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. 2016 Aug 25;2(8):e000082.
doi: 10.1099/mgen.0.000082. eCollection 2016 Aug.

Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore

Muhammad Raihan Jumat  1 , Puisan Wong  2 , Raphael Tze Chuen Lee  3 , Sebastian Maurer-Stroh  1   3 , Boon Huan Tan  2 , Richard J Sugrue  1
Affiliations

Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore

Muhammad Raihan Jumat et al. Microb Genom. .

Abstract

In this study we compared the NS1 protein of Influenza B/Lee/40 and several non-cultured Influenza B virus clinical strains detected in Singapore. In B/Lee/40 virus-infected cells and in cells expressing the recombinant B/Lee/40 NS1 protein a full-length 35 kDa NS1 protein and a 23 kDa NS1 protein species (p23) were detected. Mutational analysis of the NS1 gene indicated that p23 was generated by a novel cleavage event within the linker domain between an aspartic acid and proline at amino acid residues at positions 92 and 93 respectively (DP92-93), and that p23 contained the first 92 amino acids of the NS1 protein. Sequence analysis of the Singapore strains indicated the presence of either DP92-93 or NP92-93 in the NS1 protein, but protein expression analysis showed that p23 was only detected in NS1 proteins with DP92-93.. An additional adjacent proline residue at position 94 (P94) was present in some strains and correlated with increased p23 levels, suggesting that P94 has a synergistic effect on the cleavage of the NS1 protein. The first 145 amino acids of the NS1 protein are required for inhibition of ISG15-mediated ubiquitination, and our analysis showed that Influenza B viruses circulating in Singapore with DP92-93 expressed truncated NS1 proteins and may differ in their capacity to inhibit ISG15 activity. Thus, DP92-93 in the NS1 protein may confer a disadvantage to Influenza B viruses circulating in the human population and interestingly the low frequency of DP92-93detection in the NS1 protein since 2004 is consistent with this suggestion.

Keywords: B/Lee/40; Cleavage; Influenza B; NS1; Proteolysis.

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Figures

Fig. 1.
Fig. 1.
SDS–PAGE analysis of influenza B NS1 protein. (a). Time course infection of B/Lee/40 on HEK 293T cells. Cells were harvested at 4, 8, 12 and 16 h post infection and immunoblotted with αNS1. (b). HEK 293T cells were infected with B/Lee/40 virus (inf) or transfected with pCAGGS/NS1B-FLAG (Trans) and after 20 h the cell lysates were immunoblotted with αNS1. Mock Trans: Cells transfected with empty pCAGGS vector. Mock Inf: non-infected cells. (c). pCAGGS/NS1B-FLAG-transfected HEK 293T cell lysates were harvested at 4, 8, 12 and 16 h post transfection and immunoblotted using anti-FLAG. The full length (black arrow) and smaller (*) NS1 proteins are indicated.
Fig. 2.
Fig. 2.
Expression analysis of individual NS1 domains. (a). Schematic representation of the full-length NS1 protein and separate domains of Influenza B/Lee/40 NS1 which were cloned into pCAGGS vector with N-terminal cMyc (m) and C-terminal FLAG(f) epitope tags. (i) NS1f, (ii) mNS1f; (iii) mRB (; (iv) mRBLf; (v) EDf and (vi) mLEDf. Numbers at the top of the diagram indicate amino acid sequence positions. (b). At 16 h post transfection HEK 293T cells expressing each of the recombinant NS1 proteins indicated were immunoblotted with anti-Myc and anti-FLAG antibodies as appropriate. Protein bands corresponding to the full-length expressed protein (black arrow), p15 (**) and p23 (*) are indicated. NS1, full-length protein; RB, RNA binding domain; RBL, RNA binding domain with linker; ED, effector domain; LED, linker with effector domain.
Fig. 3.
Fig. 3.
Amino acid sequence alignment of influenza B NS1 proteins used in this study. The coding sequences of the NS1 gene from B/Lee/40 (NS1B(LEE)-FLAG) and clinical specimens DSO_090136_2004 (NS1B(136)-FLAG); DSO_040117_2006 (NS1B(117)-FLAG); DSO_020132_2007 (NS1B(132)-FLAG DSO_010147_2007); DSO_0070_2009 (NS1B(70)-FLAG) and DSO_010147_2007 (NS1B(147)-FLAG). A FLAG epitope tag at the C-terminus (highlighted in yellow) and linker domain (amino acids 91–119) (highlighted in blue) are indicated. (.) Indicates identical sequence to NS1B(LEE)-FLAG. (*) correspond to the stop codon in the nucleotide sequence.
Fig. 4.
Fig. 4.
Expression of NS1 protein from five clinical specimens. (a). Amino acid alignment of NS1B(LEE)-FLAG (Lee-NS1), NS1B(136)-FLAG (136-NS1), NS1B(117)-FLAG (117-NS1), NS1B(132)-FLAG (132-NS1), NS1B(70)-FLAG (70-NS1) and NS1B(147)-FLAG (147-NS1) at amino acid positions 70–140. The linker domain (highlighted in blue) and the flanking RNA binding domain (RNABD) and effector domain (ED) are highlighted (b and c). Expression of the NS1 constructs in HEK 293T cells. At 16 h post transfection cells expressing NS1B(LEE)-FLAG, NS1B(136)-FLAG, NS1B(117)-FLAG, NS1B(132)-FLAG, NS1B(70)-FLAG and NS1B(147)-FLAG were immunoblotted with (b) anti-FLAG antibody or (c) αNS1 Cells transfected with empty pCAGGS Vector (V) or infected with Influenza B/Lee/40 (Lee(v)) are indicated. The NS1 protein (black arrows) and p23 (*) are highlighted.
Fig. 5.
Fig. 5.
Mutational analysis of Influenza B NS1 protein. (a). Sequence alignment (between amino acids 81 and 102) of Lee-NS1 and the mutants Lee-NS1 D92N, Lee-NS1 D92A, Lee-NS1 M91A, Lee-NS1 P93A and Lee-NS1 S94P. Also shown are 117-NS1 and 117-NS1 P94S. (B–E). Protein expression profile of (b) Lee-NS1 wild-type (WT) and mutant Lee-NS1 D92A, (c). 132-NS1 wild-type (WT), 132-NS1 N92D, Lee-NS1 wild-type (WT) and Lee-NS1 D92N, (d). Lee-NS1 wild-type (WT), Lee-NS1 M91A, Lee-NS1 P93A, Lee-NS1 S94P and (e). 117-NS1 wild-type (WT) and 117-NS1 P94S. At 16 h post transfection, NS1 protein was detected by immunoblotting with anti-FLAG. pC: Cells transfected with empty pCAGGS vector. Protein bands corresponding to NS1-FLAG (black arrow) and p23-FLAG (*) are indicated.
Fig. 6.
Fig. 6.
p23 is located in the cytoplasm of NS1-expressing cells. (a). HEK 293T cells were infected with influenza B/Lee/40 virus and at 20 h post infection the cells were fractionated into cytoplasmic (Cyto) and nuclear (Nuc) fractions. Total homogenate (Total) and the Cyto and Nuc fractions were then immunoblotted with αNS1. The full-length NS1 protein (black arrow) and p23 (*) are highlighted. (b) HEK 293T cells expressing c-yc-NS1(LEE)-FLAG were fractionated into cytoplasmic (Cyto) and nuclear (Nuc) fractions, and the total homogenate (Total), Cyto and Nuc fractions were immunoblotted using (i) anti-FLAG and (ii) anti-cMyc. The full-length NS1 protein (black arrows), p23 (*) and p15(**) are highlighted. (iii) Immunoblotting with anti-lamin A/C (nuclear marker). (c). Fractionation analysis of HEK 293T cells expressing Lee-NS1, 136-NS1, 117-NS1, 132-NS1, 70-NS1 and 147-NS1. Cytoplasmic and nuclear fractions are indicated. Immunoblotting was performed with anti-FLAG to detect the NS1 protein. Anti-tubulin (cytoplasmic marker) and anti-lamin A/C (nuclear marker) are loading controls for each fraction. The full lenght NS1 protein (black arrows) and p23 (*) are highlighted in immunoblotting using anti-FLAG. The location of Tubulin and Lamin A/C is also highlighted (black arrows) in immunoblotting using anti-Tubulin and anti-Lamin A/C respectively.
Fig. 7.
Fig. 7.
The frequency of the NS1 N92D substitution from 1980 to 2015 in influenza B viruses. (a) Frequency changes of the NS1 N92D substitution and the total count of isolates in each year from 1980 to 2015. (b) Global count of residues at positions 92 and 93 of NS1 from Influenza B viruses. Count of residues from all 5285 Influenza B sequences from 1940 to 2015. The N92D substitution represents a frequency of 6.6 % in all Influenza B NS1 sequences.
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Data Bibliography

    1. Jumat, M. R., Sugrue, R. J. & Tan, B. H., GenBank, Influenza B virus nuclear export protein (NEP) gene, partial cds; and nonstructural protein 1 (NS1) gene, complete cds. PopSet: 482577243 KC844161-KC844196 (2014)
    1. Jumat, M. R., Sugrue, R. J. & Tan, B. H., GenBankGenBank, Influenza B virus (B/Singapore/DSO_090136/2004) segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) genes, complete cds. GenBankGenBank: KC844195.1 (2016)
    1. Jumat, M. R., Sugrue, R. J. & Tan, B. H., GenBankGenBank, Influenza B virus (B/Singapore/DSO_040117Variant/2006) segment 8 NS1 gene, complete sequence; nonstructural protein 1 (NEP) gene, complete cds; and nuclear export protein (NS2) gene, partial cds, GenBankGenBank: KU500812.1 (2016)
    1. Jumat, M. R., Sugrue, R. J. & Tan, B. H., GenBankGenBank, Influenza B virus (B/Singapore/DSO_010147/2007) segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) genes, complete cds, GenBankGenBank: KC844165.1 (2016)
    1. Jumat, M. R., Sugrue, R. J. & Tan, B. H., GenBankGenBank, Influenza B virus (B/Singapore/DSO_020132/2007) segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) genes, complete cds, GenBankGenBank: KC844167.1 (2016)

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