The neuraminidase of influenza virus is a surface glycoprotein that catalyzes the hydrolysis of glycosidic linkages between terminal sialic acids and adjacent sugar moieties. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated. The neuraminidase active site is conserved in all influenza type-A and type-B isolates, which makes it an excellent target for drug design. To determine the potential for resistance to develop against neuraminidase inhibitors, we have constructed mutations in seven of the conserved active-site residues of a type B (B/Lee/40) neuraminidase and analyzed the effect of the altered side chains on enzyme activity. There is a reduction in k cat in all our mutants. A transition-state analogue inhibitor shows variation in K i with the mutant neuraminidases, allowing interpretation of the effects of mutation in terms of transition-state binding and product release. The results show that Tyr409 is the most critical residue for enzyme activity, but that Asp149, Arg223, Glu275 and Arg374 also play important roles in enzyme catalysis. Based on the pH profile of neuraminidase activity of the D149E mutant protein, we conclude that Asp149 is not a proton donor, but is involved in stabilizing the transition state. If designed inhibitors are targeted to these residues where mutations are highly deleterious, particularly Tyr409, Glu275 and Asp149, the virus is unlikely to generate resistance to the drug.

Post‐transcriptional insertion, substitution or deletion of nucleotides in RNA (RNA editing) has been observed in RNAs from a number of organisms but always in messenger RNA or transfer RNA. We report here that the 17S rRNA of the mitochondrial ribosome of Physarum polycephalum is edited at 40 sites with single cytidine insertions. The locations of the editing sites are fairly evenly distributed throughout the RNA and do not correspond to any obvious feature of the primary sequence or secondary structure. In addition to these cytidine editing sites are editing sites in which a nucleotide other than cytidine is inserted. At two sites a uridine is inserted and at two sites adenosine residues are inserted. This is the first report of mixed nucleotide insertional editing. These results imply that the editing mechanism in Physarum may be different from those proposed for the kinetoplastid protozoa.

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acidbased compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.