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Cellular networks involved in the influenza virus life cycle

Tokiko Watanabe1,2,*, Shinji Watanabe1,2, and Yoshihiro Kawaoka1,2,3,4,*

1Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 575 Science Drive, Madison, WI 53711, USA2ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency,Saitama 332-0012, Japan3Division of Virology, Department of Microbiology and Immunology, University of Tokyo, Tokyo108-8639, Japan4International Research Center for Infectious Diseases, Institute of Medical Science, University ofTokyo, Tokyo 108-8639, Japan

AbstractInfluenza viruses cause epidemics and pandemics. Like all other viruses, influenza viruses rely onthe host cellular machinery to support their life cycle. Accordingly, identification of host functionsthat participate in viral replication steps is of great interest to understand the mechanisms of thevirus life cycle as well as to find new targets for the development of antiviral compounds. Multiplelaboratories have used various approaches to explore host factors involvement in the influenzavirus replication cycle. One of the most powerful approaches is an RNAi-based genome-widescreen, which has thrown new light on the search for host factors involved in virus replication. Inthis review, we examine the cellular genes identified to date as important for influenza virusreplication in genome-wide screens, assess pathways that were repeatedly identified in thesestudies, and discuss how these pathways might be involved in the individual steps of influenzavirus replication, ultimately leading to a comprehensive understanding of the virus life cycle.

IntroductionViruses, which consist of nucleic acid encased in a protein shell, are parasites of their hostorganisms. Despite their simple structures, viruses have sophisticated mechanisms toreplicate themselves in their hosts. A ‘host cellular factory’ has thousands of machines,which viruses use during each step of their life cycle. Viruses generally initiate their lifecycle by attaching to host cell surface receptors, entering the cells and uncoating their viralnucleic acid. They then replicate their viral genome. After new copies of viral proteins andgenes are synthesized, these components assemble into progeny virions, which then exit thecell. For each step, viruses use many host cellular functions. Identifying these host functionshas long been of great interest in virology to further our understanding of the precisemechanisms of the viral life cycle.

Influenza viruses cause annual epidemics and recurring pandemics with potentially severeconsequences for public health and the global economy. The first influenza pandemic of the21st century was caused by the 2009 H1N1 virus and has so far resulted in 42–86 million

*Correspondence should be addressed to Tokiko Watanabe, ERATO Infection-Induced Host Responses Project, Japan Science andTechnology Agency, Saitama 332-0012, Japan; Phone: +81(3)6459-3261, Fax: +81(3)6459-3273, [email protected] Yoshihiro Kawaoka, Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, Universityof Tokyo, Tokyo 108-8639, Japan; Phone: +81(3)5449-5310, Fax: +81(3)5449-5408, [email protected].

NIH Public AccessAuthor ManuscriptCell Host Microbe. Author manuscript; available in PMC 2011 September 6.

Published in final edited form as:Cell Host Microbe. 2010 June 25; 7(6): 427–439. doi:10.1016/j.chom.2010.05.008.

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cases of infection worldwide (http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm). Inaddition, highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia,Europe, and Africa, overcoming host species barriers to infect humans, with fatal outcomein many cases (Webster and Govorkova, 2006; Yen and Webster, 2009). Antiviral drugs,such as oseltamivir, zanamivir (Hayden, 2001), and amantadine/rimantadine (Davies et al.,1964), are available for prophylaxis and treatment of influenza virus infection; however,most human H3N2 and H1N1, including pandemic 2009 H1N1, influenza viruses areresistant to amantadine/rimantadine (Bright et al., 2005; Bright et al., 2006; Dawood et al.,2009). Moreover, the frequency with which H1N1 influenza viruses are becomingoseltamivir-resistant is a cause for concern and highlights the urgent need for new antiviraldrugs (Nicoll et al., 2008)(http://www.who.int/csr/disease/influenza/h1n1_table/en/index.html).

Influenza A viruses are enveloped negative-strand RNA viruses with eight RNA segmentsencoding at least 10 viral proteins (reviewed in (Palese, 2007)). Two additional viralproteins, PB1-F2 and PB1 N40, have been identified, although not all strains encode theseproteins (Chen et al., 2001; Wise et al., 2009). The virus particles are enclosed by a lipidenvelope, which is derived from the host cellular membrane. Three viral proteins, thesurface glycoproteins hemagglutinin (HA) and neuraminidase (NA), and the M2 ion channelprotein, are embedded in the lipid bilayer of the viral envelope. The HA protein binds tosialic acid-containing receptors on the host cell surface and mediates fusion of the viralenvelope with the endosomal membrane after receptor-mediated endocytosis (Palese, 2007;Skehel and Wiley, 2000). By contrast, the NA protein plays a crucial role late in infection byremoving sialic acid from sialyloligosaccharides, thereby releasing newly assembled virionsfrom the cell surface and preventing the self-aggregation of virus particles. Underneath thelipid bilayer lies the matrix protein (M1), a major structural protein. Within the virus shellare eight viral ribonucleoprotein (vRNP) complexes, each composed of viral RNA (vRNA)associated with the nucleoprotein NP and the three components of the viral RNApolymerase complex (PB2, PB1, and PA). The NS1 protein, which counteracts the cellularinterferon response, is synthesized from an unspliced mRNA, while a spliced mRNA yieldsthe NS2, or NEP, protein, which mediates nuclear export of vRNP complexes. In addition,the recently identified PB1-F2 protein, which is encoded by the PB1 segment, is thought toplay a role in viral pathogenicity (Chen et al., 2001; Conenello et al., 2007; McAuley et al.,2007; Zamarin et al., 2006).

While the functions of the viral proteins have been studied extensively over the last decade,relatively little is known about the cellular factors involved in influenza virus life cycle.Here, we review recent genome-wide screens that aim to close this critical gap in influenzavirus research.

Dawn of a new era: The application of genome-wide screens to identify host factorsinvolved in influenza virus replication

A number of interactions between viral components and specific host cell gene productshave now been identified. Although most host molecules remain elusive, emerging dataindicate that their identification and characterization will provide new insights into themechanisms by which viruses complete their life cycle. Moreover, such knowledge wouldilluminate potentially useful targets for therapeutic intervention. Indeed, antiviral drugstargeting host cell factors involved in viral replication have been tested for the treatment ofhuman immunodeficiency virus type I (HIV-1), with promising results (Coley et al., 2009).However, this goal would generally take several decades to achieve with conventionalgenetic screening methods and mammalian cell cultures.

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http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm

http://www.who.int/csr/disease/influenza/h1n1_table/en/index.html

Genome-wide RNAi screens—Many laboratories have expended great effort in thesearch for new host factors involved in the virus replication cycle by using variousstrategies. One of the most powerful approaches is systematic, genome-wide RNAinterference (RNAi) analysis. RNAi is a regulatory mechanism that uses double-strandedRNA (dsRNA) molecules to direct homology-dependent suppression of gene activity(reviewed in (Mello and Conte, 2004)). This powerful cellular process has been extensivelyapplied for studies of gene functions and therapeutic approaches for disease treatment. Now,this technology offers a new exciting tool for the exploration of novel host genes involved invirus replication (Brass et al., 2008; Brass et al., 2009; Cherry et al., 2005; Goff, 2008; Haoet al., 2008; Konig et al., 2010; Konig et al., 2008; Krishnan et al., 2008; Li et al., 2009;Zhou et al., 2008).

The first genome-wide screen for the identification of host factors involved in influenzavirus replication was reported by Hao et al. (2008) (Table 1). Since RNAi-based screeningwas not well-established in mammalian cells at that time, the authors used Drosophila RNAitechnology. Studies in Drosophila have made numerous fundamental contributions to ourunderstanding of mammalian cell biology because of the high degree of geneticconservation between Drosophila and vertebrates. Hao et al. (2008) first established aninfluenza virus infection system in Drosophila cells by modifying the influenza virusgenetically to infect Drosophila cells and express a reporter gene product in infected cells.This system supported influenza virus replication from post-entry to the protein expressionstep in the life cycle (Table 1). The authors tested an RNAi library against 13,071 genes(90% of the Drosophila genome) and identified 110 genes whose depletion in Drosophilacells significantly affected reporter gene expression from the influenza virus-like RNA (seeSupplementary Table S3 for the list of genes). Of those 110 candidates, they validated rolesfor the host proteins ATP6V0D1 (an ATPase), COX6A1 (a cytochrome C oxidase subunit),and NXF1 (a nuclear RNA export factor) in the replication of H5N1 and H1N1 influenza Aviruses in mammalian cells. These studies showed the feasibility and power of genome-wideRNAi screens to identify previously unrecognized host proteins required for influenza virusreplication.

The development of mammalian RNAi-based screening now enables the comprehensiveanalysis of mammalian host cell functions in influenza virus replication. Recently, Brass etal. (2009), Konig et al. (2010), and Karlas et al. (2010) reported genome-wide RNAi screensin mammalian cells that identified host proteins important for influenza virus replication(Table 1). They tested siRNAs targeting more than 17,000 human genes in humanosteosarcoma (Brass et al., 2009) and the human lung cell line A549 (Karlas et al., 2010;Konig et al., 2010), respectively. Because the readout of the assay was the measurement of aviral protein or a reporter protein encoded by the viral genome, the systems used by Brass etal. (2009) and Konig et al. (2010) allowed the identification of host gene products importantfor the early to mid stages of the influenza virus life cycle, including virus entry, uncoating,vRNP nuclear import, genome transcription, and viral protein translation. These systemsidentified 133 (Brass et al., 2009) and 295 (Konig et al., 2010) human genes whosedepletion affected the efficiency of HA cell surface expression or reporter gene expression,respectively (Table 1; see Supplementary Table S3 for the list of genes). In contrast, Karlaset al. (2010) studied the entire viral replication cycle, from viral entry to budding, bydetermining virus infectivity titers in culture supernatants from siRNA-treated, virus-infected A549 cells. This approach identified 287 human genes important for influenza virusreplication (Karlas et al., 2010) (see Supplementary Table S3 for the list of genes).

Shapira et al. (2009) employed a different tactic. They combined the results of yeast two-hybrid analyses, genome-wide transcriptional gene-expression profiling, and an RNAiscreen (Shapira et al., 2009). First, they built a physical interaction map for viral proteins



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and human cellular molecules by applying computational analysis to their comprehensiveyeast two-hybrid assay. This physical map indicated that the viral proteins of influenzainteracted with ‘a significantly greater number of human proteins than expected from thehuman interaction network, even when compared to other viruses’ (Shapira et al., 2009),suggesting influenza viruses have to maximize the diversity of functions for each protein(Shapira et al., 2009). They then examined which cellular gene products were differentiallyexpressed in primary human bronchial epithelial cells exposed to influenza virus or viralRNA. Collectively, these approaches identified 1,745 potential host factors with roles in theinfluenza virus life cycle (Shapira et al., 2009). Further validation with using siRNAstargeting to 1,745 genes narrowed this list to 616 human genes whose products affectedinfluenza virus replication.

Other approaches—Proteomics approaches and yeast two-hybrid analyses have alsobeen used to identify host molecules that interact with influenza virus components (reviewedin (Nagata et al., 2008)). Several host factors involved in the steps of viral genomereplication and transcription have been identified (Deng et al., 2006; Engelhardt et al., 2005;Huarte et al., 2001; Jorba et al., 2008; Kawaguchi and Nagata, 2007; Momose et al., 2001;Momose et al., 2002; Resa-Infante et al., 2008), as well as NS1 protein-interacting hostfactors associated with immune responses (reviewed in (Hale et al., 2008)). Using massspectrometry, Mayer et al. (2007) recently identified 41 host cellular molecules that interactwith influenza vRNPs, including nucleoplasmin which may play a role in viral RNAsynthesis.

A library encompassing about 4,800 yeast single-gene deletion strains, which covers 80% ofall yeast genes, has been used to explore host gene involvement in virus replication,especially that of several positive-strand RNA viruses (Kushner et al., 2003; Panavas et al.,2005). Naito et al. (2007) developed a system for influenza virus genome replication andtranscription in Saccharomyces cerevisiae, and conducted a screen with a sub-library of 354deletion strains, each of which lacked a gene encoding a putative nucleic acid-binding/-related functional protein. This approach resulted in the identification of several host factorsthat affected influenza RNA synthesis (Naito et al., 2007).

Sui et al. (2009) employed a new technique, called ‘Random Homozygous GenePerturbation’ (RHGP), to identify host factors involved in influenza virus replication. Thissystem uses a lentiviral-based genetic element containing a promoter (e.g., thecytomegalovirus immediate early promoter); the genetic element is designed to integrate at asingle site in one allele of the genome in either the sense or antisense orientation. Integrationof this element in the antisense orientation disrupts gene expression in one allele. Moreover,because this element contains a promoter, antisense RNA is expressed and can abrogateexpression of the same gene in the other allele. If the element is integrated in the senseorientation in one allele, either overexpression of the gene, or expression of a dominant-negative form occurs, leading to an altered phenotype. Unlike RNAi screens, the RHGPsystem can theoretically knockdown or overexpress any gene, without any prior knowledgeor annotation of that gene, resulting in alteration of the phenotype. Sui et al. (2009)generated an RHGP library in mammalian cells, followed by infection with a dose ofinfluenza virus that results in cell death. They then isolated the influenza virus-resistant cellclones and identified 110 human genes that contributed to the host cell resistance to viralinfection, demonstrating the usefulness of this RGHP system for genome-wide screening.



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Pair-wise comparison among six independent genome-wide screens for human genesinvolved in influenza virus replication

In the six independent genome-wide screens described above (Brass et al., 2009; Hao et al.,2008; Karlas et al., 2010; Konig et al., 2010; Shapira et al., 2009; Sui et al., 2009), a total of1,449 human genes were identified as potential host factors involved in influenza virusreplication (including 110 Drosophila genes that have human orthologs; SupplementaryTable S1A, B), indicating that about 5.8% of human protein-coding genes may contribute tothe life cycle of influenza virus. However, there is the possibility that these high-throughputscreens may contain false-positive. To narrow down the genes important for influenza viralreplication, we compared the candidate genes identified in these screens by pair-wisecomparison and found 128 human genes that affected influenza virus replication in at leasttwo screens (Supplementary Table S2). The number of genes common between pairs ofscreens is relatively small (from 0 to 32 genes in the 15 pair-wise comparisons). This lowincidence of overlap has also been observed in three RNAi screens designed to identify hostfactors for HIV-1 replication (Brass et al., 2008; Konig et al., 2008; Zhou et al., 2008), and itcould be due to differences in screening systems (e.g., RNAi screen vs. RHGP libraryscreen), or other conditions such as cell type, virus strains, and/or detection methods.Indeed, the number of common genes was highest in the comparison between the Konig andKarlas screens (32 human genes) (Supplementary Table S2), presumably because bothscreens were based on RNAi and used the same human lung cells. In addition, the analysisof raw data, particularly the ‘cut-off’ used to determine candidates, likely differed among thestudies. Nonetheless, a number of factors were identified in several screens (see below formore details), suggesting their importance in the influenza virus life cycle.

Network analysis of influenza-host protein interactionsFor the set of 128 human genes that were identified in two or more screens and are likelyinvolved in influenza virus replication, we next determined major gene categories. Ouranalysis with the PANTHER Classification system (Mi et al., 2005) revealed severalenriched gene categories associated with molecular functions, such as nucleic acid-bindingproteins, kinases, transcription factors, ribosomal proteins, hydrogen transporters, andproteins involved in mRNA splicing. Moreover, we found enrichment for cellular proteinsinvolved in biological processes such as protein metabolism and modification, signaltransduction, protein phosphorylation, nucleoside, nucleotide and nucleic acid metabolism,and transport. Analysis by Reactome, which is a curated knowledgebase of biologicalpathways (Matthews et al., 2009), revealed several other over-represented events, includingeukaryotic translation initiation, regulation of gene expression, processing of capped intron-containing pre-mRNAs, and Golgi to ER retrograde transport. Further, we analyzedinteractions of the set of these 128 genes with using GeneGo (GeneGo Inc, MI, USA)(Supplementary Table S4A). We then integrated this data set with information on the viraland cellular interaction partners of viral proteins as reported by Konig et al. (2010) andShapira et al. (2009) (Supplementary Table S4B), resulting in a network of host-influenzavirus interactions (Figure 2). In addition, this network included several sub-networksassociated with specific biological processes, such as translation initiation, processing ofpre-mRNA, and proton-transporting ATPase functions.

Insights into host factors involved in the influenza virus life cycleBased on the analyses described above, influenza virus-host interactions can be mapped toindividual steps of the influenza virus life cycle (Fig. 1), as outlined below.

1) Endocytosis, fusion, and uncoating—Once virus particles attach to a cell surfacereceptor, they are mainly internalized by clathrin-dependent endocytosis, although influenza



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virus also uses a non-clathrin-dependent endocytic pathway (Marsh and Helenius, 2006;Matlin et al., 1981; Rust et al., 2004; Sieczkarski and Whittaker, 2002). The importance ofboth early and late endosomes in influenza virus entry has been demonstrated withdominant-negative forms of the GTPases Rab5 and Rab7/protein kinase C β II (PKCβII),which regulate the functions of early and late endosomes, respectively (Sieczkarski et al.,2003; Sieczkarski and Whittaker, 2003). Rab10 (RAB10), which controls the movement ofendosomes (Conenello et al., 2007; Glodowski et al., 2007), was identified in two screens(Supplementary Table S3). Moreover, four of the seven subunits of the coatomer 1 (COPI)vesicular transport complex (i.e., ARCN1, COPA, COPB2, and COPG), which is requiredfor the formation of intermediate transport vesicles between the early and late endosomesand for retrograde Golgi-to-ER transport (Aniento et al., 1996; Cai et al., 2007; Whitney etal., 1995), were identified in five independent screens (Supplementary Table S3), indicatingthe biological significance of this host cell machinery in the early steps of the influenza viruslife cycle.

Uncoating of viral particles occurs upon fusion between the viral and endosomalmembranes, is mediated by HA, and leads to the release of vRNPs into the cytoplasm(Stegmann et al., 1990). The M2 ion channel protein functions in the endosomes to lowerthe pH of virion interior, resulting in the disassociation of the viral matrix M1 protein fromthe vRNP complex, which can then be imported to the nucleus (Helenius, 1992). Proton-transporting V-type ATPase functions in both the acidification and fusion of the cellularcompartments (Stevens and Forgac, 1997). Four of the six screens identified at least twosubunits of this V-type ATPase complex as host factors required for influenza virusinfection (Supplementary Table S3). Although this complex was thought to be generallyinvolved in the low pH-dependent membrane fusion between endosomes and virions,knockdown of ATP6V0D1 caused significant inhibition of influenza virus production butnot VSV production, which also requires a low pH for fusion (Hao et al., 2008). This findingimplies a specific role for the complex in influenza virus replication.

2) Transport of vRNP into the host nucleus—Replication/transcription of influenzavirus genomic RNA occurs in the nucleus (reviewed in (Engelhardt and Fodor, 2006; Palese,2007)). Therefore, the RNP complexes released from the incoming virus particles must betransported into the nucleus through the nuclear pores. The nuclear import of RNPcomplexes is dependent on the active import machinery of the host cell nuclear porecomplex (NPC) because of the size of these complexes (10–15 nm wide and 30–120 nmlong). In a classic nuclear import pathway, importin α recognizes a cargo protein containingnuclear localization signals (NLS), binds to importin β, and the cargo/importin α/β complexis transported into the nucleus through the NPC. For influenza virus, RanBP5 (orimportin-5), importin α1/α2, and importin α1/importin α5 have been shown to bind to PB1-PA dimers (Deng et al., 2006), NP (O’Neill et al., 1995; Wang et al., 1997), and PB2(Gabriel et al., 2008; Resa-Infante et al., 2008; Tarendeau et al., 2007; Tarendeau et al.,2008), respectively. Our list of 128 genes includes the importin subunit β1 (KPNB1) andnuclear pore complex proteins Nup98 and Nup153 (NUP98 and NUP153). Likely, thesefactors are involved in the nuclear import of influenza virus RNPs, as well as in the nuclearimport of newly synthesized influenza viral proteins.

3) Genome replication/transcription and translation—Once the vRNP complexesare imported into the nucleus, the vRNA of influenza virus is transcribed into mRNA andreplicated via complementary RNA (cRNA) (reviewed in (Engelhardt and Fodor, 2006;Palese, 2007)). The viral polymerase subunits (PB1, PB2 and PA) and NP catalyze bothgenome replication and transcription. Several host cellular molecules play a part in thesesteps. Host factors shown to stimulate viral RNA synthesis include BAT1 (or UAP56), heatshock protein (Hsp) 90, the minichromosome maintenance (MCM) complex, Tat-SF1, and



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DNA-dependent RNA polymerase II (PolII) (Chan et al., 2006; Engelhardt et al., 2005;Kawaguchi and Nagata, 2007; Momose et al., 2001; Momose et al., 1996; Momose et al.,2002). BAT1, which is a splicing factor for mRNA and also functions in the nuclear exportof cellular mRNA, interacts with NP to facilitate the formation of the NP-RNA complex.Similarly, Tat-SF1 facilitates NP-vRNP complex formation (Naito et al., 2007). Viral RNAsynthesis is then initiated by the viral RNA polymerase (Momose et al., 2001; Momose etal., 1996). Hsp90 stimulates viral RNA synthesis through its interaction with PB2 (Momoseet al., 1996; Momose et al., 2002). MCM, which functions as a DNA replication forkhelicase (Forsburg, 2004), plays a role in influenza viral genome replication by promotingthe association between nascent cRNA and the viral polymerase complexes during thetransition from initiation to elongation, thus stabilizing replication elongation complexes andallowing the synthesis of full-length cRNA (Kawaguchi and Nagata, 2007). As describedabove, the yeast library screen identified Tat-SF1 as a factor involved in vRNA-NP complexformation and viral RNA synthesis (Naito et al., 2007). In its active state, PolII, whichcatalyzes the synthesis of mRNA precursors and most snRNA and microRNAs, is requiredfor viral mRNA synthesis during the viral transcription step (Chan et al., 2006; Engelhardt etal., 2005). Included in the 128 genes identified in our pair-wise comparison (SupplementaryTable S2) are genes associated with pre-mRNA splicing (i.e., PTBP1, NHP2L1, SNRP70,SF3B1, SF3A1, P14, and PRPF8), suggesting the importance of the host mRNA splicingmachinery for influenza virus gene expression. Interestingly, no splicing factors were foundin the screen carried out in Drosophila cells (Hao et al., 2008), which is consistent with thefinding that influenza virus mRNAs were not spliced in this system (Hao et al, 2008).

Four screens identified NXF1 as an important host factor in influenza virus replication(Supplementary Table S3). NXF1 exports spliced, cellular mRNAs but rarely exportsunspliced cellular mRNAs (Reed and Cheng, 2005). Nonetheless, several viruses use theNXF1-pathway for the nuclear export of unspliced viral RNAs (Cullen, 2003). For influenzavirus, NP interacts with BAT1/UAP56(Momose et al., 2001), which forms a bridge betweenthe NXF1/NXT1/Ref complex and the spliced mRNA. In addition, NS1 binds NXF1 tosuppress host mRNA export (Satterly et al., 2007), perhaps to retain host mRNAs in thenucleus for ‘cap-snatching’. In addition, the requirement of NXF1 for nuclear export of viralmRNAs has recently been shown (Read and Digard, 2010). Thus, influenza virus mayexploit the NXF1-mediated RNA export pathway for multiple purposes.

Influenza virus also uses the cellular machinery for mRNA translation. Influenza virusinfection shuts-off host protein synthesis, thereby enhancing translation of viral mRNAs(Chen and Krug, 2000; Chen et al., 1999; Fortes et al., 1994; Nemeroff et al., 1998; Qiu andKrug, 1994). GRSF-1 (the cellular RNA-recognition motif containing the RNA-bindingprotein G-rich sequence factor 1) and P58IPK (the cellular inhibitor of PKR, an interferon-induced kinase that targetsthe eukaryotic translation initiation factor eIF2) appear to beinvolved in the selective synthesis of influenza viral proteins (Goodman et al., 2007; Kash etal., 2002; Park et al., 1999). Analysis with Reactome (Matthews et al., 2009) of the set of128 genes we identified above revealed several host genes involved in the formation of thetranslation initiation complex and the small ribosomal subunit (i.e., FAU, EIF3S5, RPS3A,RPS4X, RPS10, RPS14, RPS5, RPS16, EIF4A2, RPS20, and EIF3S8). Possibly, some ofthese host factors promote virus-specific translation by inhibiting host cellular mRNAtranslation and/or stimulating viral mRNA translation.

4) vRNP transport from the nucleus to the cytoplasm—Newly synthesized vRNPsare exported from the nucleus to the cytoplasm (reviewed in (Boulo et al., 2007)). The viralmatrix protein, M1, and nuclear export protein, NEP/NS2, have critical roles in this step.After M1 binds to the RNP complex, NEP/NS2 attaches to the M1/RNP complex through itsC-terminal domain (Akarsu et al., 2003). NEP/NS2 can also bind to a host cell nuclear



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export protein, CRM1 (Boulo et al., 2007; Elton et al., 2001; Neumann et al., 2000).Additionally, heat shock cognate (Hsc) 70 protein and the MAP kinase cascade may haveroles in the nuclear export of vRNP complexes (Pleschka et al., 2001; Watanabe et al.,2006).

Recently, phosphatidylinositol-3-kinase (PI3K) and its downstream effector protein (thekinase Akt) were identified as host factors involved in influenza virus-induced signaling(Ehrhardt and Ludwig, 2009; Ehrhardt et al., 2006; Hale et al., 2006; Shin et al., 2007;Zhirnov and Klenk, 2007). Consistent with this finding, three genes involved in the PI3K/AKT signaling pathway (i.e., AKT1, MDM2, IKBKE) were among the 128 genes identifiedby the screens discussed here. Roles for this pathway in antiviral activity and prevention ofpremature apoptosis have been reported (Ehrhardt et al., 2006; Ehrhardt et al., 2007; Sarkaret al., 2004). Interestingly, a PI3K inhibitor obstructs the nuclear export of vRNPs, as wellas viral RNA and protein synthesis (Shin et al., 2007).

5) Assembly and budding—In the late stage of the life cycle, the virion components,including vRNPs, the matrix protein M1, and the viral envelope proteins (HA, NA and M2)are transported to the assembly site, the apical plasma membrane in polarized epithelial cells(Nayak et al., 2004). Relatively few host factors have been identified to be involved in thisstage. Of the six genome-wide screens discussed in this review, only two studies (Karlas etal., 2010; Shapira et al., 2009) were designed to evaluate the assembly and budding steps.Depletion of subunits of the COPI complex reduced HA expression on the cell surfacerelative to the total HA level in the screen by Brass et al. (2009)(Fig. 1), suggesting that theCOPI complex plays a role in the transport of influenza viral glycoproteins (i.e., HA andNA) to the cell surface, in addition to its function in virus entry described above. The HAand NA proteins contain signals for association with lipid rafts, which are non-ionicdetergent-resistant lipid microdomains within the plasma membrane. Influenza virusespreferentially bud from these lipid raft microdomains (Barman et al., 2001; Leser and Lamb,2005; Nayak et al., 2004; Takeda et al., 2003; Zhang et al., 2000). Although the precisemechanism of M1-vRNP complex transport to the cell surface is still unclear, the viral M2protein is thought to play a role in the incorporation of M1-vRNPs into virus particles via aninteraction between M1 and the M2 cytoplasmic tail (Iwatsuki-Horimoto et al., 2006;McCown and Pekosz, 2005, 2006; Wu and Pekosz, 2008). NP and M1 interact with hostcytoskeletal components, such as actin, presumably facilitating vRNP transport to thebudding site (Avalos et al., 1997). In the final step of the life cycle, the viral membranecomponents are concentrated in the microdomains of the lipid rafts, which may provide thestage for virus particle release from the cell surface (Takeda et al., 2003). Although few hostfactors have been found that participate in the budding step, the actin cytoskeleton andRab11, which belongs to the Rab family of small GTPases, have been shown to contribute tothe budding of filamentous influenza virus particles (Bruce et al., 2010; Roberts andCompans, 1998; Simpson-Holley et al., 2002).

Perspectives and Concluding RemarksIn 2003, the human genome project completed its mission of identifying the approximately20,000–25,000 genes in human DNA. To determine host gene involvement in virusreplication, genome-wide screens, such as RNAi screens, offer an effective approach. Forinfluenza virus, among six independent genome-wide screens, 1,452 human genes, including110 human orthologs identified by a screen in Drosophila cells, have been identified as hostfactors involved in influenza virus replication; of these, 128 genes were found in at least twoscreens. Bioinformatics analysis of these 128 human genes revealed gene clusters related tohost cellular functions, such as endocytosis, translation initiation, and nuclear transport, allof which can be connected to the influenza viral life cycle. Fig. 1 illustrates influenza virus-



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host interactions mapped to individual steps of the influenza virus life cycle based on theknown functions of the identified host proteins. However, it should be borne in mind thatthese proteins may be involved in other steps of the viral life cycle with, as yet,undetermined functions.

Systematic, genome-wide RNAi screens are powerful tools for identifying host genesimportant for viral replication. However, they do have limitations; for example, the RNAilibrary does not yet covered all of the human genes, the knockdown efficiency of target geneexpression could vary among siRNAs, and genes whose siRNA knockdown leads tocytotoxicity would be eliminated from a ‘hit-list’ even though they may be important forviral replication, resulting in a number of false-positives and false-negatives. Therefore, theidentification of host gene candidates involved in viral replication by any primary screenreally represents no more than a starting point for exploration of such host factors. Moredetailed functional analyses of human genes identified in genome-wide screens will allow usto find novel cellular pathways and/or host gene sets important for influenza virusreplication, leading to a greater understanding of the precise mechanisms of influenza virusreplication.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Drs. Gabriele Neumann and Masato Hatta for comments on the manuscript, Dr. Susan Watson for editingthe manuscript. We also thank Drs. Yo Suzuki and Yoshinori Tomoyasu for valuable advice. This work wassupported, in part, by U.S. National Institute of Allergy and Infectious Diseases Public Health Service researchgrants, by a grant-in-aid for Specially Promoted Research (Japan), by funding from the Program of FoundingResearch Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture,Sports, Science and Technology (Japan), and by ERATO (Japan Science and Technology Agency).

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Figure 1. Influenza virus life cycle and host factorsUpon infection, influenza virus is internalized by receptor-mediated endocytosis. Aftermembrane fusion between the virus envelope and the endosome, the viral ribonucleoprotein(vRNP) complex is released into the cytoplasm and transported into the nucleus by theactive import machinery of the host cell nuclear pore complex. Replication and transcriptionof the viral genome take place in the nucleus and many host factors are believed to beinvolved in this process. Newly synthesized viral RNA associates with NP and forms theviral ribonucleoprotein (vRNP) complex together with viral polymerase proteins, which isthen transported from the nucleus to the cytoplasm. HA and NA are processedposttranslationally during their transport from the ER to the Golgi apparatus. Duringassembly and budding, virion components are transported to the assembly site, the lipid raftmicrodomains at the apical plasma membrane of polarized epithelial cells. Progeny virusesthen bud from the cells. The light orange rectangles indicate individual steps of the influenzavirus life cycle. The light blue rectangles indicate host cellular processes that may beinvolved in the virus life cycle. Red circles indicate host factors identified in the screensdiscussed here, while yellow circles indicate host factors identified in other previous studies.



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Figure 2. A network of host-influenza virus interactionsUsing GeneGo (GeneGo Inc, MI, USA), we analyzed the interactions of the 128 humangenes identified to be involved in influenza virus replication in at least two genome-widescreens (Supplementary Table S4A). We then integrated this information with data on hostprotein-virus interactions and interactions among viral proteins, as reported by Konig et al.(2010) and Shapira et al. (2009) (Supplementary Table S4B). Interaction networks amonghost and viral proteins (i.e., PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, NEP/NS2, andPB1-F2)(Fig 2A), virions (Fig 2B), or vRNP (Fig 2C) were visualized by using Cytoscape(http://cytoscape.org/). Yellow and lilac nodes indicate influenza viral components and hostproteins, respectively. Red circles indicate clusters associated with particular biologicalprocesses in the interaction networks, as identified by MCODE (“Molecular ComplexDetection”) analysis (Bader and Hogue, 2003): 1) translation initiation, 2) pre-mRNAprocessing, and 3) proton-transporting V-type ATPase.



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http://cytoscape.org/

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Documents

Ni Hms 319125