Darwinian selection for sites of Asn-linked glycosylation in phylogenetically disparate eukaryotes and viruses. at AS and other sites (NAS) that are located close to and distantly from the NGS in the three-dimensional structure of HA through a comparison of the rates of synonymous (and and denote the numbers of synonymous and nonsynonymous differences summed over the branches of sites included in each category, and and denote the numbers of synonymous and nonsynonymous sites averaged over the branches of sites included in each Ivermectin category with the weight proportional to the branch length. The numbers of synonymous and nonsynonymous differences as well as the numbers of synonymous and nonsynonymous sites for each branch of individual codon sites were obtained by comparing the sequences at the ancestral and descendant nodes using the modified Nei-Gojobori method with = 10. Since positions 165 and 285 appeared to be NGS throughout the evolutionary history of human A/H3N2 virus, the analysis of and for the branches where the position was an NGS and a non-NGS separately. The null hypothesis of equal and for both branches. Natural selection operating at AS10c, AS15c, AS10uc, AS15uc, NAS10c, NAS15c, NAS10uc, and NAS15uc was examined for the branches where the position under consideration was an NGS and a non-NGS separately. The proportions of and were used to obtain the expected values of and (25), and the null hypothesis of selective neutrality was Rabbit Polyclonal to RBM5 tested by the chi-square test with one degree of freedom. Positive and negative selection were inferred when the observed values of and were significantly greater than the expected values of and and NGS= 0.03 by 2 test). When the positions mentioned above became NGS, = 0.03 by binomial test). Furthermore, the reduction of 0.05 by 2 test). Table 2 NGS class,and NGS 0.05; **, 0.01). dThe 0.05). NA indicates that 0.01 by 2 test). For AS10uc and AS15uc, the NGS class,and NGS 0.05). dThe 0.05). NA indicates that em d /em N/ em d /em S was incalculable because em d /em S = 0. It should be noted that the em d /em N/ em d /em S value at AS may be influenced by natural selection operating not only on the specific function of AS but also on the HA as a whole. To eliminate the possibility that the reduction of em d /em N/ em d /em S at AS10c and AS15c observed above reflected a change in functional constraints on the entire region of HA, e.g., due to a change in the effective population size of human A/H3N2 virus, em d /em N/ em d /em S was also computed for NAS10c, NAS15c, NAS10uc, and NAS15uc.The em d /em N/ em d /em S values obtained at NAS generally were much smaller than those obtained at AS, and negative selection was detected in most cases whether the amino acid positions 45, 63, 81, 122, 126, 133, 144, 246, and 276 were non-NGS or NGS (see Tables S2 and S3 in the supplemental material). Ivermectin Although a significant difference in em d /em N/ em d /em S was observed between NGS and non-NGS branches in some cases, em d /em N/ em d /em S was usually greater in the former branches than in the latter, which was the opposite of the pattern observed at AS10c and AS15c. DISCUSSION After the emergence of A/H3N2 virus in the human population in 1968, NGS were generated at amino acid positions 63, 122, 126, 133, 144, and 246 in the globular head region of HA. Since the glycosidic linkage of oligosaccharides may be flexible, N-glycans may have multiple conformations in solution (26). In addition, the structure of N-glycans may vary among NGS in HA (13, 15). For these reasons, two different distances from NGS, 10 and 15 ?, were used as the possible ranges of amino acid positions covered by N-glycans in the present study. When positions 63, 122, 126, 133, 144, and 246 were non-NGS, the em d /em N/ em d /em S value was 1 normally for both AS10c and AS15c and positive selection was recognized at AS10c of position 144, suggesting that AS10c and AS15c of these positions contained target sites of Ab. However, the em d /em N/ em d /em S ideals at AS10c and Ivermectin AS15c decreased, and the evidence of positive selection disappeared when the positions mentioned above became NGS. In contrast, the em d Ivermectin /em N/ em d /em S ideals at AS10uc and AS15uc generally were 1 and did not switch to any large extent whether the positions mentioned above were NGS or non-NGS. In addition, the em d /em N/ em d /em S ideals at NAS10c, NAS15c, NAS10uc, and NAS15uc often were higher when these positions were NGS than Ivermectin when they were non-NGS, which was.