Viruses encoding mutant membrane protein

A method of preparing a recombinant influenza virus comprising a mutant membrane protein gene which does not encode a functional membrane protein or a functional portion thereof, comprising:

• (i) contacting a host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises:
• a) at least two vectors selected from a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to a mutant M2 cDNA linked to a transcription termination sequence, wherein the mutant M2 cDNA comprises at least two mutations relative to a corresponding M2 gene which encodes a functional membrane protein, one of which mutations is not in the transmembrane domain, the presence of which in the mutant gene, when the mutant gene is transcribed and translated in the host cell, does not yield a functional membrane protein or a functional portion thereof, and b) at least two vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2;
• and
• (ii) isolating the virus.