Amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the pH of glycoprotein transport vesicles.
Mutants of influenza Rostock virus (H7N1 subtype) were selected for resistance to amantadine hydrochloride at concentrations of the antiviral drug known to affect the function of the virus M2 transmembrane protein. Sequence analysis revealed that three mutants had no changes in M2 but contained a lysine to isoleucine substitution in the hemagglutinin (HA) membrane glycoprotein at position 58 of HA2. The mutant viruses were found to fuse membranes at a pH value 0.7 lower than wild type and to exhibit changes in the conformation of their HAs specifically at the lower pH. The homologous lysine to isoleucine substitution was introduced by site-specific mutagenesis into the HA of X-31 influenza virus (H3 subtype), which was expressed by using vaccinia virus recombinants. The expressed HA also mediated membrane fusion and changed in conformation at a pH value 0.7 lower than wild type. These results indicate that increased acid stability of the HA obviates the consequences of the inhibition of M2 function by amantadine and provide further evidence for the role of M2 in regulating the pH of vesicles involved in glycoprotein transport to the cell surface.
Steinhauer
D A
DA
National Institute for Medical Research, Mill Hill, London, United Kingdom.
Wharton
S A
SA
Skehel
J J
JJ
Wiley
D C
DC
Hay
A J
AJ
D000547:Amantadine / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004352:Drug Resistance, Microbial; D004797:Enzyme-Linked Immunosorbent Assay; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008561:Membrane Fusion; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014616:Vaccinia virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D014759:Viral Envelope Proteins; D000547:Amantadine