Charged amino acid variability related to N-glyco -sylation and epitopes in A/H3N2 influenza: Hem -agglutinin and neuraminidase

Abstract

Background: The A/H3N2 influenza viruses circulated in humans have been shown to undergo antigenic drift, a process in which amino acid mutations result from nucleotide substitutions. There are few reports regarding the charged amino acid mutations. The purpose of this paper is to explore the relations between charged amino acids, N-glycosylation and epitopes in hemagglutinin (HA) and neuraminidase (NA).

Methods: A total of 700 HA genes (691 NA genes) of A/H3N2 viruses were chronologically analyzed for the mutational variants in amino acid features, N-glycosylation sites and epitopes since its emergence in 1968.

Results: It was found that both the number of HA N-glycosylation sites and the electric charge of HA increased gradually up to 2016. The charges of HA and HA1 increased respectively 1.54-fold (+7.0 /+17.8) and 1.08-fold (+8.0/+16.6) and the number of NGS in nearly doubled (7/12). As great diversities occurred in 1990s, involving Epitope A, B and D mutations, the charged amino acids in Epitopes A, B, C and D in HA1 mutated at a high frequency in global circulating strains last decade. The charged amino acid mutations in Epitopes A (T135K) has shown high mutability in strains near years, resulting in a decrease of NGT135-135. Both K158N and K160T not only involved mutations charged in epitope B, but also caused a gain of NYT158-160. Epitope B and its adjacent N-glycosylation site NYT158-160 mutated more frequently, which might be under greater immune pressure than the rest.

Conclusions: The charged amino acid mutations in A/H3N2 Influenza play a significant role in virus evolution, which might cause an important public health issue. Variability related to both the epitopes (A and B) and N-glycosylation is beneficial for understanding the evolutionary mechanisms, disease pathogenesis and vaccine research.

Fig 1. Charged amino acid mutations on HA epitopes in variants/vaccine strains.

Nine variants/vaccine strains were analyzed and only charged amino acids included. Both acidic amino acids [Aspartic acid (D) and Glutamic acid (E)] and basic amino acids [Lysine (K), Arginine (R) and Histidine (H)] labeled in different colors. The epitopes identified referred to Reference 7, 14 and the article (Lees WD, et al. J Virol. 2011. doi: 10.1128/JVI.00579-11).

Fig 2. Spatial changing of charged amino acids on HA epitopes in variants/vaccine strains.

a. Charged amino acids of five epitopes on HA included, Epitope A labeled in blue (124, 133, 135, 142 and 145), B in red (155, 156, 158, 160, 189, 190, 193 and 197), C in green (50, 53, 275, 276, 278, 279, 307 and 310), D in yellow (172, 173, 201, 207, 208, 222, 225 and 246) and E in cyan (57, 62, 63, 75, 82, 83, 92 and 261)(a.i and a-ii). b. The charged amino acids on epitopes of variants/vaccine strains were labeled, acidic ones in red, basic ones in blue and both acid and basic ones in green. c. T₁₃₅K on Epitope A resulted in a decrease of NGT_133-135, labeled in blue (T₁₃₅K) and cyan and K₁₆₀T on Epitope B did in an increase of NYT_158-160, labeled in red (K₁₆₀T) and cyan. Other amino acids on epitopes adjacent to NGS were labeled in green.

Fig 3. Charges and numbers of NGSs in HA and NA monomer.

a. HA, b. NA. Horizontal axis marked the year and both vertical axes marked the charge values (left) and the NGS number (right). The Potential NGSs of HA and NA were analyzed using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/Net-NGlyc/). N-glycosylated sites predicted with “++” or “+++” score would include.

Fig 4. Mutations of NGSs in HA and NA of A/H3N2 on variants/vaccine strains.

The mutations of NGSs on HA and NA included respectively 14 and 11 NGSs. The original NGS apearing in 1968 and preserving was labelled in green colour, then disapearing in red. The emerging NGS preserving up to date was labelled in blue colour, then disapearing in cyan.

Fig 5. Electric charges of antigenic epitopes in HA1.

The chronological changes of charge of five HA antigenic epitopes (A to E) were displayed separately. The dashed line(-), dot(●) and block(■) showed respectively the average, maximum and minimum charge values while the average charge levels in different epitopes were in diversities. As the less genes included in GenBank in the first two decades, data from 1968 to 1987 were divided into 6 groups, including 1968-1971(n = 20), 1972-1973(n = 19), 1974-1975(n = 18), 1976-1979(n = 24), 1980–1983 (n = 25) and 1984-1987(n = 22). The data during 1988–2016 were annually analyzed. The average charge of each epitope was unweighted by the number.