Correlating novel variable and conserved motifs in the Hemagglutinin protein with significant biological functions

Abstract

Background: Variations in the influenza Hemagglutinin protein contributes to antigenic drift resulting in decreased efficiency of seasonal influenza vaccines and escape from host immune response. We performed an in silico study to determine characteristics of novel variable and conserved motifs in the Hemagglutinin protein from previously reported H3N2 strains isolated from Hong Kong from 1968-1999 to predict viral motifs involved in significant biological functions.

Results: 14 MEME blocks were generated and comparative analysis of the MEME blocks identified blocks 1, 2, 3 and 7 to correlate with several biological functions. Analysis of the different Hemagglutinin sequences elucidated that the single block 7 has the highest frequency of amino acid substitution and the highest number of co-mutating pairs. MEME 2 showed intermediate variability and MEME 1 was the most conserved. Interestingly, MEME blocks 2 and 7 had the highest incidence of potential post-translational modifications sites including phosphorylation sites, ASN glycosylation motifs and N-myristylation sites. Similarly, these 2 blocks overlap with previously identified antigenic sites and receptor binding sites.

Conclusion: Our study identifies motifs in the Hemagglutinin protein with different amino acid substitution frequencies over a 31 years period, and derives relevant functional characteristics by correlation of these motifs with potential post-translational modifications sites, antigenic and receptor binding sites.

Figure 1

Selected 14 MEME Blocks in the HA1 consensus sequence from 1968–1999. Combined block diagram of non overlapping sites with p value < 0.0001 was generated from the MEME server which are common to the entire data set, with the exception of block 14 which occurs in only 16 of the 17 sequences.

Figure 2

Number of aminoacid substitutions in each MEME block over the period from 1968–1999. (A) Bar graph of amino acid substitutions within MEME blocks for each of the years. (B) Behavior of the substitutions in MEME block 7; frequency of amino acid substitutions within MEME block 7 largely follows the occurrence pattern of substitutions within the entire protein as illustrated Table 2, reaching a peak in 1980, which corresponds to the year with the greatest number of mutations in the alignment. (C) Behavior of the substitutions in MEME block 2.

Figure 3

Entropy plot of the protein consensus ClustalW alignment. Amino acid positions that do not exhibit any changes over the years have entropy of 0, whereas positions of high variability are represented by peak in the plot. Two hot spots of variability were observed and are clustered around amino acid position 140–190, and 200–240. The entropy analysis was performed for the entire hemagglutinin sequence (560 amino acids), but at amino acid position 340 (HA2) the analysis does not exhibit much entropy.

Figure 4

Frequency of specific potential post-translational modification (prosite) motifs implicated in each of the MEME blocks. MEME block 7 has the highest number of post-translational modification sites, followed by MEME block 2, 1 and 3 respectively. High frequency of post-translational modification site was recorded when a frequency of 2 or above is observed. Frequency of potential protein kinase C phosphorylation site (PKC) in the MEME blocks reveals that MEME block 3, 2 and 7 have a high PKC sites frequency. Frequency of potential N-myristilation site in the MEME blocks reveals that MEME blocks 1, 2 and 7 have a high myristilation sites frequency. Frequency of potential N-glycosylation site in the MEME blocks reveal that MEME block 2 and 7 has a high glycosylation sites frequency. Frequency of potential CKII phosphorylation sites in the MEME blocks reveals that MEME block 1 and 2 have a high CKII sites frequency.

Figure 5

Average entropy of specific post-translational modification sites in each of the MEME blocks is demonstrated using boxplot. (A) Average entropy of potential CKII phosphorylation sites in the MEME blocks. Blocks 1, 5 and 9 have zero entropy at all CKII sites. The majority of MEME blocks 2 and 7 CKII sites have nonzero entropy. One of the MEME block 2 CKII sites (amino acid 205) has the largest entropy (1.24) among all of CKII's sites. The average entropy over MEME block 7 and 2 CKII sites is therefore higher than for any other block. MEME block 1 has a wider boxplot than the others, indicating more CKII sites in this block. (B) Average entropy of potential PKC phosphorylation site in the MEME blocks. MEME block 1 and 4 have zero entropy at all their PKC sites. The highest PKC entropy values were observed in MEME block 2 (amino acid 205) and MEME block 7 (amino acid 160) with 1.2 entropy values. MEME block 5, 7 and 11 are unusual in that very few of their PKC sites have zero entropy. MEME block 11 then 7 PKC sites have the highest average entropy. The width of the boxplots indicates that more PKC sites are observed in MEME sites 2, 3 and 7 respectively. (C) Average entropy of potential N-glycosylation site in the MEME blocks. MEME blocks 4 and 5 have zero entropy at all of their ASN sites. MEME block 2, 6 and 9 have nonzero entropy at the majority of their ASN sites. One of the ASN sites (amino acid 99) from MEME block 1 has the highest entropy (1.003) among all ASN sites. The width of the boxplots indicates that more N-glycosylation sites are observed in MEME sites 2 and 7 respectively (D) Average entropy of potential N-myristylation site in the MEME blocks. MEME blocks 1, 2, 4, and 9 have the majority of their myristylation sites possessing zero entropy. The highest myristylation sites entropy is at MEME block 9 and 7 (Amino acid 78 and 160 respectively) with an approximate entropy value of 1.2. MEME block 1 and 7 have more N-myristylation sites than any other block, although MEME block 2 also has a fairly large number of myristylation sites.

Figure 6

Graphical representation of MEME blocks and antigenic sites on the 3-D hemagglutinin structure. The HA1 and HA2 are represented in yellow and blue, respectively. A) MEME blocks on HA: MEME2 (Magenta), MEME7 (Red), MEME3 (Bright Green), MEME1 (Orange (89–129 AA)). B) Antigenic sites on HA: Antigenic Binding Site A (Green), Antigenic Binding Site B (Magenta), Antigenic Binding Site C (Red), Antigenic Binding Site D (Red).