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Abstract
Influenza is a serious illness. Variation in its surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), renders vaccines ineffective in controlling the infection. Alternative therapeutic approaches are being evaluated. Structure-based inhibitor design is becoming an important methodology for developing compounds for curing infectious diseases. The influenza protein NA, a sialidase, is an excellent target for antiviral strategies because it has a conserved active site that can be exploited for inhibitor design. The sialidase activity is essential in the virus life cycle, thus inhibiting the enzyme abrogates infection. Designing inhibitors against the NA active site necessitates a detailed understanding of the residues involved in catalysis.
In order to further our understanding of the mechanism and function of NA, we made substitutions in the NA active site residues and carried out kinetic analysis of the mutants. The kinetic parameters facilitated interpretation in terms of transition state binding and product release, aiding in defining residues essential for NA activity: Asp 149, Glu 275, and Tyr 409. Inhibitors targeted to these residues would be effective in that the virus would not be able to escape by changing these residues without losing NA activity.
Extrapolation of these observations to the in vivo situation was accomplished by selecting variants against NA active site inhibitor 4-(acetylamino)-3-guanidinobenzoic acid (BCX-140). This inhibitor binds Glu 275, an essential catalytic residue. Viruses escaped from the inhibitor, but there was no change in Glu 275 or any other NA active site residues. Instead, there were changes in the HA. This suggested that the inhibition of sialidase activity of NA could be circumvented by changes in the HA, especially in its sialic acid binding site. Thus, effective therapies targeted to NA require a further understanding of its role in the virus life cycle. To evaluate differences in the functions of type A and B NA proteins, we determined whether lack of forming a type A/B NA reassortant virus is due to differences in the function or activity of the two proteins. Using a complementation technique, we observed that the function of the cytoplasmic, anchor, head, and to a large extent the stalk domains of NA in promoting virus growth is similar in type A and B viruses. Moreover, cosedimentation of NA activity and virus in sucrose gradient fractions indicated the ability of both type A and B NAs to be packaged in a type A virus particle.
Thus, in spite of vast sequence differences, structural and functional conservation of type A and B NAs suggests that, in addition to the head domain, the anchor and cytoplasmic domains of NA should be evaluated as potential targets for inhibitor design.
