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

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

A Mutation on Influenza C Virus M1 Protein Affects Virion Morphology by Altering the Membrane Affinity of the Protein▿

Reverse genetics has been documented for influenza A, B, and Thogoto viruses belonging to the family Orthomyxoviridae. We report here the reverse genetics of influenza C virus, another member of this family. The seven viral RNA (vRNA) segments of C/Ann Arbor/1/50 were expressed in 293T cells from cloned cDNAs, together with nine influenza C virus proteins. At 48 h posttransfection, the infectious titer of the culture supernatant was determined to be 2.51 × 103 50% egg infectious doses/ml, which is lower than the number of influenza C virus-like particles (VLPs) (106/ml) generated using the same system. By generating influenza C VLPs containing a given vRNA segment, we showed that each of the vRNA segments was similarly synthesized in the plasmid-transfected cells but that some segments were less efficiently incorporated into the VLPs. This finding leads us to speculate that the differences in incorporation efficiency into VLPs between segments might be a reason for the inefficient production of infectious viruses. Second, we generated a mutant recombinant virus, rMG96A, which possesses an Ala→Thr mutation at residue 24 of the M1 protein, a substitution demonstrated to be involved in the morphology (filamentous or spherical) of the influenza C VLPs. As expected, rMG96A exhibited a spherical morphology, whereas recombinant wild-type of C/Ann Arbor/1/50, rWT, exhibited a mainly filamentous morphology. Membrane flotation analysis of the cells infected with rWT or rMG96A revealed a difference in the ratio of membrane-associated M1 proteins, suggesting that the affinity of M1 protein to the cell membrane is a determinant for virion morphology

Growth Kinetics of Influenza C Virus Antigenic Mutants That Escaped from Anti-Hemagglutinin Esterase Monoclonal Antibodies and Viral Antigenic Changes Found in Field Isolates

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability

Influenza C Virus NS1 Protein Upregulates the Splicing of Viral mRNAs▿

Pre-mRNAs of the influenza A virus M and NS genes are poorly spliced in virus-infected cells. By contrast, in influenza C virus-infected cells, the predominant transcript from the M gene is spliced mRNA. The present study was performed to investigate the mechanism by which influenza C virus M gene-specific mRNA (M mRNA) is readily spliced. The ratio of M1 encoded by a spliced M mRNA to CM2 encoded by an unspliced M mRNA in influenza C virus-infected cells was about 10 times larger than that in M gene-transfected cells, suggesting that a viral protein(s) other than M gene translational products facilitates viral mRNA splicing. RNase protection assays showed that the splicing of M mRNA in infected cells was much higher than that in M gene-transfected cells. The unspliced and spliced mRNAs of the influenza C virus NS gene encode two nonstructural (NS) proteins, NS1(C/NS1) and NS2(C/NS2), respectively. The introduction of premature translational termination into the NS gene, which blocked the synthesis of the C/NS1 and C/NS2 proteins, drastically reduced the splicing of NS mRNA, raising the possibility that C/NS1 or C/NS2 enhances viral mRNA splicing. The splicing of influenza C virus M mRNA was increased by coexpression of C/NS1, whereas it was reduced by coexpression of the influenza A virus NS1 protein (A/NS1). The splicing of influenza A virus M mRNA was also increased by coexpression of C/NS1, though it was inhibited by that of A/NS1. These results suggest that influenza C virus NS1, but not A/NS1, can upregulate viral mRNA splicing

Growth Kinetics of Influenza C Virus Antigenic Mutants That Escaped from Anti-Hemagglutinin Esterase Monoclonal Antibodies and Viral Antigenic Changes Found in Field Isolates

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability