Supplementary Figure 1. SC35M polymerase activity in the presence of Bat or SC35M NP encoded from the phw2000 rescue plasmid.
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1 Supplementary Figure 1. SC35M polymerase activity in the presence of Bat or SC35M NP encoded from the phw2000 rescue plasmid. HEK293T cells were transiently transfected with expression plasmids coding for PB2, PB1, PA of SC35M, the indicated NP proteins encoded by phw2000 plasmids harboring the packaging sequences of SC35M (see also Fig. 1b), a minigenome encoding the firefly luciferase and a Renilla luciferase expression plasmid to normalize for variations in transfection efficiency. In the negative control (Neg) PB1 was omitted. Error bars indicate the mean and SD of at least 3 independent experiments. Western blot analysis was performed to determine the expression levels of NP.
2 Supplementary Figure 2. Location and conservation of the bat influenza A-like NP amino acid in CH2 (a) Alignment (amino acid 56 to 133) of SC35M NP and CH2 harboring 18 Bat NP-specific amino acids. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n = 27,675 strains). (b) Positions of Bat-NP-specific amino acids in the modeled crystal structure of SC35M NP. The program PyMOL was used to assign the indicated positions. The body and head domains of NP are colored in dark and light grey, respectively. Bat specific amino acids are located in the body domain of NP and marked in green.
3 Supplementary Figure 3. Single cycle growth behavior of rch2, rnp7 and wt SC35M. MDCKII cells were infected at an MOI of 5 with the indicated viruses and time points post infection (p.i.). Virus titers were determined by plaque assay. Student s t test was used for two-group comparisons. Error bars indicate the mean and SD of 3 independent experiments. *P < 0.05; ***P < 0.001, ****P <
4 Supplementary Figure 4. SC35M polymerase reconstituted with CH2 efficiently replicates the NP segment. (a) HEK293T cells were transiently transfected with expression plasmids coding for PB2, PB1, PA of SC35M, the indicated NP proteins, the SC35M NP segment with a mutation in the initiation codon (ATG to ACG) to prevent synthesis of segment-encoded NP. In the negative control (Neg.) PB1 was omitted. Steady state levels of viral transcripts (mrna, crna and vrna) and 5S ribosomal RNA (5S rrna) were determined by primer extension analysis using total RNA 24 hours post transfection. (b) Signal intensities obtained in panel (a) were normalized to the signal intensities obtained with 5S rrna. Normalized values obtained with wt SC35M NP were set to 1 (all nonsignificant). Student s t test was used for two-group comparisons. Error bars indicate the mean and SD of 3 independent experiments.
5 Supplementary Figure 5. Analysis of viral particles by transmission electron microscopy (TEM). Depicted are representative micrographs of concentrated virus stocks and ultrathin sections of infected MDCK II cells. Virus particles with transversely sectioned vrna segments are indicated by arrows.
6 Supplementary Figure 6. Identification of Bat NP-specific amino acids in NP7 that cause attenuation. (a) Alignment of SC35M NP and NP7 mutant proteins. The bat NP-specific amino acids are highlighted in the color code used in Fig. 3c. Upper panel shows the sequence logo of the consensus sequence of NP of available IAV strains (n = 27,675 strains). (b) Ability of the indicated NP mutant proteins to support polymerase activity and rescue of SC35M. Relative SC35M polymerase activities in the presence of the mutant NP proteins were determined by polymerase reconstitution assays in HEK293T cells and values represent the relative polymerase activity. Numbers in parenthesis indicate the mean and SD of at least 3 independent experiments. Bat specific amino acids and nucleotides are indicated. SC35M rescue experiments were performed with NP segments encoding the indicated mutant proteins. + successful rescue. (c) MDCKII cells were infected at an MOI of with wt SC35M or mutant viruses. At the indicated time post infection (p.i.), virus titers were determined by plaque assay. Student s t test was used for two-group comparisons. Error bars indicate the mean and SD of at least 3 independent experiments. *P < 0.05; **P < 0.01.
7 Supplementary Figure 7. Effects of Bat NP-specific amino acids encoded by rnp7 on subcellular localization of viral proteins. Localization of NP, M1 or HA in MDCKII cells infected with the indicated viruses at an MOI of 5 for 4, 6 and 8 hours. For surface staining of HA cell were only fixed and not permeabilized. Scale bar: 10μM.
8 Supplementary Figure 8. Bat NP causes impaired genome packaging of SC35M Packaging efficiency mediated by SC35M NP, or Bat NP in the presence of the reporter segment IS+GFP (left panel) or IST+BS-GFP (right panel) and if indicated in the presence of the remaining 7 wild type genome segments (+7). **P < 0.01, ***P < 0.001, ****P < R, reporter genome. Error bars indicate the mean and SD of at least 3 independent experiments
9 Supplementary Figure 9. Uncropped scans of Western blot and primer extensions
10 Supplementary Table 1. NP chimeras. Name Primers used for cloning* GenBank accession numbers CH1 01;02;03;04;05;06 KU CH2 01;02;03;04;07;08 KU CH3 01;02;05;06;07;08 KU CH4 09;10;11;12;13;14 KU CH5 01;02;05;06 KX CH ;16;17;18 KU CH4.4 18;19;20;21 KU CH4.0 22;23 KU NP14 18;24;25;26 KU NP10 24;27;28;29 KU NP7 18;24;30;31 KU NP7(2-7) 18;24;32;33 KU NP7(4-5) 18;24;34;35 KU NP7(2-3,6-7) 18;24;34;36 KU NP7(1,4-5) 18;24;35;37 KU NP7(1-3,6-7) 18;24;36;37 KU NP7-R31G 18;24;38;39 KU * Numbered accordingly to Supplementary table 2.
11 Supplementary Table 2. DNA oligos used for cloning. Nr. Name Sequence (5 3 ) F GATCGCGGCCGCACCATGGCGTCTCAAG R GATCCTCGAGTTAATTFTCATATTCCTCTGCATTG F CTGATCCAAAACAGCATAACAATTGAGAGGATGG R CCATCCTCTCAATTGTTATGCTGTTTTGGATCAG F GACTTTGAAAGAGAGGGATACTCTCTAGTCGG R CCGACTAGAGAGTATCCCTCTCTTTCAAAGTC F CTGGTCTCACCCATTTGATGATCTGGCATCCAACTGAAT R ATTCAAGTTGGAGTGCCAGATCATCAAATGGGTGAGACCAG 09 5-Fr1 CCATGGCGTCTCAGGGAGCGAAAGCAGGGTAGATAATCACTCACCG AGTG 10 3-Fr1 CCATGGCGTCTCAAGCATCATTCAGGTTGGAATGCCAGAT 11 5-Fr2 CCATGGCGTCTCATGCTACCTATCAGCGAACAAGAGCCC 12 3-Fr2 ACAAGGCGTCTCAGGCATGACTTATGAGCTACTGCTCCTCGGAG 13 5-Fr3 CCATGGCGTCTCATGCCTCCCAGCCTGTGTATATGGAC 14 3-Fr3 CCATGGCGTCTCATATTAGTAGAAACAAGGGTATTTTTCTTTAATTG 15 5-Fr1 GTTGATCCGGATGATCAAAAGAGGAATGAATGACAGAAATTTCTGGAA GGGTGAACAAGGGAGAAGAACTCGAATAGCTTATGAAAGGCTCTGTA ACATCCTGAAAGGAAAGTTTCAAACAGCCGCACAG 16 3-Fr1 AGGGCAGATCTTGCAAGAAATAGTAAGTCCTCTATTTCTGCATTTCCT GGGTTTTTGCTTTCTTTGACTTGATCAACCATTGCTTTCTGTGCGGCT GTTTGAAACTTTCCTTTCAGGATGTTACAGAGCC 17 5-Fr2 CTTGCAAGATCTGCCCTGATCC 18 3-Fr2 ATCCACTGGCCACAGAAAGTCCATA 19 5-Fr1 GTTGATCCGGATGATCAAAAGAGGAATCAATGACAGAAATTTCTGGA GGGGTGAAAATGGGAGAAGAACTCGAATAGCTTATGAAAGGATGTGT AACATCCTGAAAGGAAAGTTTCAAACAGCCGCACAG 20 3-Fr1 AGGGCAGATCTTGCAAGAAATATTAAGTCCTCTATTTCTGCATTTCCT GGGTTTCTGCTTTCTCTGACTTGATCCATCATTGCTCGCTGTGCGGCT GTTTGAAACTTTCCTTTCAGGATGTTACACATCC 21 5-Fr2 ATTTCTTGCAAGATCTGCCCTGATCCTCCGAGGATCAGTAGCTCATAA 22 5-Fr1 TAATCACTCACCGAGTGACATCCACATCATGGCGTCCCAAGGCACC 23 3-Fr1 GATCATCCGGATCAACTCCATTACCATGGTCCCCACTCCTTTGACTGC TGCCCCAGCTGCTCCAGATCTTCTTGG 24 5-Fr1 ACCATGGCGTCTCAAGGCACCAAACGATCT 25 3-Fr1 CCTCGCTTGATCATCCGAATCAAT 26 5-Fr2 GATCAAGCGAGGGATGAATGATCGGAATTTCTGGAAAGGCGAACAAG GGCGGAGAACAAGAATTGCATACGAGAGACTGTGTAACATTCTCAAG GGGAAATTTCAAACAG 27 3-Fr2 CAGCACAAAAAGCAATGGTGGACCAGGTGAAGGAAAGCAAGAATCCT GGGAATGCTGAAATTGAAGATCTCCTCTTTCTTGCACGGTCTGCTCTC ATTCTGAGGGGAGCAGTGGCTCATAAGTCCTGCCTGCCTGCTTGTGT GTATGGACTTTCTGTGG 28 3-Fr1 AATTTGCGTCTCTCCATTATGAGTGTTCCAACCCCTTTCACTGCAGTT CCAGCAGCCCCAGCCCTCCTTGG 29 5-Fr2 ATTATGGCGTCTCAATGGAATTGATTCGGATGATCAAGCGAGGG 30 3-Fr1 ACCATGGCGTCTCAGATCATCCGAATCAATTCCATTACCATTGTTC 31 5-Fr2 AACCGGCGTCTCAGATCAAGCGAGGGATCAATGATCGGAATTTCTGG AGAGGCGAAAATGG 32 3-Fr1 CCATGGCGTCTCACTGTTTGAAATTTCCCCTTGAGAATGTTACACATT CTCTCGTATGCAAT 33 5-Fr2 CCATGGCGTCTCAACAGCAGCACAAAAAGCAATGGTGG 34 3-Fr1 ACCATGGCGTCTCACTGTTTGAAATTTCCCCTTGAG
12 Fr2 CCATGGCGTCTCAACAGCAGCACAACGAGCAATGATGGACCAGGTG AAGGAAAGCAAGAATCC 36 5-Fr2 CCATGGCGTCTCAACAGCAGCACAAAAAGCAATGGTGGACCAGGTGA GGGAAAGCCGGAATCCTGGG 37 3-Fr1 CCATGGCGTCTCACTGTTTGAAATTTCCCCTTGAGAATGTTACACAGT CTCTCGTATGCAAT 38 3-Fr1 AACCGGCGTCTCTAACCATTCCCCCAACAGATGCTCTG 39 5-Fr2 ACCATGGCGTCTCAGGTTGGTGGAATCGGGAGATTCT
13 Supplementary Table 3. Nucleotide composition of NP chimera CH1, CH2, CH3, CH4 and CH5 based on the positive sense of the respective genome segments. Name 5 end of SC35M NP (nt) Bat NP ORF (nt) 3 end of SC35M NP (nt) CH CH CH CH CH
14 Supplementary Table 4. DNA oligos used for primer extension analysis. Primer Sequence (5-3 ) Binding site* Size (nt) PB2 vrna TGCTAATTGGGCAAGGAGAC cmrna GCCATCATCCATTTCATCCT PB1 vrna TGACTTCGAGTCTGGACGGA cmrna TCCATGGTGTATCCTGTTCC PA vrna GCCTGATTAATGATCCCTGGG cmrna CACCCCGTTCATCAACGAAATGG HA vrna GGTTTAGCTTCGGGGCATC cmrna CCCGATATTCGCGGTTTCCAC NP vrna GATGTGTCTTTCCAGGGGCG cmrna GCCTCCCTTCATAGTCGCTG NA vrna TGGACGAGCAACAGCTTAGT cmrna CGTCGTGTTGTTCTGTGTTATGC M vrna TACGGATTGAAAAGAGGGC cmrna TTCCCTTAGTCAGAGGTGAC NS vrna GTTTGAAGAAATAAGGTGGC cmrna GCTGTTTCGATGTCCAGACC * based on 5 to 3 numbering of the template sequence in the positive sense.
15 Supplementary Table 5. SC35M segment-specific primers for quantitative RT-PCR. Segment Direction Sequence (5-3 ) F GCACAGGATGTAATCATGGAAGT PB2 R ACCAATTCTCTTTCTAGCATGTATGC F CCAGACCTATGATTGGACATTAAACA PB1 R CCGTTCGATCTGAAGACCTCTATAGT F AATGATCCCTGGGTTTTGCTTA PA R TGCCACAGCTATTTCAGTGCAT F GCTTTTGGGAGCAATTGCTGG HA R GTGCTTTTGTAGTCAGCTGCA F CTGATCCAAAACAGCATAACAATTGAGAGGATGG NP R ATTCAAGTTGGAGTGCCAGATCATCAAATGGGTGAGACCAG F CGTTCCAGAAGTGGTTTTGAGATG NA R AAACTCCCTGAGTATCCTGACC F CTTCTACCGAGGTCGAAACG M R AGGGCATTTTGGACAAAKCGTCTA F GGACCAGGCGATCATGGATAA NS R CAATTGCTCCTTCTTCGGTGAA
Supplementary Fig. S1. Schematic diagram of minigenome segments.
open reading frame 1565 (segment 5) 47 (-) 3 5 (+) 76 101 125 149 173 197 221 246 287 open reading frame 890 (segment 8) 60 (-) 3 5 (+) 172 Supplementary Fig. S1. Schematic diagram of minigenome segments.
Relative activity (%) SC35M
a 125 Bat (H17N) b 125 A/WSN (H1N1) Relative activity (%) 0 75 50 25 Relative activity (%) 0 75 50 25 0 Pos. Neg. PA PB1 Pos. Neg. NP PA PB1 PB2 0 Pos. Neg. NP PA PB1 PB2 SC35M Bat Supplementary Figure
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