Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin

Abstract

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.

FIG. 1.

Schematic drawing of the globular head of influenza A/H3N2 virus HA, showing the change in N-glycosylation sites among representative isolates (A) and the mutant HAs used in this study (B). N-glycosylation sites are indicated by solid circles. Numbers indicate Asn residues in the first position of the glycosylation sequon.

FIG. 2.

Expression of mutant HAs. COS-1 cells transfected with wild-type (WT) or mutated HA gene cDNA were labeled with [³⁵S]methionine for 15 min at 48 h posttransfection in the absence (TM−) or presence (TM+) of tunicamycin. Cells were then immunoprecipitated with antiserum against A/Aichi/2/68 virus, and the resulting immunoprecipitates were analyzed by SDS-PAGE. HA_NG, nonglycosylated HA. The arrowhead indicates the coprecipitated endoplasmic reticulum-resident chaperone binding protein BiP.

FIG. 3.

Intracellular transport of mutant HAs. COS-1 cells expressing wild-type (WT) or mutant HA were labeled with [³⁵S]methionine for 20 min at 48 h posttransfection and chased for 4 h. (A) Immediately after a pulse (P) or after a subsequent chase (C), cells were immunoprecipitated with antiserum against A/Aichi/2/68 virus. The resulting precipitates were digested (+) or mock digested (−) with endoglycosidase H and analyzed by SDS-PAGE. R and S indicate the endoglycosidase H-resistant and -sensitive forms of HA, respectively. (B) Cells were treated (+) or not (−) with TPCK-trypsin during the last 15 min of the chase and then immunoprecipitated with antiserum against A/Aichi/2/68 virus. The resulting immunoprecipitates were analyzed by SDS-PAGE on 17.5% gels.

FIG. 4.

Receptor binding activity of mutant HAs. COS-1 cells were transfected with cDNA encoding wild-type (WT) or mutant HA protein. At 48 h posttransfection, cells were subjected to hemadsorption with guinea pig erythrocytes (A) or chicken erythrocytes (B). Erythrocytes that attached to the HA-expressing cells were lysed in distilled water, and the amounts of hemoglobin released were measured by reading the absorption at 540 nm and are shown as a percentage of the wild-type HA value.

FIG. 5.

Cell fusion activity of mutant HAs. COS-1 cells expressing wild-type (WT) or mutant HA were treated with TPCK-trypsin at 48 h posttransfection, exposed to fusion medium (pH 5.0), and then incubated for 3 h in neutral-pH medium. At the end of the incubation period, the cells were stained with Giemsa solution.

FIG. 6.

Reactivity of mutant HAs with human sera. COS-1 cells transfected with wild-type (WT) or mutated HA gene cDNA were labeled with [³⁵S]methionine for 15 min at 48 h posttransfection. The cells were then immunoprecipitated separately with six human sera collected in 1976 which had hemagglutination inhibition activity against A/Aichi/2/68 virus (160 HIU/ml) but not against A/Victoria/3/75 virus (<10 HIU/ml) or A/Sydney/5/97 virus (<10 HIU/ml). The immunoprecipitates obtained were subjected to SDS-PAGE (A), and the radioactivity of the HA bands was measured (B). The relative specific reactivity of the mutant HAs was determined as a percentage of that of wild-type HA. The solid line shows the average for the six sera.