Creation of amantadine resistant clones of influenza type A virus using a new transfection procedure.

M2, the spliced segment of the matrix (M) gene of influenza A virus, is an integral membrane protein which functions as an ion channel both when the virus is in the host endosome and during protein processing in the trans-Golgi network. Amantadine inhibits replication of influenza A virus by blocking the activity of this ion channel. Reverse genetics were used to generate amantadine resistant virus mutants by introducing mutations into the M gene of cold adapted (ca) A/AA/6/60, an amantadine sensitive virus. The site directed mutagenesis involved substitutions at amino acids 27, 30 and 31, sites hypothesized to be responsible for resistance to this drug in several other influenza A viruses. This M gene was then transfected into wt A/AA/6/60, an amantadine sensitive virus, via electroporation. The desired transfectants were selected for replication in the presence of amantadine. Using this newly devised reverse genetics system to rescue a mutated gene in its homologous wild type background not only establishes the identity of amino acid mutations necessary for the establishment of amantadine resistance but will also allow us to study other mutations in the M gene without gene constellation effects. Resistance to amantadine in wt A/AA/6/60 can also occur naturally if the viruses are grown in the presence of amantadine. These spontaneously generated resistant clones contained point mutations at amino acid 30 or 31 of M2.