inter- tein metabolism. Semin Liver Dis 25: 93–104. actions between viral nonstructural proteins and host

protein hVAP-33 mediate the formation of hepatitis C virus van Leeuwen, H.C. (2009) Hepatitis C virus NS4B carboxy RNA replication complex on lipid raft. J Virol 78: 3480– terminal domain is a membrane binding domain. Virol J 6: 3488. 62. Gouttenoire, J., Castet, V., Montserret, R., Arora, N., Lundin, M., Monne, M., Widell, A., Von Heijne, G., and Raussens, V., Ruysschaert, J.M., et al. (2009) Identifica- Persson, M.A. (2003) Topology of the membrane- tion of a novel determinant for membrane association in associated hepatitis C virus protein NS4B. J Virol 77: hepatitis C virus nonstructural protein 4B. J Virol 83: 6257– 5428–5438. 6268. Lundin, M., Lindstrom, H., Gronwall, C., and Persson, M.A. Gouttenoire, J., Roingeard, P., Penin, F., and Moradpour, D. (2006) Dual topology of the processed hepatitis C virus (2010a) Amphipathic alpha-helix AH2 is a major determi- protein NS4B is influenced by the NS5A protein. J Gen nant for the oligomerization of hepatitis C virus nonstruc- Virol 87: 3263–3272. tural protein 4B. J Virol 84: 12529–12537. Manna, D., Aligo, J., Xu, C., Park, W.S., Koc, H., Heo, W.D., Gouttenoire, J., Penin, F., and Moradpour, D. (2010b) Hepa- and Konan, K.V. (2010) Endocytic Rab proteins are titis C virus nonstructural protein 4B: a journey into unex- required for hepatitis C virus replication complex formation. plored territory. Rev Med Virol 20: 117–129. Virology 398: 21–37. Hammet, A., Pike, B.L., McNees, C.J., Conlan, L.A., Tenis, Marlène Dreux, F.V.C. (2011) Impact of the autophagy N., and Heierhorst, J. (2003) FHA domains as phospho- machinery on hepatitis C virus infection. Viruses 3: 1342– threonine binding modules in cell signaling. IUBMB Life 55: 1357. 23–27. Mayer, B.J. (2001) SH3 domains: complexity in moderation. Han, Q., Aligo, J., Manna, D., Belton, K., Chintapalli, S.V., J Cell Sci 114: 1253–1263. Hong, Y., et al. (2011) Conserved GXXXG- and S/T-like Moradpour, D., Penin, F., and Rice, C.M. (2007) Replication motifs in the transmembrane domains of NS4B protein are of hepatitis C virus. Nat Rev Microbiol 5: 453–463. required for hepatitis C virus replication. J Virol 85: 6464– Moriyama, M., Kato, N., Otsuka, M., Shao, R.X., Taniguchi, 6479. H., Kawabe, T., and Omata, M. (2007) Interferon-beta is He, C., and Klionsky, D.J. (2009) Regulation mechanisms activated by hepatitis C virus NS5B and inhibited by NS4A, and signaling pathways of autophagy. Annu Rev Genet 43: NS4B, and NS5A. Hepatol Int 1: 302–310. 67–93. Naka, K., Abe, K., Takemoto, K., Dansako, H., Ikeda, M., Herceg, Z., and Paliwal, A. (2011) Epigenetic mechanisms in Shimotohno, K., and Kato, N. (2006) Epigenetic silencing hepatocellular carcinoma: how environmental factors influ- of interferon-inducible genes is implicated in interferon ence the epigenome. Mutat Res 727: 55–61. resistance of hepatitis C virus replicon-harboring cells. Hoofnagle, J.H. (1997) Hepatitis C: the clinical spectrum of J Hepatol 44: 869–878. disease. Hepatology 26: 15S–20S. Park, C.Y., Jun, H.J., Wakita, T., Cheong, J.H., and Hwang, Horton, J.D., Goldstein, J.L., and Brown, M.S. (2002) S.B. (2009) Hepatitis C virus nonstructural 4B protein SREBPs: activators of the complete program of cholesterol modulates sterol regulatory element-binding protein signal- and fatty acid synthesis in the liver. J Clin Invest 109: ing via the AKT pathway. J Biol Chem 284: 9237–9246. 1125–1131. Park, J.S., Yang, J.M., and Min, M.K. (2000) Hepatitis C virus Hugle, T., Fehrmann, F., Bieck, E., Kohara, M., Krausslich, nonstructural protein NS4B transforms NIH3T3 cells in H.G., Rice, C.M., et al. (2001) The hepatitis C virus non- cooperation with the Ha-ras oncogene. Biochem Biophys structural protein 4B is an integral endoplasmic reticulum Res Commun 267: 581–587. membrane protein. Virology 284: 70–81. Popescu, C.I., and Dubuisson, J. (2010) Role of lipid metabo- Jove, R. (2000) Preface: STAT signaling. Oncogene 19: lism in hepatitis C virus assembly and entry. Biol Cell 102: 2466–2467. 63–74. Kato, J., Kato, N., Yoshida, H., Ono-Nita, S.K., Shiratori, Y., Rai, R., and Deval, J. (2011) New opportunities in anti- and Omata, M. (2002) Hepatitis C virus NS4A and NS4B hepatitis C virus drug discovery: targeting NS4B. Antiviral proteins suppress translation in vivo. J Med Virol 66: 187– Res 90: 93–101. 199. Rice, C.M. (2011) New insights into HCV replication: potential Kok, K.F., te Morsche, R.H., van Oijen, M.G., and Drenth, J.P. antiviral targets. Top Antivir Med 19: 117–120. (2010) Prevalence of genetic polymorphisms in the pro- Schroder, M., and Kaufman, R.J. (2005) ER stress and the moter region of the alpha-1 antitrypsin (SERPINA1) gene unfolded protein response. Mutat Res 569: 29–63. in chronic liver disease: a case control study. BMC Gas- Shimoike, T., Mimori, S., Tani, H., Matsuura, Y., and Miya- troenterol 10: 22. mura, T. (1999) Interaction of hepatitis C virus core protein Kuroiwa-Trzmielina, J., de Conti, A., Scolastici, C., Pereira, with viral sense RNA and suppression of its translation. D., Horst, M.A., Purgatto, E., et al. (2009) Chemopreven- J Virol 73: 9718–9725. tion of rat hepatocarcinogenesis with histone deacetylase Stone, M., Jia, S., Heo, W.D., Meyer, T., and Konan, K.V. inhibitors: efficacy of tributyrin, a butyric acid prodrug. Int J (2007) Participation of rab5, an early endosome protein, in Cancer 124: 2520–2527. hepatitis C virus RNA replication machinery. J Virol 81: Li, S., Ye, L., Yu, X., Xu, B., Li, K., Zhu, X., et al. (2009) 4551–4563. Hepatitis C virus NS4B induces unfolded protein response Su, A.I., Pezacki, J.P., Wodicka, L., Brideau, A.D., Supekova, and endoplasmic reticulum overload response-dependent L., Thimme, R., et al. (2002) Genomic analysis of the host NF-kappaB activation. Virology 391: 257–264. response to hepatitis C virus infection. Proc Natl Acad Sci Liefhebber, J.M., Brandt, B.W., Broer, R., Spaan, W.J., and USA 99: 15669–15674.

Su, W.C., Chao, T.C., Huang, Y.L., Weng, S.C., Jeng, K.S., Waris, G., Sarker, S., and Siddiqui, A. (2004) Two-step affin- and Lai, M.M. (2011) Rab5 and class III PI-3-kinase Vps34 ity purification of the hepatitis C virus ribonucleoprotein are involved in hepatitis C virus NS4B-induced autophagy. complex. RNA 10: 321–329. J Virol 85: 10561–10571. Waris, G., Felmlee, D.J., Negro, F., and Siddiqui, A. (2007) Syed, G.H., Amako, Y., and Siddiqui, A. (2010) Hepatitis C Hepatitis C virus induces proteolytic cleavage of sterol virus hijacks host lipid metabolism. Trends Endocrinol regulatory element binding proteins and stimulates their Metab 21: 33–40. phosphorylation via oxidative stress. J Virol 81: 8122– Tasaka, M., Sakamoto, N., Itakura, Y., Nakagawa, M., Itsui, 8130. Y., Sekine-Osajima, Y., et al. (2007) Hepatitis C virus Welsch, C., Albrecht, M., Maydt, J., Herrmann, E., Welker, non-structural proteins responsible for suppression of the M.W., Sarrazin, C., et al. (2007) Structural and functional RIG-I/Cardif-induced interferon response. J Gen Virol 88: comparison of the non-structural protein 4B in flaviviridae. 3323–3333. J Mol Graph Model 26: 546–557. Tatebe, H., Shimizu, M., Shirakami, Y., Sakai, H., Yasuda, Y., Yu, G.Y., Lee, K.J., Gao, L., and Lai, M.M. (2006) Palmitoy- Tsurumi, H., and Moriwaki, H. (2009) Acyclic retinoid lation and polymerization of hepatitis C virus NS4B protein. synergises with valproic acid to inhibit growth in human J Virol 80: 6013–6023. hepatocellular carcinoma cells. Cancer Lett 285: 210– Zheng, Y., Ye, L.B., Liu, J., Jing, W., Timani, K.A., Yang, X.J., 217. et al. (2005) Gene expression profiles of HeLa cells Thompson, A.A., Zou, A., Yan, J., Duggal, R., Hao, W., impacted by hepatitis C virus non-structural protein NS4B. Molina, D., et al. (2009) Biochemical characterization J Biochem Mol Biol 38: 151–160. of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity. Biochemistry 48: 906–916. Supporting information Tong, W.Y., Nagano-Fujii, M., Hidajat, R., Deng, L., Takigawa, Y., and Hotta, H. (2002) Physical interaction between hepa- Additional Supporting Information may be found in the online titis C virus NS4B protein and CREB-RP/ATF6beta. version of this article: Biochem Biophys Res Commun 299: 366–372. Regulation of interaction networks by covalent modifications. Tripathi, L.P., Kataoka, C., Taguwa, S., Moriishi, K., Mori, Y., Table S1. Predicted Interaction Motifs within HCV NS4B. Matsuura, Y., and Mizuguchi, K. (2010) Network based Table S2. Summary of protein domains and covalent modifica- analysis of hepatitis C virus core and NS4B protein inter- tions in NS4B. actions. Mol Biosyst 6: 2539–2553. te Velthuis, A.J., Sakalis, P.A., Fowler, D.A., and Bagowski, Please note: Wiley-Blackwell are not responsible for the content C.P. (2011) Genome-wide analysis of PDZ domain binding or functionality of any supporting materials supplied by the reveals inherent functional overlap within the PDZ interac- authors. Any queries (other than missing material) should be tion network. PLoS ONE 6: e16047. directed to the corresponding author for the article.