Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein.
We established a reverse genetics system for the M gene of influenza A virus, using amantadine resistance as a selection criterion. Transfection of an artificial M ribonucleoprotein complex of A/Puerto Rico/8/34 (H1N1), a naturally occurring amantadine-resistant virus, and superinfection with amantadine-sensitive A/equine/Miami/1/63 (H3N8), followed by cultivation in the presence of the drug, led to the generation of a transfectant virus with the A/Puerto Rico/8/34 (H1N1) M gene. With this system, we attempted to generate a virus containing a deletion in an M-gene product (M2 protein). Viruses lacking the carboxyl-terminal Glu of M2, but not those lacking 5 or 10 carboxyl-terminal residues, were rescued in the presence of amantadine. These findings indicate that carboxyl-terminal residues of the M2 protein play an important role in influenza virus replication. The M-gene-based reverse genetics system will allow the study of different M-gene mutations to achieve a balance between attenuation and virus replication, thus facilitating the production of live vaccine strains.
Castrucci
M R
MR
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Kawaoka
Y
Y
D000547:Amantadine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004285:Dogs; D004352:Drug Resistance, Microbial / Q000235:genetics; D005814:Genes, Viral; D009980:Influenza A virus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D007473:Ion Channels / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017384:Sequence Deletion; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication / Q000235:genetics / Q000502:physiology; D000086082:Viroporin Proteins
D000547:Amantadine; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D007473:Ion Channels; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins