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.

FIG. 1.

Distribution of HA protein that did not show hemadsorption activity. After transfection of mutant HAs which were incapable of hemadsorption, cells were fixed with ethanol-acetone or 4% formamide as described in Materials and Methods. pME18SHA(2/0)#101-transfected cells were fixed with ethanol-acetone (A) or 4% formamide (C), and pME18SHA(2/0)#34-transfected cells were fixed with ethanol-acetone (B) or 4% formamide (D). In panel B, the open triangle indicates staining of the perinuclear region, and the solid triangle indicates staining of ER.

FIG. 2.

Single amino acid substitutions and their effect on hemadsorption activity. HA proteins changed by a single amino acid, which were derived from the HA gene of A/Aichi/68, were analyzed by hemadsorption activity and cellular location, as described in Materials and Methods. Substituted amino acids are shown under the sequence using the one-letter system. Red letters indicate a negative change, and green letters indicate a positive change. The location of the HA of these single HA mutants is also shown under the substitution. An “s” indicates that the mutant HA had moved to the cell surface mainly, and “g” or “e” indicates that the mutant HA remained mainly in the Golgi or ER, respectively.

FIG. 3.

Possible mainstream amino acid changes of the H3 HA1 from 1968 to 2000. Possible mainstream amino acid changes are shown from A/Aichi/2/68 to A/Panama/2007/99. The numbers on the right side of the mainstream line indicate positions that underwent the second (✽) and the third (✽✽) amino acid changes. The number symbol (#) indicates the changed amino acids of positions 190, 260, and 276 that were different compared to those in Fig. 2. Site-directed mutagenesis was carried out at these sites as described in the text. The position of A/Panama/2007/99 was determined by analyzing many strains isolated during 1999 and 2002.

FIG. 4.

Two-point amino acid substitutions and their effect on hemadsorption activity. These two-point amino acid-substituted HA cDNAs were constructed as described in Materials and Methods. Transfection and hemadsortion assays were as described in Materials and Methods. After incubation of transfected COS cells with CRBCs, cells were washed with PBS. Adsorbed RBCs were measured by determining the absorbance at 540 nm after disruption of these cells with 100 μl of water. The results are shown for vector, 144HA, and (144+x)HA.

FIG. 5.

Fine structure of antigenic sites. Observed amino acid positions in natural isolates (green) and negatively changed positions (red) in Table 2 are shown on a three-dimensional structure of the HA monomer.