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Cellular Networks Involved in the Influenza Cycle

Tokiko Watanabe,1,2,* Shinji Watanabe,1,2 and Yoshihiro Kawaoka1,2,3,4,* 1Department of Pathobiological , School of Veterinary , University of Wisconsin-Madison, 575 Drive, Madison, WI 53711, USA 2ERATO -Induced Host Responses Project, Japan Science and Technology Agency, Saitama 332-0012, Japan 3Division of , Department of and 4International Research Center for Infectious Diseases Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan *Correspondence: [email protected] (T.W.), [email protected] (Y.K.) DOI 10.1016/j.chom.2010.05.008

Influenza cause and . Like all viruses, influenza viruses rely on the host cellular machinery to support their life cycle. Accordingly, identification of the host functions co-opted for viral repli- cation is of interest to understand the mechanisms of the virus life cycle and to find new targets for the devel- opment of antiviral compounds. Among the various approaches used to explore host factor involvement in the influenza virus replication cycle, perhaps the most powerful is RNAi-based -wide screening, which has shed new light on the search for host factors involved in virus replication. In this review, we examine the cellular identified to date as important for influenza virus replication in genome-wide screens, assess pathways that were repeatedly identified in these studies, and discuss how these pathways might be involved in the individual steps of influenza virus replication, ultimately leading to a comprehensive understanding of the virus life cycle.

Introduction oseltamivir resistant is a cause for concern and highlights the Viruses, which consist of encased in a shell, urgent need for new antiviral drugs (Nicoll et al., 2008)(http:// are parasites of their host . Despite their simple struc- www.who.int/csr/disease/influenza/h1n1_table/en/index.html). tures, viruses employ sophisticated mechanisms to replicate Influenza A viruses are enveloped negative-strand RNA within their hosts. A ‘‘host cellular factory’’ has thousands of viruses with eight RNA segments encoding at least 10 viral machines, which viruses co-opt or subvert for each step of their (reviewed in Palese, 2007). Two additional viral proteins, life cycle. Viruses generally initiate their life cycle by attaching to PB1-F2 and PB1 N40, have been identified, although not all host cell surface receptors, entering the cells, uncoating the viral strains encode these proteins (Chen et al., 2001; Wise et al., nucleic acid, and replicating their genome. After new copies of 2009). The virus are enclosed by a envelope, which viral proteins and genes are synthesized, these components is derived from the host cellular membrane. Three viral proteins, assemble into progeny virions, which exit the cell. For each the surface glycoproteins (HA) and step, viruses need to use many host cellular functions, and iden- (NA) and the M2 ion channel protein, are embedded in the lipid tifying these host functions has long been of great interest in bilayer of the . The HA protein binds to sialic virology to further our understanding of the precise mechanisms acid-containing receptors on the host cell surface and mediates of the . fusion of the viral envelope with the endosomal membrane Influenza viruses cause annual epidemics and recurring after -mediated (Palese, 2007; Skehel and pandemics with potentially severe consequences for public Wiley, 2000). By contrast, the NA protein plays a crucial role health and the global economy. The first influenza of late in infection by removing sialic acid from sialyloligosacchar- the 21st century was caused by the 2009 H1N1 virus and has ides, thereby releasing newly assembled virions from the cell so far resulted in 42–86 million cases of infection worldwide surface and preventing the self-aggregation of virus particles. (http://www.cdc.gov/h1n1flu/estimates_2009_h1n1.htm). In addi- Underneath the lies the matrix protein (M1), a major tion, highly pathogenic avian H5N1 influenza A viruses have structural protein. Within the virus shell are eight viral ribonucleo- spread throughout Asia, Europe, and Africa, overcoming host protein (vRNP) complexes, each composed of viral RNA (vRNA) barriers to infect humans, with fatal outcome in many associated with the NP and the three components cases (Webster and Govorkova, 2006; Yen and Webster, 2009). of the viral RNA complex (PB2, PB1, and PA). The Antiviral drugs, such as oseltamivir, zanamivir (Hayden, 2001), NS1 protein, which counteracts the cellular response, and amantadine/ (Davies et al., 1964), are available is synthesized from an unspliced mRNA, and a spliced mRNA for prophylaxis and treatment of influenza virus infection; yields the NS2, or NEP, protein, which mediates nuclear export however, most human H3N2 and H1N1, including pandemic of vRNP complexes. In addition, the recently identified PB1-F2 2009 H1N1, influenza viruses are resistant to amantadine/riman- protein, which is encoded by the PB1 segment, is thought to tadine (Bright et al., 2005, 2006; Dawood et al., 2009). Moreover, play a role in viral pathogenicity (Chen et al., 2001; Conenello the frequency with which H1N1 influenza viruses are becoming et al., 2007; McAuley et al., 2007; Zamarin et al., 2006).

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Whereas the functions of the viral proteins have been studied These studies showed the feasibility and power of genome- extensively during the last decade, relatively little is known about wide RNAi screens to identify previously unrecognized host the cellular factors involved in influenza virus life cycle. Here, we proteins required for influenza virus replication. review recent genome-wide screens that aim to close this critical The development of mammalian RNAi-based screening now gap in influenza virus research. enables the comprehensive analysis of mammalian host cell functions in influenza virus replication. Recently, Brass et al. Dawn of a New Era: The Application of Genome-wide (2009), Ko¨ nig et al. (2010), and Karlas et al. (2010) reported Screens to Identify Host Factors Involved in Influenza genome-wide RNAi screens in mammalian cells that identified Virus Replication host proteins important for influenza virus replication (Table 1). A number of interactions between viral components and specific They tested siRNAs targeting more than 17,000 human genes host cell products have now been identified. Although most in human osteosarcoma (Brass et al., 2009) and the human host remain elusive, emerging data indicate that their cell line A549 (Karlas et al., 2010; Ko¨ nig et al., 2010), respec- identification and characterization will provide new insights into tively. Because the readout of the was the measurement of the mechanisms by which viruses complete their life cycle. a or a reporter protein encoded by the viral genome, Moreover, such knowledge would illuminate potentially useful the systems used by Brass et al. (2009) and Ko¨ nig et al. (2010) targets for therapeutic intervention. Indeed, antiviral drugs tar- allowed the identification of host gene products important for geting host cell factors involved in have been the early to mid stages of the influenza virus life cycle, including tested for the treatment of human immunodeficiency virus type virus entry, uncoating, vRNP nuclear import, genome transcrip- I (HIV-1) with promising results (Coley et al., 2009). However, tion, and viral protein . These systems identified 133 this goal would generally take several decades to achieve with (Brass et al., 2009) and 295 (Ko¨ nig et al., 2010) human genes conventional genetic screening methods and mammalian cell whose depletion affected the efficiency of HA cell surface cultures. expression or reporter , respectively (Table 1; Genome-wide RNAi Screens see Table S3 for the list of genes). In contrast, Karlas et al. Many laboratories have expended great effort in the search (2010) studied the entire viral replication cycle, from for new host factors involved in the virus replication cycle by to , by determining virus titers in culture super- using various strategies. A powerful approach is systematic, natants from siRNA-treated, virus-infected A549 cells. This genome-wide RNA interference (RNAi) analysis. RNAi is a approach identified 287 human genes important for influenza regulatory mechanism that uses double-stranded RNA (dsRNA) virus replication (Karlas et al., 2010) (see Table S3 for the list of molecules to direct homology-dependent suppression of gene genes). activity (reviewed in Mello and Conte, 2004). This powerful Shapira et al. (2009) employed a different tactic. They cellular process has been extensively applied for studies of combined the results of yeast two-hybrid analyses, genome- gene functions and therapeutic approaches for disease treat- wide transcriptional gene expression profiling, and an RNAi ment. Now, this technology offers a new exciting tool for the screen (Shapira et al., 2009). First, they built a physical interac- exploration of novel host genes involved in virus replication tion map for viral proteins and human cellular molecules by (Brass et al., 2008, 2009; Cherry et al., 2005; Goff, 2008; Hao applying computational analysis to their comprehensive yeast et al., 2008; Ko¨ nig et al., 2008, 2010; Krishnan et al., 2008; Li two-hybrid assay. This physical map indicated that the viral et al., 2009; Zhou et al., 2008). proteins of influenza interacted with ‘‘a significantly greater The first genome-wide screen for the identification of host number of human proteins than expected from the human inter- factors involved in influenza virus replication was reported by action network, even when compared to other viruses’’ (Shapira Hao et al. (2008) (Table 1). Because RNAi-based screening et al., 2009), suggesting that influenza viruses have to maximize was not established in mammalian cells at that , the the diversity of functions for each protein (Shapira et al., 2009). authors used Drosophila RNAi technology. Studies in Drosophila They then examined which cellular gene products were differen- have made numerous fundamental contributions to our under- tially expressed in primary human bronchial epithelial cells standing of mammalian because of the high degree exposed to influenza virus or viral RNA. Collectively, these of genetic conservation between Drosophila and . approaches identified 1745 potential host factors with roles in Hao et al. (2008) first established an influenza virus infection the influenza virus life cycle (Shapira et al., 2009). Further valida- system in Drosophila cells by modifying the influenza virus tion using siRNAs targeting to 1745 genes narrowed this list genetically to infect Drosophila cells and express a reporter to 616 human genes whose products affected influenza virus gene product in infected cells. This system supported influenza replication. virus replication from post-entry to the protein expression step Other Approaches in the life cycle (Table 1). The authors tested an RNAi library Proteomics approaches and yeast two-hybrid analyses have against 13,071 genes (90% of the Drosophila genome) and iden- also been used to identify host molecules that interact with influ- tified 110 genes whose depletion in Drosophila cells significantly enza virus components (reviewed in Nagata et al., 2008). Several affected reporter gene expression from the influenza virus-like host factors involved in the steps of viral genome replication and RNA (see Table S3 available online for the list of genes). Of those have been identified (Deng et al., 2006; Engelhardt 110 candidates, they validated roles for the host proteins et al., 2005; Huarte et al., 2001; Jorba et al., 2008; Kawaguchi ATP6V0D1 (an ATPase), COX6A1 (a cytochrome c oxidase and Nagata, 2007; Momose et al., 2001, 2002; Resa-Infante subunit), and NXF1 (a nuclear RNA export factor) in the replica- et al., 2008), as well as NS1 protein-interacting host factors asso- tion of H5N1 and H1N1 influenza A viruses in mammalian cells. ciated with immune responses (reviewed in Hale et al., 2008).

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Table 1. Summary of Genome-wide Screens for the Identification of Host Factors Involved in Influenza Virus Replication Steps in the Viral Number of Hits/ Methods Cells Used for Life Cycle Covered Number of Reference (Approach) RNAi Screen Virus Detection Methods by the Screen Genes Tested Brass et al. RNAi screen Osteosarcoma A/Puerto Rico/8/34 HA expression entry, uncoating, 133/17,877 (2009) cells (U2OS) (PR8; H1N1) on cell surface nuclear import, transcription, translation, HA trafficking to cell surface Hao et al. RNAi screen Drosophila Recombinant luciferase activity uncoating, nuclear 110/13,071 (2008) cells A/WSN/33 (WSN; import, transcription, H1N1) virus translation possessing vesicular stomatitis virus glycoprotein G and NA-Luciferase (Luc) genes Ko¨ nig et al. RNAi screen human lung recombinant WSN luciferase activity entry, uncoating, 295/19,628 (2010) cells (A549) possessing HA-Luc nuclear import, reporter gene transcription, translation Karlas et al. RNAi screen human lung WSN (H1N1); virus yield entry, uncoating, 287/22,843 (2010) cells (A549) H1N1 2009 determined by nuclear import, NP and transcription, luciferase activity translation, nuclear export, protein transportation/ processing, assembly, budding Shapira et al. Y2H; microarray; HBEC PR8 (H1N1); virus yield entry, uncoating, 616/1,745 (2009) RNAi screen A/Udorn/307/72 determined by nuclear import, (RNAi screen) (H3N2) luciferase activity transcription, translation, nuclear export, protein transportation/ processing assembly, budding Sui et al. RHGP MDCK cells Udorn (H3N2) cloning of cells entry, uncoating, 110 clones, (2009) resistant to influenza nuclear import, sequenced virus infection transcription, translation, nuclear export, protein transportation/ processing assembly, budding

Using mass spectrometry, Mayer et al. (2007) recently identified protein. This approach resulted in the identification of several 41 host cellular molecules that interact with influenza vRNPs, host factors that affected influenza RNA synthesis (Naito et al., including nucleoplasmin, which may play a role in viral RNA 2007). synthesis. Sui et al. (2009) employed a new technique called ‘‘random A library encompassing about 4800 yeast single-gene deletion homozygous gene perturbation’’ (RHGP) to identify host factors strains, which covers 80% of all yeast genes, has been used to involved in influenza virus replication. This system uses a lentivi- explore host gene involvement in virus replication, especially ral-based genetic element containing a promoter (e.g., the that of several positive-strand RNA viruses (Kushner et al., immediate early promoter). The genetic 2003; Panavas et al., 2005). Naito et al. (2007) developed element is designed to integrate at a single site in one allele of a system for influenza virus genome replication and transcription the genome in either the sense or antisense orientation. Integra- in Saccharomyces cerevisiae and conducted a screen with a tion of this element in the antisense orientation disrupts gene sublibrary of 354 deletion strains, each of which lacked a gene expression in one allele. Moreover, because this element con- encoding a putative nucleic acid-binding/-related functional tains a promoter, antisense RNA is expressed and can abrogate

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expression of the same gene in the other allele. If the element is tion, protein phosphorylation, nucleoside, nucleotide and nucleic integrated in the sense orientation in one allele, either overex- acid , and transport. Analysis by Reactome, which is pression of the gene or expression of a dominant-negative a curated knowledgebase of biological pathways (Matthews form occurs, leading to an altered phenotype. Unlike RNAi et al., 2009), revealed several other overrepresented events, screens, the RHGP system can theoretically knock down or including eukaryotic translation initiation, regulation of gene overexpress any gene without any prior knowledge or annotation expression, processing of capped -containing pre-mRNAs, of that gene, resulting in alteration of the phenotype. Sui et al. and Golgi-to-ER retrograde transport. Further, we analyzed inter- (2009) generated an RHGP library in mammalian cells, followed actions of the set of these 128 genes by using GeneGo (GeneGo by infection with a of influenza virus that results in cell Inc, MI, USA) (Table S4A). We then integrated this data set with . They then isolated the influenza virus-resistant cell clones information on the viral and cellular interaction partners of viral and identified 110 human genes that contributed to the host cell proteins as reported by Ko¨ nig et al. (2010) and Shapira et al. resistance to viral infection, demonstrating the usefulness of this (2009) (Table S4B), resulting in a network of host-influenza virus RGHP system for genome-wide screening. interactions (Figures 1–3). In addition, this network included several subnetworks associated with specific biological pro- Pair-Wise Comparison among Six Independent cesses, such as translation initiation, processing of pre-mRNA, Genome-wide Screens for Human Genes Involved and proton-transporting ATPase functions. in Influenza Virus Replication In the six independent genome-wide screens described above Insights into Host Factors Involved in the Influenza Virus (Brass et al., 2009; Hao et al., 2008; Karlas et al., 2010; Ko¨ nig Life Cycle et al., 2010; Shapira et al., 2009; Sui et al., 2009), a total of Based on the analyses described above, influenza virus-host 1449 human genes were identified as potential host factors interactions can be mapped to individual steps of the influenza involved in influenza virus replication (including 110 Drosophila virus life cycle (Figure 4), as outlined below. genes that have human orthologs; Tables S1A and S1B), indi- Endocytosis, Fusion, and Uncoating cating that about 5.8% of human protein-coding genes may Once virus particles attach to a cell surface receptor, they are contribute to the life cycle of influenza virus. However, there is mainly internalized by clathrin-dependent endocytosis, although the possibility that these high-throughput screens contain false influenza virus also uses a nonclathrin-dependent endocytic positives. To narrow down the genes important for influenza viral pathway (Marsh and Helenius, 2006; Matlin et al., 1981; Rust replication, we analyzed the candidate genes identified in these et al., 2004; Sieczkarski and Whittaker, 2002). The importance screens by pair-wise comparison and found 128 human genes of both early and late endosomes in influenza virus entry has that affected influenza virus replication in at least two screens been demonstrated with dominant-negative forms of the (Table S2). The number of genes common between pairs of GTPases Rab5 and Rab7/protein kinase C b II (PKCbII), which screens is relatively small (from 0 to 32 genes in the 15 pair- regulate the functions of early and late endosomes, respectively wise comparisons). This low incidence of overlap has also (Sieczkarski et al., 2003; Sieczkarski and Whittaker, 2003). been observed in three RNAi screens designed to identify host Rab10 (RAB10), which controls the movement of endosomes factors for HIV-1 replication (Brass et al., 2008; Ko¨ nig et al., (Glodowski et al., 2007), was identified in two screens (Table S3). 2008; Zhou et al., 2008), and it could be due to differences in Moreover, four of the seven subunits of the coatomer 1 (COPI) screening systems (e.g., RNAi screen versus RHGP library vesicular transport complex (i.e., ARCN1, COPA, COPB2, and screen) or other conditions such as cell type, virus strains, and/ COPG), which is required for the formation of intermediate trans- or detection methods. Indeed, the number of common genes port vesicles between the early and late endosomes and for was highest in the comparison between the Ko¨ nig and Karlas retrograde Golgi-to-ER transport (Aniento et al., 1996; Cai screens (32 human genes) (Table S2), presumably because et al., 2007; Whitney et al., 1995), were identified in five indepen- both screens were based on RNAi and used the same human dent screens (Table S3), indicating the biological significance lung cells. In addition, the analysis of raw data, particularly the of this host cell machinery in the early steps of the influenza virus ‘‘cut-off’’ used to determine candidates, likely differed among life cycle. the studies. Nonetheless, a number of factors were identified in Uncoating of viral particles occurs upon fusion between the several screens (see below for more details), suggesting their viral and endosomal membranes, is mediated by HA, and leads importance in the influenza virus life cycle. to the release of vRNPs into the (Stegmann et al., 1990). The M2 ion channel protein functions in the endosomes Network Analysis of Influenza-Host Protein Interactions to lower the pH of virion interior, resulting in the disassociation For the set of 128 human genes that were identified in two or of the viral matrix from the vRNP complex, which more screens and are likely involved in influenza virus replication, can then be imported to the nucleus (Helenius, 1992). Proton- we next determined major gene categories. Our analysis with transporting V-type ATPase functions in both the acidification the PANTHER Classification System (Mi et al., 2005) revealed and fusion of the cellular compartments (Stevens and Forgac, several enriched gene categories associated with molecular 1997). Four of the six screens identified at least two subunits functions, such as nucleic acid-binding proteins, kinases, tran- of this V-type ATPase complex as host factors required for scription factors, ribosomal proteins, hydrogen transporters, influenza virus infection (Table S3). Although this complex was and proteins involved in mRNA splicing. Moreover, we found thought to be generally involved in the low pH-dependent enrichment for cellular proteins involved in biological processes membrane fusion between endosomes and virions, knockdown such as protein metabolism and modification, signal transduc- of ATP6V0D1 caused significant inhibition of influenza virus

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Figure 1. A Network of Host-Influenza Virus Interactions: Host and Viral Proteins Using GeneGo (GeneGo Inc, MI, USA), we analyzed the interactions of the 128 human genes identified to be involved in influenza virus replication in at least two genome-wide screens (Table S4A). We then integrated this information with data on host protein-virus interactions and interactions among viral proteins, as reported by Ko¨ nig et al. (2010) and Shapira et al. (2009) (Table S4B). An interaction network between host and viral proteins (i.e., PA, PB1, PB2, NP, HA, NA, M1, M2, NS1, NEP/NS2, and PB1-F2) was visualized by using Cytoscape (http://cytoscape.org/). Yellow and lilac nodes indicate influenza viral components and host proteins, respectively. Red circles indicate clusters associated with particular biological processes in the interaction networks, as identified by MCODE (‘‘Molecular Complex Detection’’) analysis (Bader and Hogue, 2003): (1) translation initiation, (2) pre-mRNA processing, and (3) proton-transporting V-type ATPase. production, but not VSV production, which also requires a low Transport of vRNP into the Host Nucleus pH for fusion (Hao et al., 2008). This finding implies a specific Replication/transcription of influenza virus genomic RNA occurs role for the complex in influenza virus replication. in the nucleus (reviewed in Engelhardt and Fodor, 2006; Palese,

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Figure 2. A Network of Host-Influenza Virus Interactions: Host and vRNP An interaction network between host and vRNP was analyzed and visualized in the same way described in Figure 1. Yellow and lilac nodes indicate vRNP of influenza virions and host proteins, respectively. Red circles indicate clusters associated with particular biological processes in the interaction networks, as iden- tified by MCODE analysis (Bader and Hogue, 2003): (1) translation initiation, (2) pre-mRNA processing, and (3) proton-transporting V-type ATPase.

2007). Therefore, the RNP complexes released from the tin b, and the cargo/importin a/b complex is transported into the incoming virus particles must be transported into the nucleus nucleus through the NPC. For influenza virus, RanBP5 (or im- through the nuclear pores. The nuclear import of RNP complexes portin 5), importin a1/a2, and importin a1/importin a5 have is dependent on the active import machinery of the host cell been shown to bind to PB1-PA dimers (Deng et al., 2006), NP nuclear pore complex (NPC) because of the size of these (O’Neill et al., 1995; Wang et al., 1997), and PB2 (Gabriel et al., complexes (10–15 nm wide and 30–120 nm long). In a classic 2008; Resa-Infante et al., 2008; Tarendeau et al., 2007, 2008), nuclear import pathway, importin a recognizes a cargo protein respectively. Our list of 128 genes includes the importin subunit containing nuclear localization signals (NLS) and binds to impor- b1(KPNB1) and nuclear pore complex proteins Nup98 and

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Nup153 (NUP98 and NUP153). Likely, these factors are involved synthesis, thereby enhancing translation of viral mRNAs (Chen in the nuclear import of influenza virus RNPs, as well as in the and Krug, 2000; Chen et al., 1999; Fortes et al., 1994; Nemeroff nuclear import of newly synthesized influenza viral proteins. et al., 1998; Qiu and Krug, 1994). GRSF-1 (the cellular RNA Genome Replication/Transcription and Translation recognition motif containing the RNA-binding protein G-rich Once the vRNP complexes are imported into the nucleus, the sequence factor 1) and P58IPK (the cellular inhibitor of PKR, an vRNA of influenza virus is transcribed into mRNA and replicated interferon-induced kinase that targets the eukaryotic translation via complementary RNA (cRNA) (reviewed in Engelhardt and initiation factor eIF2) appear to be involved in the selective Fodor, 2006; Palese, 2007). The viral polymerase subunits synthesis of influenza viral proteins (Goodman et al., 2007; (PB1, PB2, and PA) and NP catalyze both genome replication Kash et al., 2002; Park et al., 1999). Analysis with Reactome and transcription. Several host cellular molecules play a part in (Matthews et al., 2009) of the set of 128 genes that we identified these steps. Host factors shown to stimulate viral RNA synthesis above revealed several host genes involved in the formation of include BAT1 (or UAP56), heat shock protein (Hsp) 90, the mini- the translation initiation complex and the small ribosomal subunit maintenance (MCM) complex, Tat-SF1, and DNA- (i.e., FAU, EIF3S5, RPS3A, RPS4X, RPS10, RPS14, RPS5, dependent RNA polymerase II (Pol II) (Chan et al., 2006; RPS16, EIF4A2, RPS20, and EIF3S8). It is possible that some Engelhardt et al., 2005; Kawaguchi and Nagata, 2007; Momose of these host factors promote virus-specific translation by et al., 1996, 2001, 2002; Naito et al., 2007). BAT1, which is a inhibiting host cellular mRNA translation and/or stimulating viral splicing factor for mRNA and also functions in the nuclear export mRNA translation. of cellular mRNA, interacts with NP to facilitate the formation of vRNP Transport from the Nucleus to the Cytoplasm the NP-RNA complex. Similarly, Tat-SF1 facilitates NP-vRNP Newly synthesized vRNPs are exported from the nucleus to the complex formation (Naito et al., 2007). Viral RNA synthesis is cytoplasm (reviewed in Boulo et al., 2007). The viral matrix then initiated by the viral RNA polymerase (Momose et al., 1996, protein, M1, and nuclear export protein, NEP/NS2, have critical 2001). Hsp90 stimulates viral RNA synthesis through its interac- roles in this step. After M1 binds to the RNP complex, NEP/ tion with PB2 (Momose et al., 1996, 2002). MCM, which functions NS2 attaches to the M1/RNP complex through its C-terminal as a DNA replication fork helicase (Forsburg, 2004), plays a role in (Akarsu et al., 2003). NEP/NS2 can also bind to a host influenza viral genome replication by promoting the association cell nuclear export protein, CRM1 (Boulo et al., 2007; Elton between nascent cRNA and the viral polymerase complexes et al., 2001; Neumann et al., 2000). In addition, heat shock during the transition from initiation to elongation, thus stabilizing cognate (Hsc) 70 protein and the MAP kinase cascade may replication elongation complexes and allowing the synthesis of have roles in the nuclear export of vRNP complexes (Pleschka full-length cRNA (Kawaguchi and Nagata, 2007). As described et al., 2001; Watanabe et al., 2006). above, the yeast library screen identified Tat-SF1 as a factor Recently, phosphatidylinositol 3-kinase (PI3K) and its down- involved in vRNA-NP complex formation and viral RNA synthesis stream effector protein (the kinase Akt) were identified as host (Naito et al., 2007). In its active state, Pol II, which catalyzes the factors involved in influenza virus-induced signaling (Ehrhardt synthesis of mRNA precursors and most snRNA and microRNAs, and Ludwig, 2009; Ehrhardt et al., 2006; Hale et al., 2006; Shin is required for viral mRNA synthesis during the viral transcription et al., 2007; Zhirnov and Klenk, 2007). Consistent with this step (Chan et al., 2006; Engelhardt et al., 2005). Included in the finding, three genes involved in the PI3K/AKT signaling pathway 128 genes identified in our pair-wise comparison (Table S2) are (AKT1, MDM2, and IKBKE) were among the 128 genes identified genes associated with pre-mRNA splicing (i.e., PTBP1, NHP2L1, by the screens discussed here. Roles for this pathway in antiviral SNRP70, SF3B1, SF3A1, P14, and PRPF8), suggesting the activity and prevention of premature have been importance of the host mRNA splicing machinery for influenza reported (Ehrhardt et al., 2006, 2007; Sarkar et al., 2004). Of virus gene expression. Of interest, no splicing factors were found interest, a PI3K inhibitor obstructs the nuclear export of vRNPs, in the screen carried out in Drosophila cells (Hao et al., 2008), as well as viral RNA and protein synthesis (Shin et al., 2007). which is consistent with the finding that influenza virus mRNAs Assembly and Budding were not spliced in this system (Hao et al., 2008). In the late stage of the life cycle, the virion components, including Four screens identified NXF1 as an important host factor in vRNPs, the matrix protein M1, and the viral envelope proteins influenza virus replication (Table S3). NXF1 exports spliced (HA, NA, and M2) are transported to the assembly site, the apical cellular mRNAs but rarely exports unspliced cellular mRNAs plasma membrane in polarized epithelial cells (Nayak et al., (Reed and Cheng, 2005). Nonetheless, several viruses use the 2004). Relatively few host factors have been identified to be NXF1 pathway for the nuclear export of unspliced viral involved in this stage. Of the six genome-wide screens dis- (Cullen, 2003). For influenza virus, NP interacts with BAT1/ cussed in this review, only two studies (Karlas et al., 2010; UAP56 (Momose et al., 2001), which forms a bridge between Shapira et al., 2009) were designed to evaluate the assembly the NXF1/NXT1/Ref complex and the spliced mRNA. In addition, and budding steps. Depletion of subunits of the COPI complex NS1 binds NXF1 to suppress host mRNA export (Satterly et al., reduced HA expression on the cell surface relative to the total 2007), perhaps to retain host mRNAs in the nucleus for ‘‘cap- HA level in the screen by Brass et al. (2009) (Figure 4), suggesting snatching.’’ In addition, the requirement of NXF1 for nuclear that the COPI complex plays a role in the transport of influenza export of viral mRNAs has recently been shown (Read and viral glycoproteins (i.e., HA and NA) to the cell surface, in addition Digard, 2010). Thus, influenza virus may exploit the NXF1-medi- to its function in virus entry described above. The HA and NA ated RNA export pathway for multiple purposes. proteins contain signals for association with lipid rafts, which Influenza virus also uses the cellular machinery for mRNA are nonionic detergent-resistant lipid microdomains within the translation. Influenza virus infection shuts off host protein plasma membrane. Influenza viruses preferentially bud from

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Lipid raft

RAB11 RAB5 RAB7

RAB10 Endocytosis Protein transport PKCβII vRNP+M1+NS2, HA, NA, M2 ATP6AP1 ATP6AP2 COPI vesicular ATP6V0B V-type ATPase transport ATP6V0C proton transport Posttranslational ATP6V0D1 COPA modification COPB2 COPG ARCN1 ATP6V1A Processing of HA, NA ATP6V1B2 Endosome GRSF-1 P58IPK Golgi NUP98 NUP153 ER Translation

Nuclear pore complex Translation initiation Ribosome KPNB1 Nuclear import (vRNP) HSP90 MCM Tat-SF1 RPS16 Importin-5 PolII BAT1 EIF4A2 Importin alpha1/alpha2 NUP153 Importin alpha1/alpha5 RPS20 Genome replication EIF3S8 /transcription Nuclear pore complex FAU Pre-mRNA splicing Nuclear export EIF3S5 (viral mRNA, RNP) RPS3A PTBP1 RPS4X NHP2L1 PRPF8 P14 NXF1 SNRP70 SF3A1 SF3B1 AKT1 MDM2 IKBKE RPS10 RPS14 Nucleus Hsc70 CRM1 RPS5

Known host factors Host cellular biological process Flow of virus life cycle Host factors identified in screens Step of viral life cycle

Figure 4. Influenza Virus Life Cycle and Host Factors Upon infection, influenza virus is internalized by receptor-mediated endocytosis. After membrane fusion between the virus envelope and the endosome, the viral ribonucleoprotein (vRNP) complex is released into the cytoplasm and transported into the nucleus by the active import machinery of the host cell nuclear pore complex. Replication and transcription of the viral genome take place in the nucleus, and many host factors are believed to be involved in this process. Newly synthesized viral RNA associates with NP and forms the viral ribonucleoprotein (vRNP) complex together with viral polymerase proteins, which is then transported from the nucleus to the cytoplasm. HA and NA are processed posttranslationally during their transport from the ER to the Golgi apparatus. During assembly and budding, virion components are transported to the assembly site, the lipid raft microdomains at the apical plasma membrane of polarized epithelial cells. Progeny viruses then bud from the cells. The light-orange rectangles indicate individual steps of the influenza virus life cycle. The light-blue rectangles indicate host cellular processes that may be involved in the virus life cycle. Red circles indicate host factors identified in the screens discussed here, and yellow circles indicate host factors identified in other previous studies.

these lipid raft microdomains (Barman et al., 2001; Leser and McCown and Pekosz, 2005, 2006; Wu and Pekosz, 2008). NP Lamb, 2005; Nayak et al., 2004; Takeda et al., 2003; Zhang and M1 interact with host cytoskeletal components, such as et al., 2000). Although the precise mechanism of M1-vRNP actin, presumably facilitating vRNP transport to the budding complex transport to the cell surface is still unclear, the viral site (Avalos et al., 1997). In the final step of the life cycle, the viral M2 protein is thought to play a role in the incorporation of membrane components are concentrated in the microdomains M1-vRNPs into virus particles via an interaction between M1 of the lipid rafts, which may provide the stage for virus and the M2 cytoplasmic tail (Iwatsuki-Horimoto et al., 2006; release from the cell surface (Takeda et al., 2003). Although

Figure 3. A Network of Host-Influenza Virus Interactions: Host and Virions An interaction network between host and virions was analyzed and visualized in the same way described in Figure 1. Yellow and lilac nodes indicate influenza virions and host proteins, respectively. Red circles indicate clusters associated with particular biological processes in the interaction networks, as identified by MCODE analysis (Bader and Hogue, 2003): (1) translation initiation, (2) pre-mRNA processing, and (3) proton-transporting V-type ATPase.

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few host factors have been found that participate in the budding REFERENCES step, the actin cytoskeleton and Rab11, which belongs to the Rab of small GTPases, have been shown to contribute Akarsu, H., Burmeister, W.P., Petosa, C., Petit, I., Mu¨ ller, C.W., Ruigrok, R.W., and Baudin, F. (2003). structure of the M1 protein-binding domain to the budding of filamentous influenza virus particles (Bruce of the influenza A virus nuclear export protein (NEP/NS2). EMBO J. 22, et al., 2010; Roberts and Compans, 1998; Simpson-Holley 4646–4655. et al., 2002). Aniento, F., Gu, F., Parton, R.G., and Gruenberg, J. (1996). An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J. Cell Biol. 133, 29–41.

Perspectives and Concluding Remarks Avalos, R.T., Yu, Z., and Nayak, D.P. (1997). Association of influenza virus NP In 2003, the human completed its mission of and M1 proteins with cellular cytoskeletal elements in influenza virus-infected identifying the 20,000–25,000 genes in human DNA. To deter- cells. J. Virol. 71, 2947–2958. mine host gene involvement in virus replication, genome-wide Bader, G.D., and Hogue, C.W. (2003). An automated method for finding molec- screens, such as RNAi screens, offer an effective approach. ular complexes in large protein interaction networks. BMC Bioinformatics 4,2. For influenza virus, among six independent genome-wide Barman, S., Ali, A., Hui, E.K., Adhikary, L., and Nayak, D.P. (2001). Transport of screens, 1449 human genes, including 110 human orthologs viral proteins to the apical membranes and interaction of matrix protein with identified by a screen in Drosophila cells, have been identified glycoproteins in the assembly of influenza viruses. Virus Res. 77, 61–69. as host factors involved in influenza virus replication; of these, Boulo, S., Akarsu, H., Ruigrok, R.W., and Baudin, F. (2007). Nuclear traffic of 128 genes were found in at least two screens. Bioinformatics influenza virus proteins and ribonucleoprotein complexes. Virus Res. 124, analysis of these 128 human genes revealed gene clusters 12–21. related to host cellular functions, such as endocytosis, transla- Brass, A.L., Dykxhoorn, D.M., Benita, Y., Yan, N., Engelman, A., Xavier, R.J., tion initiation, and nuclear transport, all of which can be con- Lieberman, J., and Elledge, S.J. (2008). Identification of host proteins required nected to the influenza viral life cycle. Figure 4 illustrates for HIV infection through a functional genomic screen. Science 319, 921–926. influenza virus-host interactions mapped to individual steps of Brass, A.L., Huang, I.C., Benita, Y., John, S.P., Krishnan, M.N., Feeley, E.M., the influenza virus life cycle based on the known functions of Ryan, B.J., Weyer, J.L., van der Weyden, L., Fikrig, E., et al. (2009). The IFITM the identified host proteins. However, it should be borne in proteins mediate cellular resistance to influenza A H1N1 virus, , and . Cell 139, 1243–1254. mind that these proteins may be involved in other steps of the viral life cycle with as yet undetermined functions. Bright, R.A., Medina, M.J., Xu, X., Perez-Oronoz, G., Wallis, T.R., Davis, X.M., Povinelli, L., Cox, N.J., and Klimov, A.I. (2005). Incidence of adamantane resis- Systematic, genome-wide RNAi screens are powerful tools for tance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: identifying host genes important for viral replication. However, a cause for concern. Lancet 366, 1175–1181. they do have limitations; for example, the RNAi library does not Bright, R.A., Shay, D.K., Shu, B., Cox, N.J., and Klimov, A.I. (2006). Adaman- yet cover all of the human genes, the knockdown efficiency of tane resistance among influenza A viruses isolated early during the 2005-2006 target gene expression could vary among siRNAs, and genes influenza season in the United States. JAMA 295, 891–894. whose siRNA knockdown leads to cytotoxicity would be elimi- Bruce, E.A., Digard, P., and Stuart, A.D. (2010). The Rab11 pathway is required nated from a ‘‘hit list’’ even though they may be important for viral for influenza A viral budding and filament formation. J. Virol. 84, 5848–5859. replication, resulting in a number of false positives and false negatives. Therefore, the identification of host gene candidates Cai, H., Reinisch, K., and Ferro-Novick, S. (2007). Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport involved in viral replication by any primary screen really repre- vesicle. Dev. Cell 12, 671–682. sents no more than a starting point for exploration of such host Chan, A.Y., Vreede, F.T., Smith, M., Engelhardt, O.G., and Fodor, E. (2006). factors. More detailed functional analyses of human genes iden- Influenza virus inhibits RNA polymerase II elongation. Virology 351, 210–217. tified in genome-wide screens will allow us to find novel cellular pathways and/or host gene sets important for influenza virus Chen, Z., and Krug, R.M. (2000). Selective nuclear export of viral mRNAs in influenza-virus-infected cells. Trends Microbiol. 8, 376–383. replication, leading to a greater understanding of the precise mechanisms of influenza virus replication. Chen, Z., Li, Y., and Krug, R.M. (1999). Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 30-end processing machinery. EMBO J. 18, 2273–2283.

SUPPLEMENTAL INFORMATION Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O’Neill, R., Schickli, J., Palese, P., et al. (2001). A novel influenza A virus mito- Supplemental Information includes four tables and can be found with this chondrial protein that induces cell death. Nat. Med. 7, 1306–1312. article online at doi:10.1016/j.chom.2010.05.008. Cherry, S., Doukas, T., Armknecht, S., Whelan, S., Wang, H., Sarnow, P., and Perrimon, N. (2005). Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. Genes Dev. 19, ACKNOWLEDGMENTS 445–452.

Coley, W., Kehn-Hall, K., Van Duyne, R., and Kashanchi, F. (2009). Novel HIV-1 We thank Drs. Gabriele Neumann and Masato Hatta for comments on the therapeutics through targeting altered host cell pathways. Expert Opin. Biol. manuscript and Dr. Susan Watson for editing the manuscript. We also thank Ther. 9, 1369–1382. Drs. Yo Suzuki and Yoshinori Tomoyasu for valuable advice. This work was supported, in part, by U.S. National Institute of and Infectious Diseases Conenello, G.M., Zamarin, D., Perrone, L.A., Tumpey, T., and Palese, P. (2007). Public Health Service research grants, by a grant-in-aid for Specially A single in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses Promoted Research (Japan), by funding from the Program of Founding contributes to increased . PLoS Pathog. 3, 1414–1421. Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology (Japan), Cullen, B.R. (2003). Nuclear mRNA export: insights from virology. Trends Bio- and by ERATO (Japan Science and Technology Agency). chem. Sci. 28, 419–424.

436 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. Cell Host & Microbe Review

Davies, W.L., Grunert, R.R., Haff, R.F., McGahen, J.W., Neumayer, E.M., Paul- Iwatsuki-Horimoto, K., Horimoto, T., Noda, T., Kiso, M., Maeda, J., Watanabe, shock, M., Watts, J.C., Wood, T.R., Hermann, E.C., and Hoffmann, C.E. (1964). S., Muramoto, Y., Fujii, K., and Kawaoka, Y. (2006). The cytoplasmic tail of the Antiviral Activity of 1-Adamantanamine (Amantadine). Science 144, 862–863. influenza A virus M2 protein plays a role in viral assembly. J. Virol. 80, 5233–5240. Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., Lindstrom, S., Garten, R.J., Gu- bareva, L.V., Xu, X., Bridges, C.B., and Uyeki, T.M.; Novel Swine-Origin Influ- Jorba, N., Juarez, S., Torreira, E., Gastaminza, P., Zamarren˜ o, N., Albar, J.P., enza A (H1N1) Virus Investigation Team. (2009). Emergence of a novel swine- and Ortı´n, J. (2008). Analysis of the interaction of influenza virus polymerase origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 360, 2605–2615. complex with human cell factors. Proteomics 8, 2077–2088.

Deng, T., Engelhardt, O.G., Thomas, B., Akoulitchev, A.V., Brownlee, G.G., Karlas, A., Machuy, N., Shin, Y., Pleissner, K.P., Artarini, A., Heuer, D., Becker, and Fodor, E. (2006). Role of ran binding protein 5 in nuclear import and D., Khalil, H., Ogilvie, L.A., Hess, S., et al. (2010). Genome-wide RNAi screen assembly of the influenza virus RNA polymerase complex. J. Virol. 80, identifies human host factors crucial for influenza virus replication. 11911–11919. 463, 818–822.

Ehrhardt, C., and Ludwig, S. (2009). A new player in a deadly game: influenza Kash, J.C., Cunningham, D.M., Smit, M.W., Park, Y., Fritz, D., Wilusz, J., and viruses and the PI3K/Akt signalling pathway. Cell. Microbiol. 11, 863–871. Katze, M.G. (2002). Selective translation of eukaryotic mRNAs: functional molecular analysis of GRSF-1, a positive regulator of influenza virus protein Ehrhardt, C., Marjuki, H., Wolff, T., Nu¨ rnberg, B., Planz, O., Pleschka, S., and synthesis. J. Virol. 76, 10417–10426. Ludwig, S. (2006). Bivalent role of the phosphatidylinositol-3-kinase (PI3K) during influenza virus infection and host cell defence. Cell. Microbiol. 8, Kawaguchi, A., and Nagata, K. (2007). De novo replication of the influenza virus 1336–1348. RNA genome is regulated by DNA replicative helicase, MCM. EMBO J. 26, 4566–4575. Ehrhardt, C., Wolff, T., Pleschka, S., Planz, O., Beermann, W., Bode, J.G., Schmolke, M., and Ludwig, S. (2007). Influenza A virus NS1 protein activates Ko¨ nig, R., Zhou, Y., Elleder, D., Diamond, T.L., Bonamy, G.M., Irelan, J.T., the PI3K/Akt pathway to mediate antiapoptotic signaling responses. J. Virol. Chiang, C.Y., Tu, B.P., De Jesus, P.D., Lilley, C.E., et al. (2008). Global analysis 81, 3058–3067. of host- interactions that regulate early-stage HIV-1 replication. Cell 135, 49–60. Elton, D., Simpson-Holley, M., Archer, K., Medcalf, L., Hallam, R., McCauley, J., and Digard, P. (2001). Interaction of the influenza virus nucleoprotein with Ko¨ nig, R., Stertz, S., Zhou, Y., Inoue, A., Hoffmann, H.H., Bhattacharyya, S., the cellular CRM1-mediated nuclear export pathway. J. Virol. 75, 408–419. Alamares, J.G., Tscherne, D.M., Ortigoza, M.B., Liang, Y., et al. (2010). Human host factors required for influenza virus replication. Nature 463, Engelhardt, O.G., and Fodor, E. (2006). Functional association between viral 813–817. and cellular transcription during influenza virus infection. Rev. Med. Virol. 16, 329–345. Krishnan, M.N., Ng, A., Sukumaran, B., Gilfoy, F.D., Uchil, P.D., Sultana, H., Brass, A.L., Adametz, R., Tsui, M., Qian, F., et al. (2008). RNA interference Engelhardt, O.G., Smith, M., and Fodor, E. (2005). Association of the influenza screen for human genes associated with West Nile virus infection. Nature A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. 455, 242–245. J. Virol. 79, 5812–5818. Kushner, D.B., Lindenbach, B.D., Grdzelishvili, V.Z., Noueiry, A.O., Paul, S.M., Forsburg, S.L. (2004). Eukaryotic MCM proteins: beyond replication initiation. and Ahlquist, P. (2003). Systematic, genome-wide identification of host genes Microbiol. Mol. Biol. Rev. 68, 109–131. affecting replication of a positive-strand RNA virus. Proc. Natl. Acad. Sci. USA 100, 15764–15769. Fortes, P., Beloso, A., and Ortı´n, J. (1994). Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. Leser, G.P., and Lamb, R.A. (2005). Influenza virus assembly and budding in 13, 704–712. raft-derived microdomains: a quantitative analysis of the surface distribution of HA, NA and M2 proteins. Virology 342, 215–227. Gabriel, G., Herwig, A., and Klenk, H.D. (2008). Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of Li, Q., Brass, A.L., Ng, A., Hu, Z., Xavier, R.J., Liang, T.J., and Elledge, S.J. influenza A virus. PLoS Pathog. 4, e11. (2009). A genome-wide genetic screen for host factors required for C virus propagation. Proc. Natl. Acad. Sci. USA 106, 16410–16415. Glodowski, D.R., Chen, C.C., Schaefer, H., Grant, B.D., and Rongo, C. (2007). RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent Marsh, M., and Helenius, A. (2006). Virus entry: open sesame. Cell 124, endocytosis pathway. Mol. Biol. Cell 18, 4387–4396. 729–740.

Goff, S.P. (2008). Knockdown screens to knockout HIV-1. Cell 135, 417–420. Matlin, K.S., Reggio, H., Helenius, A., and Simons, K. (1981). Infectious entry pathway of influenza virus in a canine kidney cell line. J. Cell Biol. 91, Goodman, A.G., Smith, J.A., Balachandran, S., Perwitasari, O., Proll, S.C., 601–613. Thomas, M.J., Korth, M.J., Barber, G.N., Schiff, L.A., and Katze, M.G. (2007). The cellular protein P58IPK regulates influenza virus mRNA translation Matthews, L., Gopinath, G., Gillespie, M., Caudy, M., Croft, D., de Bono, B., and replication through a PKR-mediated mechanism. J. Virol. 81, 2221–2230. Garapati, P., Hemish, J., Hermjakob, H., Jassal, B., et al. (2009). Reactome knowledgebase of human biological pathways and processes. Nucleic Acids Hale, B.G., Jackson, D., Chen, Y.H., Lamb, R.A., and Randall, R.E. (2006). Res. 37(Database issue), D619–D622. Influenza A virus NS1 protein binds p85beta and activates phosphatidylinosi- tol-3-kinase signaling. Proc. Natl. Acad. Sci. USA 103, 14194–14199. Mayer, D., Molawi, K., Martı´nez-Sobrido, L., Ghanem, A., Thomas, S., Baginsky, S., Grossmann, J., Garcı´a-Sastre, A., and Schwemmle, M. (2007). Hale, B.G., Randall, R.E., Ortı´n, J., and Jackson, D. (2008). The multifunctional Identification of cellular interaction partners of the influenza virus ribo- NS1 protein of influenza A viruses. J. Gen. Virol. 89, 2359–2376. nucleoprotein complex and polymerase complex using proteomic-based approaches. J. Res. 6, 672–682. Hao, L., Sakurai, A., Watanabe, T., Sorensen, E., Nidom, C.A., Newton, M.A., Ahlquist, P., and Kawaoka, Y. (2008). Drosophila RNAi screen identifies host McAuley, J.L., Hornung, F., Boyd, K.L., Smith, A.M., McKeon, R., Bennink, J., genes important for influenza virus replication. Nature 454, 890–893. Yewdell, J.W., and McCullers, J.A. (2007). Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial Hayden, F.G. (2001). Perspectives on antiviral use during pandemic influenza. . Cell Host Microbe 2, 240–249. Philos. Trans. R. Soc. Lond. B Biol. Sci. 356, 1877–1884. McCown, M.F., and Pekosz, A. (2005). The influenza A virus M2 cytoplasmic Helenius, A. (1992). Unpacking the incoming influenza virus. Cell 69, 577–578. tail is required for infectious virus production and efficient genome packaging. J. Virol. 79, 3595–3605. Huarte, M., Sanz-Ezquerro, J.J., Roncal, F., Ortı´n, J., and Nieto, A. (2001). PA subunit from influenza virus polymerase complex interacts with a cellular McCown, M.F., and Pekosz, A. (2006). Distinct domains of the influenza a virus protein with homology to a family of transcriptional activators. J. Virol. 75, M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate 8597–8604. infectious virus production. J. Virol. 80, 8178–8189.

Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. 437 Cell Host & Microbe Review

Mello, C.C., and Conte, D., Jr. (2004). Revealing the world of RNA interference. Rust, M.J., Lakadamyali, M., Zhang, F., and Zhuang, X. (2004). Assembly of Nature 431, 338–342. endocytic machinery around individual influenza viruses during viral entry. Nat. Struct. Mol. Biol. 11, 567–573. Mi, H., Lazareva-Ulitsky, B., Loo, R., Kejariwal, A., Vandergriff, J., Rabkin, S., Guo, N., Muruganujan, A., Doremieux, O., Campbell, M.J., et al. (2005). The Sarkar, S.N., Peters, K.L., Elco, C.P., Sakamoto, S., Pal, S., and Sen, G.C. PANTHER database of protein families, subfamilies, functions and pathways. (2004). Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in Nucleic Acids Res. 33(Database issue), D284–D288. double-stranded RNA signaling. Nat. Struct. Mol. Biol. 11, 1060–1067.

Momose, F., Handa, H., and Nagata, K. (1996). Identification of host factors Satterly, N., Tsai, P.L., van Deursen, J., Nussenzveig, D.R., Wang, Y., Faria, that regulate the influenza virus RNA polymerase activity. Biochimie 78, P.A., Levay, A., Levy, D.E., and Fontoura, B.M. (2007). Influenza virus targets 1103–1108. the mRNA export machinery and the nuclear pore complex. Proc. Natl. Acad. Sci. USA 104, 1853–1858. Momose, F., Basler, C.F., O’Neill, R.E., Iwamatsu, A., Palese, P., and Nagata, K. (2001). Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with Shapira, S.D., Gat-Viks, I., Shum, B.O., Dricot, A., de Grace, M.M., Wu, L., the influenza virus nucleoprotein and enhances viral RNA synthesis. J. Virol. Gupta, P.B., Hao, T., Silver, S.J., Root, D.E., et al. (2009). A physical and regu- 75, 1899–1908. latory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 139, 1255–1267. Momose, F., Naito, T., Yano, K., Sugimoto, S., Morikawa, Y., and Nagata, K. (2002). Identification of Hsp90 as a stimulatory host factor involved in influenza Shin, Y.K., Liu, Q., Tikoo, S.K., Babiuk, L.A., and Zhou, Y. (2007). Effect of the virus RNA synthesis. J. Biol. Chem. 277, 45306–45314. phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation. J. Gen. Virol. 88, 942–950. Nagata, K., Kawaguchi, A., and Naito, T. (2008). Host factors for replication and transcription of the influenza virus genome. Rev. Med. Virol. 18, 247–260. Sieczkarski, S.B., and Whittaker, G.R. (2002). Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J. Virol. 76, Naito, T., Kiyasu, Y., Sugiyama, K., Kimura, A., Nakano, R., Matsukage, A., and 10455–10464. Nagata, K. (2007). An influenza virus system in yeast identified Tat- SF1 as a stimulatory host factor for viral RNA synthesis. Proc. Natl. Acad. Sieczkarski, S.B., and Whittaker, G.R. (2003). Differential requirements of Sci. USA 104, 18235–18240. Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic 4, 333–343. Nayak, D.P., Hui, E.K., and Barman, S. (2004). Assembly and budding of influ- enza virus. Virus Res. 106, 147–165. Sieczkarski, S.B., Brown, H.A., and Whittaker, G.R. (2003). Role of protein kinase C betaII in influenza virus entry via late endosomes. J. Virol. 77, Nemeroff, M.E., Barabino, S.M., Li, Y., Keller, W., and Krug, R.M. (1998). Influ- 460–469. enza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 30end formation of cellular pre-mRNAs. Mol. Cell 1, 991–1000. Simpson-Holley, M., Ellis, D., Fisher, D., Elton, D., McCauley, J., and Digard, P. (2002). A functional link between the actin cytoskeleton and lipid rafts during Neumann, G., Hughes, M.T., and Kawaoka, Y. (2000). Influenza A virus NS2 budding of filamentous influenza virions. Virology 301, 212–225. protein mediates vRNP nuclear export through NES-independent interaction Skehel, J.J., and Wiley, D.C. (2000). Receptor binding and membrane fusion in with hCRM1. EMBO J. 19, 6751–6758. virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569. Nicoll, A., Ciancio, B., and Kramarz, P.; Influenza Project Team. (2008). Stegmann, T., White, J.M., and Helenius, A. (1990). Intermediates in influenza Observed oseltamivir resistance in seasonal influenza viruses in Europe inter- induced membrane fusion. EMBO J. 9, 4231–4241. pretation and potential implications. Euro Surveill. 31, 13. Stevens, T.H., and Forgac, M. (1997). Structure, function and regulation of the O’Neill, R.E., Jaskunas, R., Blobel, G., Palese, P., and Moroianu, J. (1995). vacuolar (H+)-ATPase. Annu. Rev. Cell Dev. Biol. 13, 779–808. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J. Biol. Chem. 270, Sui, B., Bamba, D., Weng, K., Ung, H., Chang, S., Van Dyke, J., Goldblatt, M., 22701–22704. Duan, R., Kinch, M.S., and Li, W.B. (2009). The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza. Virology Palese P., ed. (2007). Orthomixoviridae, Fifth Edition (Philadelphia, PA: Lippin- 387, 473–481. cott Williams & Wilkins). Takeda, M., Leser, G.P., Russell, C.J., and Lamb, R.A. (2003). Influenza virus Panavas, T., Serviene, E., Brasher, J., and Nagy, P.D. (2005). Yeast genome- hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. wide screen reveals dissimilar sets of host genes affecting replication of RNA Proc. Natl. Acad. Sci. USA 100, 14610–14617. viruses. Proc. Natl. Acad. Sci. USA 102, 7326–7331. Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M., Boulo, S., Park, Y.W., Wilusz, J., and Katze, M.G. (1999). Regulation of eukaryotic protein Baudin, F., Ruigrok, R.W., Daigle, N., Ellenberg, J., et al. (2007). Structure and synthesis: selective influenza viral mRNA translation is mediated by the cellular nuclear import function of the C-terminal domain of influenza virus polymerase RNA-binding protein GRSF-1. Proc. Natl. Acad. Sci. USA 96, 6694–6699. PB2 subunit. Nat. Struct. Mol. Biol. 14, 229–233.

Pleschka, S., Wolff, T., Ehrhardt, C., Hobom, G., Planz, O., Rapp, U.R., and Tarendeau, F., Crepin, T., Guilligay, D., Ruigrok, R.W., Cusack, S., and Hart, Ludwig, S. (2001). Influenza virus propagation is impaired by inhibition of the D.J. (2008). Host determinant residue lysine 627 lies on the surface of Raf/MEK/ERK signalling cascade. Nat. Cell Biol. 3, 301–305. a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog. 4, e1000136. Qiu, Y., and Krug, R.M. (1994). The influenza virus NS1 protein is a poly(A)- binding protein that inhibits nuclear export of mRNAs containing poly(A). Wang, P., Palese, P., and O’Neill, R.E. (1997). The NPI-1/NPI-3 (karyopherin J. Virol. 68, 2425–2432. alpha) binding site on the influenza a virus nucleoprotein NP is a nonconven- tional nuclear localization signal. J. Virol. 71, 1850–1856. Read, E.K., and Digard, P. (2010). Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export. Watanabe, K., Fuse, T., Asano, I., Tsukahara, F., Maru, Y., Nagata, K., J. Gen. Virol. 91, 1290–1301. Kitazato, K., and Kobayashi, N. (2006). Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle. FEBS Reed, R., and Cheng, H. (2005). TREX, SR proteins and export of mRNA. Curr. Lett. 580, 5785–5790. Opin. Cell Biol. 17, 269–273. Webster, R.G., and Govorkova, E.A. (2006). H5N1 influenza—continuing Resa-Infante, P., Jorba, N., Zamarren˜ o, N., Ferna´ ndez, Y., Jua´ rez, S., and and spread. N. Engl. J. Med. 355, 2174–2177. Ortı´n, J. (2008). The host-dependent interaction of alpha-importins with influenza PB2 polymerase subunit is required for virus RNA replication. Whitney, J.A., Gomez, M., Sheff, D., Kreis, T.E., and Mellman, I. (1995). Cyto- PLoS ONE 3, e3904. plasmic coat proteins involved in endosome function. Cell 83, 703–713.

Roberts, P.C., and Compans, R.W. (1998). Host cell dependence of viral Wise, H.M., Foeglein, A., Sun, J., Dalton, R.M., Patel, S., Howard, W., morphology. Proc. Natl. Acad. Sci. USA 95, 5746–5751. Anderson, E.C., Barclay, W.S., and Digard, P. (2009). A complicated

438 Cell Host & Microbe 7, June 17, 2010 ª2010 Elsevier Inc. Cell Host & Microbe Review

message: Identification of a novel PB1-related protein translated from influ- Zhang, J., Pekosz, A., and Lamb, R.A. (2000). Influenza virus assembly and enza A virus segment 2 mRNA. J. Virol. 83, 8021–8031. lipid raft microdomains: a role for the cytoplasmic tails of the spike glycopro- teins. J. Virol. 74, 4634–4644. Wu, W.H., and Pekosz, A. (2008). Extending the cytoplasmic tail of the influ- enza a virus M2 protein leads to reduced virus replication in vivo but not Zhirnov, O.P., and Klenk, H.D. (2007). Control of apoptosis in influenza virus- in vitro. J. Virol. 82, 1059–1063. infected cells by up-regulation of Akt and signaling. Apoptosis 12, 1419–1432. Yen, H.L., and Webster, R.G. (2009). Pandemic influenza as a current threat. Curr. Top. Microbiol. Immunol. 333, 3–24. Zhou, H., Xu, M., Huang, Q., Gates, A.T., Zhang, X.D., Castle, J.C., Stec, E., Ferrer, M., Strulovici, B., Hazuda, D.J., and Espeseth, A.S. (2008). Genome- Zamarin, D., Ortigoza, M.B., and Palese, P. (2006). Influenza A virus PB1-F2 scale RNAi screen for host factors required for HIV replication. Cell Host protein contributes to in mice. J. Virol. 80, 7976–7983. Microbe 4, 495–504.

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