The M2 protein of live, attenuated influenza vaccine encodes a mutation that reduces replication in human nasal epithelial cells
Introduction
Influenza A virus (IAV) is a member of the Orthomyxoviridae family and contains an 8-segment, negative-sense RNA genome encoding 10–14 proteins [1]. The live attenuated influenza vaccine (LAIV) is a 6:2 reassortant vaccine virus containing the PB2, PB1, PA, NP, M, and NS genome segments of a live-attenuated donor virus that was selected for its attenuated (att) and temperature-sensitivity (ts; replication at ≤32 °C but not ≥39 °C) phenotypes. There are 11 amino acid differences between the LAIV strain and the IAV strain from which it was derived [2]. The att and ts LAIV phenotypes have been mapped to the PB1 (E391, G581, T661), PB2 (S265), and NP (G34) genes by experiments in immortalized cell lines [3], [4], [5], [6], [7]. These experiments did not demonstrate a contribution of the M2-A86S mutation to temperature dependent LAIV replication. However, in vivo experiments in hamsters, ferrets and even humans have implicated the LAIV mutation M2-A86S as important for the att phenotype [3], [7], [8], [9]. Few studies have been performed to understand the molecular mechanisms conferring these phenotypes. During single-step replication in Madin-Darby canine kidney (MDCK) cells at 39 °C, viral RNA synthesis and vRNP export from the nucleus are reduced. Furthermore, incorporation of the viral matrix protein M1 into virus particles was reduced, causing heterogeneous and irregular virion morphology [6], [7]. LAIV replication is restricted within physiological ranges of temperature (32–37 °C) during infection of primary, differentiated human nasal epithelial cell (hNEC) cultures [10], [11], suggesting that other mutations in the LAIV genome may contribute to reduced virus replication in primary respiratory epithelial cell cultures.
The M2 protein is a 97 amino acid integral membrane protein with a 54 amino acid cytoplasmic tail [12]. It is a highly conserved protein required for virus entry [13], membrane scission [14] and the production of infectious virus particles [15], [16], [17], [18], [19]. M2 has been shown to form disulfide-linked homo-tetramers with pH-gated, proton-selective ion channel activity [20], [21]. The ion channel activity is critical for virus uncoating - by allowing protons to enter the virion interior, permitting the vRNP to dissociate from M1 [1], [22], [23], [24] - and preventing the premature cleavage of HA by neutralizing the pH in the late Golgi [25]. M2 has also been shown to be important for inhibition of autophagy and preventing autophagosome fusion with lysosomes [26], [27], [28].
The distal region of the M2 cytoplasmic tail is essential for IAV replication and is involved in vRNP incorporation into progeny virions [15], [16], [17], [18], [19]. When the M2 cytoplasmic tail is truncated by deleting the last 16 amino acids there is decreased release of infectious virus particles, which is not observed when only the last 8 amino acids are deleted [17]. When the region (M2 82–89) was mutated to alanine residues, infectious particle production was not altered. However, the original alanine residues at M2 positions 83 and 86 were never mutated and their contribution to the M2 protein functions were never investigated. The A83 residue is conserved across a wide range of IAV strains while A86 is highly conserved except for pandemic 2009 H1N1 viruses (Val) and LAIV (Ser).
To investigate the role amino acids at positions 83 and 86 of M2 may play in IAV replication and attenuation, we generated recombinant viruses with substitutions at these amino acids. This allowed us to show that both amino acid positions contributed to efficient virus replication. Both glutamic acid and serine substitutions at position 86 caused an approximately 2-log decrease in virus replication in primary human nasal epithelial cells (hNEC) but not MDCK cells. The magnitude of decrease was dependent on the temperature (greater reduction at 37 °C than at the permissive temperature of 32 °C) of the cells during infection. Introducing an M2-S86A substitution increased LAIV replication and altered interferon lambda (IFN-λ) production in hNEC cultures, indicating a role for the M2-86S amino acid change in both the att and ts phenotypes of LAIV.
Section snippets
Plasmids
The plasmid pHH21 expressing full length influenza virus gene segments was used to generate recombinant viruses [16] as described below. All mutations were introduced using the QuikChange Lightning site-directed mutagenesis kit (Agilent). All inserts and mutations were confirmed by sequencing. Primer sequences are available upon request.
Cell culture
MDCK cells and HEK 293 T (293 T) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL
Rescue of recombinant viruses with mutations in the M2 cytoplasmic tail
Previous work showed that the region of M2 containing amino acids 82–89 was important for virus replication and efficient genome packaging, but scanning alanine mutagenesis failed to reveal any single residue responsible for the phenotype [16]. However, substitutions at M2 amino acid positions 83 and 86 were not made. Since an Ala residue is present at M2 position 83 in nearly 100% of North American human influenza virus isolates, we replaced Ala83 with a number of amino acids to assess the
Discussion
The LAIV associated M segment has been shown to contribute, in part, to LAIV’s attenuation phenotype [3], [7], [8]. The only M segment mutation between the WT and cold-adapted strains of A/Ann Arbor/6/1960 is a missense mutation at M2 position 86 from an alanine in the WT stain to a serine in the cold-adapted vaccine stain [2]. Our data indicate that the M2-A86S mutation acquired during LAIV cold adaption contributes to reduced virus replication in hNEC cultures but not MDCK cells, in a
Acknowledgements
We thank the members of the Pekosz laboratory, Sabra Klein, and members of the Klein laboratory for useful and critical discussions of the data. The work was supported by the Shikani/El Hibri Prize for Discovery and Innovation (AP), R01 AI097417 (AP), HHSN272201400007C (AP), R01 AI072502 (APL) and T32 AI007417 (NW).
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These authors contributed equally to the manuscript.