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Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Review
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Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity
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Ariel Rodriguez-Frandsen a,b,1,2 , Roberto Alfonso a,b,1,3 , Amelia Nieto a,b,∗ a b
Centro Nacional de Biotecnología. C.S.I.C. Darwin 3, Cantoblanco, 28049 Madrid, Spain Ciber de Enfermedades Respiratorias, Mallorca, Illes Balears, Spain
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Article history: Received 14 September 2014 Received in revised form 25 March 2015 Accepted 26 March 2015 Available online xxx
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Keywords: Influenza virus Host adaptation Pathogenicity Viral polymerase RNA polymerase II Virulence
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The viral polymerase is an essential complex for the influenza virus life cycle as it performs the viral RNA transcription and replication processes. To that end, the polymerase carries out a wide array of functions and associates to a large number of cellular proteins. Due to its importance, recent studies have found numerous mutations in all three polymerase protein subunits contributing to virus host range and pathogenicity. In this review, we will point out viral polymerase polymorphisms that have been associated with virus adaptation to mammalian hosts, increased viral polymerase activity and virulence. Furthermore, we will summarize the current knowledge regarding the new set of proteins expressed from the viral polymerase genes and their contribution to infection. In addition, the mechanisms used by the virus to counteract the cellular immune response in which the viral polymerase complex or its subunits are involved will be highlighted. Finally, the degradative process induced by the viral polymerase on the cellular transcription machinery and its repercussions on virus pathogenicity will be of particular interest. © 2015 Published by Elsevier B.V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Structure and function of the influenza polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Polymerase associated host factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host range and pathogenicity determinants on influenza polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. PB2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Residue 627 as a determinant of host adaptation and transmissibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Correlation between adaptation and polymerase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Role of other PB2 residues in adaptation, transmissibility and pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. PB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. PA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recently discovered protein expressed from IAV polymerase genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. PB1-F2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. N40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. PA-derived proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of cellular response to infection by IAV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Inhibition of IFN responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗ Corresponding author at: Centro Nacional de Biotecnología, C.S.I.C., Darwin 3, Cantoblanco, 28049 Madrid, Spain. Tel.: +34 91 5854914; fax: +34 91 5854506. E-mail address: [email protected] (A. Nieto). 1 Both author equally contributed to this work. 2 Present address: Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America. 3 Present address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. http://dx.doi.org/10.1016/j.virusres.2015.03.017 0168-1702/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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4.2. Inhibition of cellular transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
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The influenza A virus (IAV) is an important respiratory human pathogen causing yearly recurrent seasonal epidemics with an average global burden of >600 million cases (www.who.int). In rare instances IAV can also spread from its natural zoonotic reservoirs (aquatic birds) to cross species barriers and transmit to humans where it can evolve into strains that cause diseases ranging from mild to severe, with occasional widespread distribution known as pandemic. In the past century, the most devastating pandemic took place in 1918–1920 (also known as the “1918 flu” or “Spanish flu”), infecting hundreds of millions and killing between 20 and 50 million people worldwide (Johnson and Mueller, 2002; Taubenberger and Morens, 2006). Two additional pandemic events originating in Asia took place during the second half of the 20th century, the so-called Asian H2N2 pandemic of 1957 (1 million deaths) and the Hong Kong H3N2 pandemic in 1968 (1 million deaths). Although of relatively low virulence, the recent H1N1 influenza 2009 pandemic of swine origin emerged unexpectedly in Mexico to spread around the world in just a few months. IAV naturally infects wild aquatic birds making up an extremely heterogeneous population which includes many possible combinations between the two surfaces glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA); a total of 18 different HA and 11 NA have been recognized. At present there are concerns that avian influenza strains, including the highly pathogenic influenza viruses H5N1 and the novel H7N9 subtypes not yet capable of spreading from one human to another, could adapt and become more easily transmissible among humans. The ability of influenza A viruses to infect a variety of hosts is based on their genetic diversity due to two main reasons: (i) their RNA polymerase is error-prone, and (ii) they contain a segmented genome, which allows for exchange of RNA segments between genotypically diverse influenza viruses. These features lead to the rapid generation of novel strains and subtypes and thus contribute to the constant threat that newly emerging and re-emerging influenza viruses pose to the human population.
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1.1. Structure and function of the influenza polymerase
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IAV, a member of the Orthomyxoviridae family, possesses a negative-sense single-stranded RNA genome (vRNA) divided into eight segments. vRNAs are protected in a structure known as viral ribonucleoprotein (vRNP), in which the RNA strand is wrapped by the nucleoprotein (NP), and a single viral polymerase complex interacts with the complementary 3 and 5 genomic end sequences. Recent cryo-EM reconstructions of vRNPs obtained from different sources show a double-helical stem structure in which the NP proteins and the protected viral RNA form two anti-parallel strands (Arranz et al., 2012; Moeller et al., 2012). Both strands are connected by a short loop at one end of the particle and interact with the viral polymerase at the other end. The incoming parental vRNPs are released into the cytoplasm of the infected host cell and then quickly transported into the nucleus. IAV is a rare RNA-genome virus in that expression and amplification of viral genomes take place inside the infected cell nucleus and it therefore heavily depends on host nucleocytoplasmic trafficking and nuclear functions. Within the host nucleus, cellular insoluble fractions, such as nuclear matrix and chromatin structures, have been shown to encompass part of the viral polymerase transcription and
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replication activity and act as a platform for the release of progeny vRNPs outside the nucleus (Chase et al., 2011; Garcia-Robles et al., 2005; Lopez-Turiso et al., 1990; Takizawa et al., 2006). The IAV genome encodes for 10 major proteins, although alternative protein products have been characterized from several genome segments (see below). The complete coding capacity of the IAV genome is far from known and processes like splicing, protein truncations, and the use of alternative initiation codons or overlapping frames are known to increase the diversity of proteins generated during infection. The widespread synthesis of small viral non-coding RNAs with largely unknown roles in the outcome of infection further complicates the understanding of IAV full expression capacity (Perez et al., 2010, 2012). The three largest viral genome segments encode for the polymerase heterotrimeric complex, responsible for the RNA-dependent RNA polymerase activity of the virus (Fig. 1). The three polymerase subunits, named PA, PB1 and PB2, together with the nucleoprotein, form the minimum set of viral proteins required for viral RNA transcription and replication (reviewed in (Fodor, 2013; Resa-Infante et al., 2011)). Viral transcription depends on primers of host origin obtained through a cap-snatching process targeting newly synthesized host pre-mRNAs (Krug et al., 1979). The viral positive-sense mRNA also includes a 3 polyA tail generated by repetitive polymerization of a polyU track on the genomic vRNP, thus creating a transcript that is structurally undistinguished from host mRNAs. The vRNP undergoing transcription is processed by a cis-acting polymerase (Jorba et al., 2009). The requirement of newly synthesized cellular pre-mRNAs as a source of cap-olignonucleotides for viral transcription initiation involves a functional association between the viral and the cellular transcription machineries, which has significant consequences for viral outcome (see below). A switch from viral transcription to replication is required during a successful infectious cycle. The action of viral short virion RNAs (svRNAs) and the availability of cellular nucleotides and newly synthesized viral polymerase and NP, have been involved in this process (Jorba et al., 2009; Perez et al., 2010; Vreede and Brownlee, 2007; Vreede et al., 2004). The viral replication step is initiated early during infection with the synthesis of the cRNA, a complete unpolyadenylated positive-polarity copy of the vRNA (Hay et al., 1977). cRNAs within the infected host cell form cRNPs structures, and serve as templates for the synthesis of new vRNPs which are ready for either further rounds of viral gene expression or their transportation outside the nucleus and subsequent encapsidation. As opposed to what happens with the viral mRNA, the production of cRNAs is only started after viral protein synthesis has begun and seems to require a soluble trans-acting polymerase different from the one resident in the RNP (Hay et al., 1977; Jorba et al., 2009). Basic protein 1, PB1, is the core of the complex, the most conserved of the polymerase subunits, and contains the enzymatic motifs needed for RNA polymerization activity (Kobayashi et al., 1996). The PB2 subunit has a key role in viral transcription due to its recognition and binding of host 5 mRNA cap structures generated by the cellular transcription machinery (Blaas et al., 1982; Braam et al., 1983). Acidic protein PA has an endonucleolytic activity needed for the viral cap-snatching process (Dias et al., 2009; Yuan et al., 2009). In recent years some parts of the three proteins have been structurally characterized including important
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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Fig. 1. Influenza A virus polymerase complex. PA, PB1 and PB2 subunits are depicted as bars with the amino and carboxy-terminal ends indicated. The putative functional domains (Endonuclease, RdRP and Cap-binding) and the interaction regions between subunits are also highlighted. The NLS regions described for each protein are represented by blue boxes.
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functional domains, such as the cap-binding site on PB2 (Guilligay et al., 2008), the carboxy-terminal part of PB2 that contains the nuclear localization sequence (NLS) domain and important host range determinants (Tarendeau et al., 2007; Tarendeau et al., 2008), and the endonuclease domain at the N-terminus of PA (Dias et al., 2009; Yuan et al., 2009). Additionally, crystal structures of the interactions between the carboxy-terminal part of PA and the first 15 amino-terminal amino acids of PB1 (He et al., 2008), and between the last 72 amino acids of carboxy-terminal PB1 and the first 35 amino-terminal amino acids of protein PB2 (Sugiyama et al., 2009), have been obtained. Furthermore the cryoEM reconstructions of the vRNP, as well as other structural analyses of the viral polymerase out of the context of the RNP, illustrate this heterotrimer complex as a compact structure with some indications of flexibility (Area et al., 2004; Arranz et al., 2012; Coloma et al., 2009; Moeller et al., 2012; Resa-Infante et al., 2010; Torreira et al., 2007). In agreement with that, the crystal structures of the complete RNA polymerase complexes from influenza A and B viruses show that the three subunits have multiple interactions with each other and all of them participate in the binding of the two strands of the RNA promoter (Pflug et al., 2014; Reich et al., 2014). Overall, the viral polymerase is a major component of the virus as it provides the necessary machinery to translate its genome and replicate itself. In addition to its basic functions on viral genome expression, the viral polymerase has a key role in transmission between hosts and pathogenesis. For instance, a viral polymerase with higher activity usually correlates with an increased ability of transmission and adaptability, as well as higher pathogenicity (Naffakh et al., 2008). In this review, we highlight those mutations in IAV polymerase that have been associated with higher virus adaptability to a new host, increased viral polymerase activity and pathogenicity. Additionally, some of the host cellular mechanisms that have been linked to these processes are discussed. Special attention is paid to the consequences derived from the functional association between viral and cellular transcription machineries as well as the detrimental effect caused by the degradative process induced by the viral polymerase upon the cellular transcription apparatus and its implications for virus pathogenicity.
1.2. Polymerase associated host factors While most of the basic aspects of virus replication and transcription have been known for decades, the study of hostassociated factors and the cellular pathways involved during these processes represent an emerging research field. Numerous proteomic approaches and yeast two-hybrid analysis have vastly extended the number of interacting partners for the viral RNPs and polymerase components (Bradel-Tretheway et al., 2011; Jorba et al., 2008; Mayer et al., 2007; Munier et al., 2013; Shapira et al., 2009; Tafforeau et al., 2011). Further, several RNAi-based screenings have uncovered numerous host proteins involved in the IAV replication cycle (Brass et al., 2009; Hao et al., 2008; Karlas et al., 2010; Konig et al., 2010; Shapira et al., 2009). All these studies describe a complex and dynamic interrelationship between the virus and its host, including associations between the viral polymerase and several cell structures and components, such as the mitochondria, the nuclear pore and nucleocytoplasmic transport machinery, signaling pathways, protein translation components, RNA binding and splicing related proteins, and cellular RNA polymerase subunits and accessory factors. Although most of the RNAi-based screenings use common H1N1 or H3N2 laboratory virus strains infecting a particular mammalian cell line, other studies have been carried out pinpointing polymerase-associated host factors that show virus-strain and host-cell specificity functions (Bortz et al., 2011; Gabriel et al., 2011; Resa-Infante et al., 2008). The different association of viral proteins to host cell factors depending on the virus strain and type and origin of the infected cell, combined with host response analysis through transcriptome and proteome approaches, contribute to a better understanding of the complex mechanisms involved in virus host adaptability and pathogenicity. Few viral polymerase-associated host proteins have been mechanistically described in some detail. Polymerase interacting partners known to facilitate virus RNA expression include: the minichromosome maintenance (MCM) complex, a cellular DNA replication fork helicase that interacts with the PA subunit of the viral polymerase and promotes the elongation of nascent cRNA in vitro (Kawaguchi and Nagata, 2007); the heat shock protein
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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(Hsp) 90 and the nuclear import RanBP5 and Importin-␣ proteins which play important roles in the assembly and nuclear import of the viral polymerase (Deng et al., 2006; Momose et al., 2002; Resa-Infante et al., 2008); and the RNA polymerase II (RNAPII) and transcription related proteins CDK9, SFPQ/PSF, CLE/C14orf166, RED, and DDX17 thought to play important roles in several steps of the viral RNA synthesis (Bortz et al., 2011; Engelhardt et al., 2005; Fournier et al., 2014; Huarte et al., 2001; Landeras-Bueno et al., 2011; Perez-Gonzalez et al., 2006; Rodriguez et al., 2011; Zhang et al., 2010). On the contrary, several cellular proteins that interact with the viral polymerase and have repressive roles in its activity have also been characterized. Among these are the protein chaperone Hsp70, the dsRNA-binding protein NF90 and the retinoblastoma-like protein RBL2, all known to interact with components of the viral RNPs and block virus transcription and replication activities (Kakugawa et al., 2009; Li et al., 2011; Wang et al., 2009); HAX1, a protein with antiapoptotic activity that interacts with the PA subunit in the cytoplasm and blocks its entry into the nucleus (Hsu et al., 2013); and CHD6, a cellular transcription related chromatin remodeling protein that acts as a suppressor of the viral polymerase transcription and replication activities (Alfonso et al., 2013). The interaction between IAV proteins and specific host proteins can also involve a complex dynamic interplay as shown for cellular DDX21, an RNA helicase implicated in innate immunity that binds to PB1 and inhibits polymerase assembly at early stages of infection (Chen et al., 2014; Zhang et al., 2011). However, at later times the viral non-structural protein 1 (NS1) overcomes this inhibition by binding to DDX21 and displacing PB1. Thus, it seems that by using these sequential interactions IAV not only overcomes a cellular restriction, but also temporally regulates viral gene expression (Chen et al., 2014). Collectively, there are a large number of host factors that cooperate to modulate infection severity and outcome, and the viral polymerase plays a crucial role as a major contributor to viral-host cell interactions.
2. Host range and pathogenicity determinants on influenza polymerase IAV faces the challenge of dealing with an unknown environment when infecting a new host. Three crucial steps need to be accomplished efficiently for successful infection and propagation: (i) entry into the host cell, (ii) efficient replication of the viral genome, and (iii) dissemination of progeny virions into a new host (Sorrell et al., 2011). Preferential interaction of the hemagglutinin with different sialic acid cell receptors constitutes an important host range determinant for influenza viruses (reviewed in (de Graaf and Fouchier, 2014)). In addition, several recently described mutations in the viral polymerase subunits are known to enhance virus replication in mammalian cells and play an essential role in virus adaptability to various hosts. The genetic diversity of IAV and the ability to undergo genome reassortment and infect a large variety of species make the genetic study of IAV and the identification of host range and virulence markers a daunting task. Reverse genetics and high-throughput technologies assist in the recognition of specific mutations crucial for disease outcome caused by IAV, as well as in the study of general virus dynamics during IAV infection. Mice, ferrets, and guinea pigs, each with their own advantages and disadvantages, are the three most frequently used animal models (reviewed in (Thangavel and Bouvier, 2014)). Together, new technical achievements and the use of in vivo models have provided very useful data in the identification of viral determinants for specific functions.
2.1. PB2 The influenza virus polymerase proteins, and in particular PB2, have been shown to be important determinants of viral pathogenicity and virus adaptation to mammalian species (Table 1). 2.1.1. Residue 627 as a determinant of host adaptation and transmissibility Early analysis using reassortant viruses highlighted the polymerase PB2 subunit as a key factor in virus infection adaptability (Almond, 1977). Subsequent work proved that a change in residue 627 of PB2 was an essential host range determinant in a mutational analysis of a single gene reassortant H3N2 human virus with an incorporated avian PB2 (Subbarao et al., 1993). This virus showed host range restriction and could efficiently replicate in avian tissue, but failed to do so in mammalian cells. The genetic analysis of mutant viruses that restore the capacity to infect mammalian cells revealed a glutamic acid (E)-to-lysine (K) substitution in PB2-627 as the only change responsible for this adaptation (Subbarao et al., 1993). It soon became evident that amino acid 627 behaves as a host-associated genetic signature, being generally a glutamic acid residue in avian viruses and a lysine residue in human viruses. Direct evidence showing E627K mutation having a role in avian virus adaptation into mammalian hosts has been provided. This mutation has been regularly detected in fatal cases of human infections with highly pathogenic (HPAI) and low pathogenic (LPAI) avian influenza viruses, including virus isolates from H5N1, H9N2, H7N9 and H7N7 infections (Fouchier et al., 2004; Kageyama et al., 2013; Lam et al., 2013; Li et al., 2012; Subbarao et al., 1998; Zhang et al., 2014). It is also found in the avian-like PB2 from the rescued 1918 H1N1 virus (Taubenberger et al., 2005), and the E627K change quickly appears in mouse adaptation experiments (de Jong et al., 2013; Gabriel et al., 2005; Min et al., 2013). Furthermore, a prominent role of this mutation in transmissibility using ferrets and guinea pig models has been reported (Gao et al., 2009; Herfst et al., 2012; Linster et al., 2014; Steel et al., 2009). This could be, at least in part, due to the ability of avian viruses with a lysine at position 627 to efficiently replicate in the upper and lower respiratory tracts of mammals (Hatta et al., 2007). Nevertheless, there are a number of indications that residue PB2-627 is a strong, yet non-essential, determinant of mammalian adaptation and that a more complex inter-relationship between several residues in PB2 as well as the effect of other viral proteins such as NP need to be considered. For instance, it has been shown that only a reassortant virus with PB2-627E that included a NP protein of the same origin could successfully mutate into PB2-627K in H5N1 viruses infecting mammalian MDCK cells (Bogs et al., 2011). Other influenza viruses, such as the 2009 H1N1 pandemic virus (A(H1N1)pdm09) which contains a PB2 subunit of avian origin retaining several avian signatures including residue PB2-627E, do not follow the normal PB2-627 distribution. The lack of PB2627K change in the highly transmissible A(H1N1)pdm09 virus is explained by the acquisition of a combination of changes in residues 271 and 590/591 (Liu et al., 2012; Mehle and Doudna, 2009; Yamada et al., 2010). Moreover, some of the H5N1 human isolates, generally those that showed less virulence, do not carry PB2-627K and not all mammalian viruses incorporate a lysine in this residue, including equine viruses and some swine viruses (Gao et al., 1999; Schnitzler and Schnitzler, 2009; Shinya et al., 2007). Exceptions to the main PB2-627 avian to mammal mutation distribution are also found in some avian viruses (Russell et al., 2012). 2.1.2. Correlation between adaptation and polymerase activity Many of the virus adaptation studies show that this process arises with a simultaneous increase in replication capacity and pathogenicity, thus establishing an obvious correlation between
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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Table 1 Major pathogenicity determinants of influenza virus polymerase proteins in mammalian hosts. Protein
Mutation
Function
Reference
PA
F35L A36T
Seyer et al., 2012 Zhu et al., 2012
G631S
> polymerase activity and virulence in mice. Serially passaged pdm in mice > polymerase activity and growth in human and porcine cells. Mouse adapted pdm > polymerase activity and virus replication in human cells. pdm > polymerase activity and virulence. Serially passaged H5N2 in mice > polymerase activity and virulence (combined with A70V mutation). pdm in mice > polymerase activity and replication. Mouse adapted pdm > polymerase activity and virulence. pdm in mice > polymerase activity, viral growth and pathogenesis. H7N9 in mice > polymerase activity, RNAPII degradation and pathogenicity. H1N1 & pdm in mice > polymerase activity, virus replication and pathogenicity. Avian & human viruses > virulence. H5N1 in mice
PB1
H99Y L473V L598P
> polymerase activity and airborne transmission. H5N1 in ferrets > polymerase activity and viral growth. H5N1 & pdm in mice > polymerase activity and viral growth. H5N1 & pdm in mice
Linster et al., 2014 Xu et al., 2012 Xu et al., 2012
PB1-F2
P62L
Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice > levels of cytokines in lung, viral replication and virulence. > binding to MAVS and < Type I IFN response. H5N1& 1918 in mice > lung inflammation and predisposition to secondary bacterial infection. H1N1 in mice > lung inflammation and predisposition to secondary bacterial infection. H1N1 in mice > lung inflammation and predisposition to secondary bacterial infection. H1N1 in mice Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice
Alymova et al., 2011
T85I T97I P224S L295P L336M N409S I550L T552S
N66S T68I Q69L G70V H75R Q79R S82L PB2
D9N M147L E158G/A D256G H357N I504V T588I G590S Q591R E627K
Mitochondrial localization. < IFN expression. > pathogenicity. H1N1 & H5N1 in mice > viral replication and virulence (combined with E627K mutation). H9N2 in mice > polymerase activity and replication. Mouse adapted pdm > polymerase activity in mammalian cells and replication in pigs. H5N1 in pigs > polymerase activity and pathogenicity. Mouse adapted pdm > polymerase activity, RNAPII degradation and pathogenicity. H1N1 & pdm in mice > polymerase activity, replication and virulence.> binding and inhibition to MAVS. pdm in mice > polymerase activity and viral replication. Pdm > polymerase activity, viral replication and pathogenicity. pdm & H5N1 in mice. > polymerase activity, viral protein expression, viral growth in mammalian cells, brain invasiveness and virulence. Confers avian viruses efficient growth in mammalian upper and lower respiratory tracts. H5N1, H7N7, H9N2, H7N9 in mice and ferrets
D701N
> polymerase activity, cap-1 RNA binding activity, viral growth, transmission and virulence. Mouse adapted H7N7, H5N1, H3N2, H7N9 in mice
S714R
> polymerase activity, cap-1 RNA binding activity and virulence. Synergistic effect with 701N. H5N1 & H7N7 in mice
Bussey et al., 2011 Song et al., 2009 Sun et al., 2014 Ilyushina et al., 2010 Bussey et al., 2011 Yamayoshi et al., 2014 Llompart et al., 2014; Rolling et al., 2009 Mehle et al., 2012 Hiromoto et al., 2000
Conenello et al., 2007; Conenello et al., 2011; Schmolke et al., 2011; Varga et al., 2012 Alymova et al., 2014 Alymova et al., 2014 Alymova et al., 2014 Alymova et al., 2011 Alymova et al., 2011 Alymova et al., 2011 Graef et al., 2010; Kim et al., 2010 Wang et al., 2012 Ilyushina et al., 2010 Manzoor et al., 2009 Zhu et al., 2012 Llompart et al., 2014; Rolling et al., 2009 Zhao et al., 2014 Mehle and Doudna, 2009 Mehle and Doudna, 2009; Yamada et al., 2010 de Jong et al., 2013; Hatta et al., 2001; Hatta et al., 2007; Li et al., 2012; Mok et al., 2014; Munster et al., 2007; Shinya et al., 2004; Shinya et al., 2007; Subbarao et al., 1993; Zhang et al., 2014 Czudai-Matwich et al., 2014; Gabriel et al., 2005; Li et al., 2005; Mok et al., 2014; Ping et al., 2010; Zhang et al., 2012 Czudai-Matwich et al., 2014; Gabriel et al., Zhang et al., 2012
(>) Increasing effect; (
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Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Review
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Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity
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Ariel Rodriguez-Frandsen a,b,1,2 , Roberto Alfonso a,b,1,3 , Amelia Nieto a,b,∗ a b
Centro Nacional de Biotecnología. C.S.I.C. Darwin 3, Cantoblanco, 28049 Madrid, Spain Ciber de Enfermedades Respiratorias, Mallorca, Illes Balears, Spain
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Article history: Received 14 September 2014 Received in revised form 25 March 2015 Accepted 26 March 2015 Available online xxx
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Keywords: Influenza virus Host adaptation Pathogenicity Viral polymerase RNA polymerase II Virulence
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Contents
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The viral polymerase is an essential complex for the influenza virus life cycle as it performs the viral RNA transcription and replication processes. To that end, the polymerase carries out a wide array of functions and associates to a large number of cellular proteins. Due to its importance, recent studies have found numerous mutations in all three polymerase protein subunits contributing to virus host range and pathogenicity. In this review, we will point out viral polymerase polymorphisms that have been associated with virus adaptation to mammalian hosts, increased viral polymerase activity and virulence. Furthermore, we will summarize the current knowledge regarding the new set of proteins expressed from the viral polymerase genes and their contribution to infection. In addition, the mechanisms used by the virus to counteract the cellular immune response in which the viral polymerase complex or its subunits are involved will be highlighted. Finally, the degradative process induced by the viral polymerase on the cellular transcription machinery and its repercussions on virus pathogenicity will be of particular interest. © 2015 Published by Elsevier B.V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Structure and function of the influenza polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Polymerase associated host factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host range and pathogenicity determinants on influenza polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. PB2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Residue 627 as a determinant of host adaptation and transmissibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Correlation between adaptation and polymerase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Role of other PB2 residues in adaptation, transmissibility and pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. PB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. PA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recently discovered protein expressed from IAV polymerase genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. PB1-F2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. N40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. PA-derived proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of cellular response to infection by IAV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Inhibition of IFN responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗ Corresponding author at: Centro Nacional de Biotecnología, C.S.I.C., Darwin 3, Cantoblanco, 28049 Madrid, Spain. Tel.: +34 91 5854914; fax: +34 91 5854506. E-mail address: [email protected] (A. Nieto). 1 Both author equally contributed to this work. 2 Present address: Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America. 3 Present address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. http://dx.doi.org/10.1016/j.virusres.2015.03.017 0168-1702/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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4.2. Inhibition of cellular transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
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The influenza A virus (IAV) is an important respiratory human pathogen causing yearly recurrent seasonal epidemics with an average global burden of >600 million cases (www.who.int). In rare instances IAV can also spread from its natural zoonotic reservoirs (aquatic birds) to cross species barriers and transmit to humans where it can evolve into strains that cause diseases ranging from mild to severe, with occasional widespread distribution known as pandemic. In the past century, the most devastating pandemic took place in 1918–1920 (also known as the “1918 flu” or “Spanish flu”), infecting hundreds of millions and killing between 20 and 50 million people worldwide (Johnson and Mueller, 2002; Taubenberger and Morens, 2006). Two additional pandemic events originating in Asia took place during the second half of the 20th century, the so-called Asian H2N2 pandemic of 1957 (1 million deaths) and the Hong Kong H3N2 pandemic in 1968 (1 million deaths). Although of relatively low virulence, the recent H1N1 influenza 2009 pandemic of swine origin emerged unexpectedly in Mexico to spread around the world in just a few months. IAV naturally infects wild aquatic birds making up an extremely heterogeneous population which includes many possible combinations between the two surfaces glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA); a total of 18 different HA and 11 NA have been recognized. At present there are concerns that avian influenza strains, including the highly pathogenic influenza viruses H5N1 and the novel H7N9 subtypes not yet capable of spreading from one human to another, could adapt and become more easily transmissible among humans. The ability of influenza A viruses to infect a variety of hosts is based on their genetic diversity due to two main reasons: (i) their RNA polymerase is error-prone, and (ii) they contain a segmented genome, which allows for exchange of RNA segments between genotypically diverse influenza viruses. These features lead to the rapid generation of novel strains and subtypes and thus contribute to the constant threat that newly emerging and re-emerging influenza viruses pose to the human population.
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1.1. Structure and function of the influenza polymerase
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IAV, a member of the Orthomyxoviridae family, possesses a negative-sense single-stranded RNA genome (vRNA) divided into eight segments. vRNAs are protected in a structure known as viral ribonucleoprotein (vRNP), in which the RNA strand is wrapped by the nucleoprotein (NP), and a single viral polymerase complex interacts with the complementary 3 and 5 genomic end sequences. Recent cryo-EM reconstructions of vRNPs obtained from different sources show a double-helical stem structure in which the NP proteins and the protected viral RNA form two anti-parallel strands (Arranz et al., 2012; Moeller et al., 2012). Both strands are connected by a short loop at one end of the particle and interact with the viral polymerase at the other end. The incoming parental vRNPs are released into the cytoplasm of the infected host cell and then quickly transported into the nucleus. IAV is a rare RNA-genome virus in that expression and amplification of viral genomes take place inside the infected cell nucleus and it therefore heavily depends on host nucleocytoplasmic trafficking and nuclear functions. Within the host nucleus, cellular insoluble fractions, such as nuclear matrix and chromatin structures, have been shown to encompass part of the viral polymerase transcription and
00 00 00 00
replication activity and act as a platform for the release of progeny vRNPs outside the nucleus (Chase et al., 2011; Garcia-Robles et al., 2005; Lopez-Turiso et al., 1990; Takizawa et al., 2006). The IAV genome encodes for 10 major proteins, although alternative protein products have been characterized from several genome segments (see below). The complete coding capacity of the IAV genome is far from known and processes like splicing, protein truncations, and the use of alternative initiation codons or overlapping frames are known to increase the diversity of proteins generated during infection. The widespread synthesis of small viral non-coding RNAs with largely unknown roles in the outcome of infection further complicates the understanding of IAV full expression capacity (Perez et al., 2010, 2012). The three largest viral genome segments encode for the polymerase heterotrimeric complex, responsible for the RNA-dependent RNA polymerase activity of the virus (Fig. 1). The three polymerase subunits, named PA, PB1 and PB2, together with the nucleoprotein, form the minimum set of viral proteins required for viral RNA transcription and replication (reviewed in (Fodor, 2013; Resa-Infante et al., 2011)). Viral transcription depends on primers of host origin obtained through a cap-snatching process targeting newly synthesized host pre-mRNAs (Krug et al., 1979). The viral positive-sense mRNA also includes a 3 polyA tail generated by repetitive polymerization of a polyU track on the genomic vRNP, thus creating a transcript that is structurally undistinguished from host mRNAs. The vRNP undergoing transcription is processed by a cis-acting polymerase (Jorba et al., 2009). The requirement of newly synthesized cellular pre-mRNAs as a source of cap-olignonucleotides for viral transcription initiation involves a functional association between the viral and the cellular transcription machineries, which has significant consequences for viral outcome (see below). A switch from viral transcription to replication is required during a successful infectious cycle. The action of viral short virion RNAs (svRNAs) and the availability of cellular nucleotides and newly synthesized viral polymerase and NP, have been involved in this process (Jorba et al., 2009; Perez et al., 2010; Vreede and Brownlee, 2007; Vreede et al., 2004). The viral replication step is initiated early during infection with the synthesis of the cRNA, a complete unpolyadenylated positive-polarity copy of the vRNA (Hay et al., 1977). cRNAs within the infected host cell form cRNPs structures, and serve as templates for the synthesis of new vRNPs which are ready for either further rounds of viral gene expression or their transportation outside the nucleus and subsequent encapsidation. As opposed to what happens with the viral mRNA, the production of cRNAs is only started after viral protein synthesis has begun and seems to require a soluble trans-acting polymerase different from the one resident in the RNP (Hay et al., 1977; Jorba et al., 2009). Basic protein 1, PB1, is the core of the complex, the most conserved of the polymerase subunits, and contains the enzymatic motifs needed for RNA polymerization activity (Kobayashi et al., 1996). The PB2 subunit has a key role in viral transcription due to its recognition and binding of host 5 mRNA cap structures generated by the cellular transcription machinery (Blaas et al., 1982; Braam et al., 1983). Acidic protein PA has an endonucleolytic activity needed for the viral cap-snatching process (Dias et al., 2009; Yuan et al., 2009). In recent years some parts of the three proteins have been structurally characterized including important
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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Fig. 1. Influenza A virus polymerase complex. PA, PB1 and PB2 subunits are depicted as bars with the amino and carboxy-terminal ends indicated. The putative functional domains (Endonuclease, RdRP and Cap-binding) and the interaction regions between subunits are also highlighted. The NLS regions described for each protein are represented by blue boxes.
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functional domains, such as the cap-binding site on PB2 (Guilligay et al., 2008), the carboxy-terminal part of PB2 that contains the nuclear localization sequence (NLS) domain and important host range determinants (Tarendeau et al., 2007; Tarendeau et al., 2008), and the endonuclease domain at the N-terminus of PA (Dias et al., 2009; Yuan et al., 2009). Additionally, crystal structures of the interactions between the carboxy-terminal part of PA and the first 15 amino-terminal amino acids of PB1 (He et al., 2008), and between the last 72 amino acids of carboxy-terminal PB1 and the first 35 amino-terminal amino acids of protein PB2 (Sugiyama et al., 2009), have been obtained. Furthermore the cryoEM reconstructions of the vRNP, as well as other structural analyses of the viral polymerase out of the context of the RNP, illustrate this heterotrimer complex as a compact structure with some indications of flexibility (Area et al., 2004; Arranz et al., 2012; Coloma et al., 2009; Moeller et al., 2012; Resa-Infante et al., 2010; Torreira et al., 2007). In agreement with that, the crystal structures of the complete RNA polymerase complexes from influenza A and B viruses show that the three subunits have multiple interactions with each other and all of them participate in the binding of the two strands of the RNA promoter (Pflug et al., 2014; Reich et al., 2014). Overall, the viral polymerase is a major component of the virus as it provides the necessary machinery to translate its genome and replicate itself. In addition to its basic functions on viral genome expression, the viral polymerase has a key role in transmission between hosts and pathogenesis. For instance, a viral polymerase with higher activity usually correlates with an increased ability of transmission and adaptability, as well as higher pathogenicity (Naffakh et al., 2008). In this review, we highlight those mutations in IAV polymerase that have been associated with higher virus adaptability to a new host, increased viral polymerase activity and pathogenicity. Additionally, some of the host cellular mechanisms that have been linked to these processes are discussed. Special attention is paid to the consequences derived from the functional association between viral and cellular transcription machineries as well as the detrimental effect caused by the degradative process induced by the viral polymerase upon the cellular transcription apparatus and its implications for virus pathogenicity.
1.2. Polymerase associated host factors While most of the basic aspects of virus replication and transcription have been known for decades, the study of hostassociated factors and the cellular pathways involved during these processes represent an emerging research field. Numerous proteomic approaches and yeast two-hybrid analysis have vastly extended the number of interacting partners for the viral RNPs and polymerase components (Bradel-Tretheway et al., 2011; Jorba et al., 2008; Mayer et al., 2007; Munier et al., 2013; Shapira et al., 2009; Tafforeau et al., 2011). Further, several RNAi-based screenings have uncovered numerous host proteins involved in the IAV replication cycle (Brass et al., 2009; Hao et al., 2008; Karlas et al., 2010; Konig et al., 2010; Shapira et al., 2009). All these studies describe a complex and dynamic interrelationship between the virus and its host, including associations between the viral polymerase and several cell structures and components, such as the mitochondria, the nuclear pore and nucleocytoplasmic transport machinery, signaling pathways, protein translation components, RNA binding and splicing related proteins, and cellular RNA polymerase subunits and accessory factors. Although most of the RNAi-based screenings use common H1N1 or H3N2 laboratory virus strains infecting a particular mammalian cell line, other studies have been carried out pinpointing polymerase-associated host factors that show virus-strain and host-cell specificity functions (Bortz et al., 2011; Gabriel et al., 2011; Resa-Infante et al., 2008). The different association of viral proteins to host cell factors depending on the virus strain and type and origin of the infected cell, combined with host response analysis through transcriptome and proteome approaches, contribute to a better understanding of the complex mechanisms involved in virus host adaptability and pathogenicity. Few viral polymerase-associated host proteins have been mechanistically described in some detail. Polymerase interacting partners known to facilitate virus RNA expression include: the minichromosome maintenance (MCM) complex, a cellular DNA replication fork helicase that interacts with the PA subunit of the viral polymerase and promotes the elongation of nascent cRNA in vitro (Kawaguchi and Nagata, 2007); the heat shock protein
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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(Hsp) 90 and the nuclear import RanBP5 and Importin-␣ proteins which play important roles in the assembly and nuclear import of the viral polymerase (Deng et al., 2006; Momose et al., 2002; Resa-Infante et al., 2008); and the RNA polymerase II (RNAPII) and transcription related proteins CDK9, SFPQ/PSF, CLE/C14orf166, RED, and DDX17 thought to play important roles in several steps of the viral RNA synthesis (Bortz et al., 2011; Engelhardt et al., 2005; Fournier et al., 2014; Huarte et al., 2001; Landeras-Bueno et al., 2011; Perez-Gonzalez et al., 2006; Rodriguez et al., 2011; Zhang et al., 2010). On the contrary, several cellular proteins that interact with the viral polymerase and have repressive roles in its activity have also been characterized. Among these are the protein chaperone Hsp70, the dsRNA-binding protein NF90 and the retinoblastoma-like protein RBL2, all known to interact with components of the viral RNPs and block virus transcription and replication activities (Kakugawa et al., 2009; Li et al., 2011; Wang et al., 2009); HAX1, a protein with antiapoptotic activity that interacts with the PA subunit in the cytoplasm and blocks its entry into the nucleus (Hsu et al., 2013); and CHD6, a cellular transcription related chromatin remodeling protein that acts as a suppressor of the viral polymerase transcription and replication activities (Alfonso et al., 2013). The interaction between IAV proteins and specific host proteins can also involve a complex dynamic interplay as shown for cellular DDX21, an RNA helicase implicated in innate immunity that binds to PB1 and inhibits polymerase assembly at early stages of infection (Chen et al., 2014; Zhang et al., 2011). However, at later times the viral non-structural protein 1 (NS1) overcomes this inhibition by binding to DDX21 and displacing PB1. Thus, it seems that by using these sequential interactions IAV not only overcomes a cellular restriction, but also temporally regulates viral gene expression (Chen et al., 2014). Collectively, there are a large number of host factors that cooperate to modulate infection severity and outcome, and the viral polymerase plays a crucial role as a major contributor to viral-host cell interactions.
2. Host range and pathogenicity determinants on influenza polymerase IAV faces the challenge of dealing with an unknown environment when infecting a new host. Three crucial steps need to be accomplished efficiently for successful infection and propagation: (i) entry into the host cell, (ii) efficient replication of the viral genome, and (iii) dissemination of progeny virions into a new host (Sorrell et al., 2011). Preferential interaction of the hemagglutinin with different sialic acid cell receptors constitutes an important host range determinant for influenza viruses (reviewed in (de Graaf and Fouchier, 2014)). In addition, several recently described mutations in the viral polymerase subunits are known to enhance virus replication in mammalian cells and play an essential role in virus adaptability to various hosts. The genetic diversity of IAV and the ability to undergo genome reassortment and infect a large variety of species make the genetic study of IAV and the identification of host range and virulence markers a daunting task. Reverse genetics and high-throughput technologies assist in the recognition of specific mutations crucial for disease outcome caused by IAV, as well as in the study of general virus dynamics during IAV infection. Mice, ferrets, and guinea pigs, each with their own advantages and disadvantages, are the three most frequently used animal models (reviewed in (Thangavel and Bouvier, 2014)). Together, new technical achievements and the use of in vivo models have provided very useful data in the identification of viral determinants for specific functions.
2.1. PB2 The influenza virus polymerase proteins, and in particular PB2, have been shown to be important determinants of viral pathogenicity and virus adaptation to mammalian species (Table 1). 2.1.1. Residue 627 as a determinant of host adaptation and transmissibility Early analysis using reassortant viruses highlighted the polymerase PB2 subunit as a key factor in virus infection adaptability (Almond, 1977). Subsequent work proved that a change in residue 627 of PB2 was an essential host range determinant in a mutational analysis of a single gene reassortant H3N2 human virus with an incorporated avian PB2 (Subbarao et al., 1993). This virus showed host range restriction and could efficiently replicate in avian tissue, but failed to do so in mammalian cells. The genetic analysis of mutant viruses that restore the capacity to infect mammalian cells revealed a glutamic acid (E)-to-lysine (K) substitution in PB2-627 as the only change responsible for this adaptation (Subbarao et al., 1993). It soon became evident that amino acid 627 behaves as a host-associated genetic signature, being generally a glutamic acid residue in avian viruses and a lysine residue in human viruses. Direct evidence showing E627K mutation having a role in avian virus adaptation into mammalian hosts has been provided. This mutation has been regularly detected in fatal cases of human infections with highly pathogenic (HPAI) and low pathogenic (LPAI) avian influenza viruses, including virus isolates from H5N1, H9N2, H7N9 and H7N7 infections (Fouchier et al., 2004; Kageyama et al., 2013; Lam et al., 2013; Li et al., 2012; Subbarao et al., 1998; Zhang et al., 2014). It is also found in the avian-like PB2 from the rescued 1918 H1N1 virus (Taubenberger et al., 2005), and the E627K change quickly appears in mouse adaptation experiments (de Jong et al., 2013; Gabriel et al., 2005; Min et al., 2013). Furthermore, a prominent role of this mutation in transmissibility using ferrets and guinea pig models has been reported (Gao et al., 2009; Herfst et al., 2012; Linster et al., 2014; Steel et al., 2009). This could be, at least in part, due to the ability of avian viruses with a lysine at position 627 to efficiently replicate in the upper and lower respiratory tracts of mammals (Hatta et al., 2007). Nevertheless, there are a number of indications that residue PB2-627 is a strong, yet non-essential, determinant of mammalian adaptation and that a more complex inter-relationship between several residues in PB2 as well as the effect of other viral proteins such as NP need to be considered. For instance, it has been shown that only a reassortant virus with PB2-627E that included a NP protein of the same origin could successfully mutate into PB2-627K in H5N1 viruses infecting mammalian MDCK cells (Bogs et al., 2011). Other influenza viruses, such as the 2009 H1N1 pandemic virus (A(H1N1)pdm09) which contains a PB2 subunit of avian origin retaining several avian signatures including residue PB2-627E, do not follow the normal PB2-627 distribution. The lack of PB2627K change in the highly transmissible A(H1N1)pdm09 virus is explained by the acquisition of a combination of changes in residues 271 and 590/591 (Liu et al., 2012; Mehle and Doudna, 2009; Yamada et al., 2010). Moreover, some of the H5N1 human isolates, generally those that showed less virulence, do not carry PB2-627K and not all mammalian viruses incorporate a lysine in this residue, including equine viruses and some swine viruses (Gao et al., 1999; Schnitzler and Schnitzler, 2009; Shinya et al., 2007). Exceptions to the main PB2-627 avian to mammal mutation distribution are also found in some avian viruses (Russell et al., 2012). 2.1.2. Correlation between adaptation and polymerase activity Many of the virus adaptation studies show that this process arises with a simultaneous increase in replication capacity and pathogenicity, thus establishing an obvious correlation between
Please cite this article in press as: Rodriguez-Frandsen, A., et al., Influenza virus polymerase: Functions on host range, inhibition of cellular response to infection and pathogenicity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.03.017
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Table 1 Major pathogenicity determinants of influenza virus polymerase proteins in mammalian hosts. Protein
Mutation
Function
Reference
PA
F35L A36T
Seyer et al., 2012 Zhu et al., 2012
G631S
> polymerase activity and virulence in mice. Serially passaged pdm in mice > polymerase activity and growth in human and porcine cells. Mouse adapted pdm > polymerase activity and virus replication in human cells. pdm > polymerase activity and virulence. Serially passaged H5N2 in mice > polymerase activity and virulence (combined with A70V mutation). pdm in mice > polymerase activity and replication. Mouse adapted pdm > polymerase activity and virulence. pdm in mice > polymerase activity, viral growth and pathogenesis. H7N9 in mice > polymerase activity, RNAPII degradation and pathogenicity. H1N1 & pdm in mice > polymerase activity, virus replication and pathogenicity. Avian & human viruses > virulence. H5N1 in mice
PB1
H99Y L473V L598P
> polymerase activity and airborne transmission. H5N1 in ferrets > polymerase activity and viral growth. H5N1 & pdm in mice > polymerase activity and viral growth. H5N1 & pdm in mice
Linster et al., 2014 Xu et al., 2012 Xu et al., 2012
PB1-F2
P62L
Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice > levels of cytokines in lung, viral replication and virulence. > binding to MAVS and < Type I IFN response. H5N1& 1918 in mice > lung inflammation and predisposition to secondary bacterial infection. H1N1 in mice > lung inflammation and predisposition to secondary bacterial infection. H1N1 in mice > lung inflammation and predisposition to secondary bacterial infection. H1N1 in mice Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice Proinflammatory motif causing morbidity, mortality and pulmonary inflammation. H3N2 in mice
Alymova et al., 2011
T85I T97I P224S L295P L336M N409S I550L T552S
N66S T68I Q69L G70V H75R Q79R S82L PB2
D9N M147L E158G/A D256G H357N I504V T588I G590S Q591R E627K
Mitochondrial localization. < IFN expression. > pathogenicity. H1N1 & H5N1 in mice > viral replication and virulence (combined with E627K mutation). H9N2 in mice > polymerase activity and replication. Mouse adapted pdm > polymerase activity in mammalian cells and replication in pigs. H5N1 in pigs > polymerase activity and pathogenicity. Mouse adapted pdm > polymerase activity, RNAPII degradation and pathogenicity. H1N1 & pdm in mice > polymerase activity, replication and virulence.> binding and inhibition to MAVS. pdm in mice > polymerase activity and viral replication. Pdm > polymerase activity, viral replication and pathogenicity. pdm & H5N1 in mice. > polymerase activity, viral protein expression, viral growth in mammalian cells, brain invasiveness and virulence. Confers avian viruses efficient growth in mammalian upper and lower respiratory tracts. H5N1, H7N7, H9N2, H7N9 in mice and ferrets
D701N
> polymerase activity, cap-1 RNA binding activity, viral growth, transmission and virulence. Mouse adapted H7N7, H5N1, H3N2, H7N9 in mice
S714R
> polymerase activity, cap-1 RNA binding activity and virulence. Synergistic effect with 701N. H5N1 & H7N7 in mice
Bussey et al., 2011 Song et al., 2009 Sun et al., 2014 Ilyushina et al., 2010 Bussey et al., 2011 Yamayoshi et al., 2014 Llompart et al., 2014; Rolling et al., 2009 Mehle et al., 2012 Hiromoto et al., 2000
Conenello et al., 2007; Conenello et al., 2011; Schmolke et al., 2011; Varga et al., 2012 Alymova et al., 2014 Alymova et al., 2014 Alymova et al., 2014 Alymova et al., 2011 Alymova et al., 2011 Alymova et al., 2011 Graef et al., 2010; Kim et al., 2010 Wang et al., 2012 Ilyushina et al., 2010 Manzoor et al., 2009 Zhu et al., 2012 Llompart et al., 2014; Rolling et al., 2009 Zhao et al., 2014 Mehle and Doudna, 2009 Mehle and Doudna, 2009; Yamada et al., 2010 de Jong et al., 2013; Hatta et al., 2001; Hatta et al., 2007; Li et al., 2012; Mok et al., 2014; Munster et al., 2007; Shinya et al., 2004; Shinya et al., 2007; Subbarao et al., 1993; Zhang et al., 2014 Czudai-Matwich et al., 2014; Gabriel et al., 2005; Li et al., 2005; Mok et al., 2014; Ping et al., 2010; Zhang et al., 2012 Czudai-Matwich et al., 2014; Gabriel et al., Zhang et al., 2012
(>) Increasing effect; (
