SUPPLEMENTARY INFORMATION

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1 doi: /nature14008 Supplementary Figure 1. Sequence alignment of A/little yellow-shouldered bat/guatemala/060/2010 (H17N10) polymerase with that of human strain A/Victoria/3/75(H3N2). The secondary structure of the bat polymerase is indicated above. White on red background indicates identical residues. Red lettering indicates similar residues. Figure prepared with Espript 3.0 ( a. PA subunit b. PB1 subunit c. PB2 subunit 1

2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 1. a. PA subunit 2

3 RESEARCH b. PB1 subunit 3

4 RESEARCH SUPPLEMENTARY INFORMATION c. PB2 subunit 4

5 RESEARCH 1. Discussion of previous studies of the vrna binding site on influenza polymerase. Various studies have sought to identify the vrna 3 and 5 end binding sites. Using PB1 deletion mutants it was found that the N-terminal 83 residues and C-terminal residues beyond 493 of PB1 were involved in 5 end binding 1. Using a crosslinking approach with thio-u substituted RNA, region (notably 571-RR-572) of PB1 was identified to be involved in 5 end binding (thio-u at position 15) and region (notably F251, F254) in 3 end binding (thio-u at position 10) 2. In another study, the previous results on 3 end binding were not confirmed and region (notably R233, R238) of PB1 was proposed to be involved in 5 end binding and R239, R249 in duplex binding 3. This study noted that PA also binds the promoter and PB2 is only involved in 3 end binding. Mutations of PB1 C- terminal residues K669, R670 and R672 were shown to be important in regulation of transcription by reducing promoter binding 4. All numbering is for human/avian A strains. In the light of the current structure it is interesting that very few of the residues cited above are directly involved in promotor binding. PB1 R571 is 16 Å from the promoter duplex region. It is possible that this could cross-link to thio-u at position 15 on 5 end since of 5 end would be very flexible in the absence of 3 end to form the duplex. PB1 F251 and F254 are buried and their mutation would likely disrupt the fold. R233, R238 are on the finger-tips loop close, but not directly interacting, with the 5 end hook (Extended Data Figure 6a); their mutation would likely disrupt 5 end binding. R239 (finger-tips loop) is in the NTP channel (Extended Data Figure 6a) and R249 likely binds the template in the PB1 cavity (Figure 4a). PB1 R672 is involved in 3 end binding (Extended Data Figure 6). Although in the structure 5 adenosine A1 only has a monophosphate, conserved basic residues PA K328 and PB1 K360 and H363 (bat numbering) could plausibly interact with a triphosphate on this nucleotide. Interestingly, the recently reported 5 end derived small virus 5

6 RESEARCH SUPPLEMENTARY INFORMATION generated RNAs 5 were found to depend on PA R566 (bat R561, Fig. 5d) and to a lesser extent on PA K328 (bat K328) and K539 (bat K534) for polymerase binding suggesting that these svrnas bind in the same manner as the genomic 5 end. 2. Discussion of putative host-specific residues in influenza polymerase. There is considerable interest in understanding the role of polymerase residues that have been implicated in host adaptation, notably between avian and human influenza A strains (reviewed in 6 ). Residue substitutions potentially involved in inter-species transmission have been identified from naturally occurring sequences by bioinformatics analysis 7,8 or by host adaption experiments using cells 9-11 or mice 12. Such mutations typically have a neutral effect in avian cells but enhance polymerase activity in mammalian cells, without necessarily augmenting pathogenicity. The most well characterised host adaptation residues occur in the PB2 627-NLS domain with avian/mammalian preferences V613T, E627K, D701N, K702R and S714R. Other implicated PB2 residues are E158G and T271A. In PA, substitutions T85I, G186S, L336M, P400L, M423I, V476A, T552S and V630E have been implicated and in PB1 N375S. All numbering is for human/avian A strains. It is fair to say that the mechanism by which these substitutions cause observed altered viral tropism is unknown for most of these substitutions 6 although those on the PB2-NLS domain have been associated with interactions with host importins 13,14. Since the positions of implicated residues can henceforth be mapped onto the full polymerase structure, an initial distinction that can now be made is between those residues (type 1) that are more likely to affect the intrinsic rate of polymerase functions (which could be important for species dependent physiological reasons) and others (type 2), which because of their surface location, possibly act through direct interaction with other viral or host factors (of which there are numerous putative contenders ). For the PB2 627-NLS domain 6

7 RESEARCH residues the complete structure does not add much new insight except to say that the double domain and residue 627 in particular (S627 in bat, K627 in human A and B strains, E in avian) is distant from the polymerase catalytic center but possibly on the exit path of newly synthesised RNA 20. Neither PB2 residues 158 (a conserved glutamate in A and B strains) and 271 (I in bat, S in human, T in avian) are in surface exposed positions and thus could be of type 1. Residue 158 is in the PB2 helical lid domain at the N-terminus of helix α9. The lid domain is implicated in channelling of capped primer into the polymerase active site but is also likely to change conformation during elongation 20. Residue 271 is at the interface between the PB2 mid and 627 domains. Thus both these substitutions could affect polymerase activity through altering dynamic interactions of polymerase sub-domains domains or with RNA. This is similarly the case for PA positions 85 (at the endonuclaease- PB1Cter interface), 186 (at the beginning of the flexible PA-linker), 400 (in the PA-arch near the 5 end binding site) and 476 (close the separation point of 3 and 5 strands). On the other hand, a clear example of a potential type 2 residue is PA 552 (T in bat, and avian, S in human) which is on the very prominent and exposed PA 550-loop at the bottom of the polymerase (Fig. 1a) and similarly PA residues 336 and 630 are surface exposed. PB1 residue 375 (D in bat and S in most other flu strains) is in an exposed surface position not far from the promoter binding site. Clearly further biochemical and structural studies of polymerase in different conformations and eventually with host factors is required to determine the exact role of these putative host-specific residues. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary References. 1 Gonzalez, S. & Ortin, J. Characterization of influenza virus PB1 protein binding to viral RNA: two separate regions of the protein contribute to the interaction domain. Journal of virology 73, (1999). 2 Li, M. L., Ramirez, B. C. & Krug, R. M. RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. The EMBO journal 17, (1998). 3 Jung, T. E. & Brownlee, G. G. A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase. The Journal of general virology 87, (2006). 4 Kerry, P. S., Willsher, N. & Fodor, E. A cluster of conserved basic amino acids near the C-terminus of the PB1 subunit of the influenza virus RNA polymerase is involved in the regulation of viral transcription. Virology 373, , doi: /j.virol (2008). 5 Perez, J. T. et al. Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proceedings of the National Academy of Sciences of the United States of America 107, , doi: /pnas (2010). 6 Cauldwell, A. V., Long, J. S., Moncorge, O. & Barclay, W. S. Viral determinants of influenza A virus host range. The Journal of general virology 95, , doi: /vir (2014). 7 Taubenberger, J. K. et al. Characterization of the 1918 influenza virus polymerase genes. Nature 437, (2005). 8 Tamuri, A. U., dos Reis, M., Hay, A. J. & Goldstein, R. A. Identifying Changes in Selective Constraints: Host Shifts in Influenza. PLoS Comput Biol 5, e (2009). 9 Bussey, K. A. et al. PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells. Journal of virology 85, , doi: /jvi (2011). 10 Bussey, K. A., Bousse, T. L., Desmet, E. A., Kim, B. & Takimoto, T. PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. Journal of virology 84, , doi: /jvi (2010). 11 Mehle, A., Dugan, V. G., Taubenberger, J. K. & Doudna, J. A. Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers. Journal of virology 86, , doi: /jvi (2012). 12 Gabriel, G. et al. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proceedings of the National Academy of Sciences of the United States of America 102, (2005). 13 Tarendeau, F. et al. Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nature structural & molecular biology 14, (2007). 14 Resa-Infante, P. & Gabriel, G. The nuclear import machinery is a determinant of influenza virus host adaptation. BioEssays : news and reviews in molecular, cellular and developmental biology 35, 23-27, doi: /bies (2013). 15 Jorba, N. et al. Analysis of the interaction of influenza virus polymerase complex with human cell factors. Proteomics 8, (2008). 8

9 RESEARCH 16 Munier, S., Rolland, T., Diot, C., Jacob, Y. & Naffakh, N. Exploration of binary virus-host interactions using an infectious protein complementation assay. Molecular & cellular proteomics : MCP 12, , doi: /mcp.m (2013). 17 Karlas, A. et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463, , doi: /nature08760 (2010). 18 Mehle, A. & Doudna, J. A. A host of factors regulating influenza virus replication. Viruses 2, , doi: /v (2010). 19 Bradel-Tretheway, B. G. et al. Comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and RNA polymerase accessory factors. Journal of virology 85, , doi: /jvi (2011). 20 Reich, S. et al. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Submitted (2014). 9

Correlated mutations in the four influenza proteins essential for viral RNA synthesis, host adaptation, and virulence: NP, PA, PB1, and PB2

Vol.2, No.10, 1138-1147 (2010) http://dx.doi.org/10.4236/ns.2010.210141 Natural Science Correlated mutations in the four influenza proteins essential for viral RNA synthesis, host adaptation, and virulence: