pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion.
The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66-76, 1996, and J. Virol. 71:4062-4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the alpha-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound.
Cianci
C
C
Departments of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492, USA.
Yu
K L
KL
Dischino
D D
DD
Harte
W
W
Deshpande
M
M
Luo
G
G
Colonno
R J
RJ
Meanwell
N A
NA
Krystal
M
M
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping; D019920:Photoaffinity Labels / Q000378:metabolism*; D011487:Protein Conformation; D011807:Quinolizines / Q000494:pharmacology