Identification of a Novel Splice Variant Form of the Influenza A Virus M2 Ion Channel with an Antigenically Distinct Ectodomain

Abstract

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

“…Moreover, several other novel viral proteins have been shown to be expressed by splicing, alternative initiation, or ribosomal frameshifts. For example, M42 and NS3 are translated from spliced mRNAs transcribed from the M and NS segments, respectively (15,16). M42 functions in place of M2 as a proton channel (15), and NS3 is associated with the adaptation of avian influenza A virus to new mammalian hosts (16).…”

“…For example, M42 and NS3 are translated from spliced mRNAs transcribed from the M and NS segments, respectively (15,16). M42 functions in place of M2 as a proton channel (15), and NS3 is associated with the adaptation of avian influenza A virus to new mammalian hosts (16). Approximately 30 of Ͼ18,000 isolates likely express M42 and NS3, based on nucleotide sequence analyses (15,16).…”

Identification of Conserved Peptides Comprising Multiple T Cell Epitopes of Matrix 1 Protein in H1N1 Influenza Virus

“…The M1 protein is transcribed and further translated as one of the proteins coded by segment seven of the influenza A genome, along with other two splice variant: M2 ion channel and M42 (53). The M1 monomer (252 amino acid) is 60 Å long (40), carrying two globular regions-the N-terminal (1-164) and C-terminal (165-252)-linked by a proteasesensitive loop.…”

Role and application of RNA interference in replication of influenza viruses

“…The genome of influenza viruses consists of eight molecules of single-stranded RNA of negative polarity that encode the three subunits of the viral polymerase complex, polymerase basic protein 1(PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA); the nucleoprotein (NP); the glycoproteins hemagglutinin (HA) and neuraminidase (NA); the matrix protein M1; the ion channel protein M2; the non-structural protein NS1; the nuclear export protein (NEP); and the recently discovered PB1-F2, NP40, PA-X and M42 proteins (Chen et al, 2001;Wise et al, 2009Wise et al, , 2011Wise et al, , 2012Jagger et al, 2012). The ability of influenza viruses to continuously mutate has made control strategies based on vaccination difficult.…”