Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

Abstract

We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.

Comparison of Epitope Structures of H3HAs through Protein Modeling of Influenza A Virus Hemagglutinin: Mechanism for Selection of Antigenic Variants in the Presence of a Monoclonal Antibody

“…In escape mutants selected by mAb203, the nine positions with changes were clustered in A2 and A3 subsites of antigenic site A (17,18). Generally speaking, about 16 amino acid residues at an antigenic site are involved in antibody-antigen binding (3).…”

“…We compared the positions in escape mutants selected by mAb203 to the mainstream amino acid changes that occurred from 1968 to 1999 (17 …”

Effect of the Addition of Oligosaccharides on the Biological Activities and Antigenicity of Influenza A/H3N2 Virus Hemagglutinin

“…Nobusawa et al (10) reported that a change in amino acid residue 190 from Glu to Asp was responsible for this characteristic, as determined in studies with HA cDNA of A/Aichi/ 51/92 virus. Nakajima et al (8) demonstrated that an amino acid change from Glu to Asp at residue 190 of A/Aichi/2/68 virus HA did not inhibit its hemadsorbing activity with chicken erythrocytes. Therefore, they suggested that multiple amino acid substitutions on the HA of A/Aichi/2/68 virus are necessary to change the receptor specificity.…”

“…Based on analysis of the HA sequences of A/H3N2 viruses isolated from 1968 to 2002, the oligosaccharide chains on the globular head show large variations in number among different A/H3N2 isolates, although five glycosylation sites at Asn residues 8,22,38,285, and 483 on the stem region are strictly conserved. Most of the A/H3N2 viruses that circulated between 1968 and 1974 (represented by A/Aichi/2/68) had only two oligosaccharides at residues 81 and 165 on the globular head of the HA (Fig.…”

Chemical Insight Into the Influenza A Virus Hemagglutinin Binding to the Sialoside Revealed by the Fragment Molecular Orbital Method

“…On the sequence alignment for the known human H3s, the Ser136 preservation is significant for the recognition of 1-COO Neu5Ac. Actually, each of the one-point substitutions Ser136Thr [68], Ser136Ala [68], Ser136Cys [69], Ser136Asn [70] decreased the human erythrocyte binding. The Asn145 and Lys145 sometimes required a hydrophobic Tyr and Phe at position 137 [61].…”