The recurrence of influenza virus infection in man is attributed primarily to changes occurring in the antigenic structure of the viral surface glycoproteins, especially of the haemagglutinin (HA) molecule. Comparative antigenic analysis of epidemic influenza virus strains has allowed the description of 'strain-specific' and 'cross-reactive' antigenic determinants. However, the interpretation of these findings remained ambiguous, because the specificity of the applied antisera was insufficiently defined and because the antigenic differences among the HA molecules of various epidemic virus strains resulted presumably from a large number of amino acid substitutions. Thus, in characterizing the antigenic structure of the HA molecule, our approach has been (1) to generate a panel of monoclonal anti-HA hybridoma antibodies, (2) to use some of these antibodies to select mutants of the influenza A/PR/8/34 (PR8) virus expressing antigenically altered HA molecules, and (3) to construct an operational antigenic map of the HA molecule by comparative antigenic analysis of the mutant viruses with the monoclonal antibodies. As we report here, analysis of the 34 mutant viruses selected has enabled us to define four antigenic sites on the HA molecule. Our observation that these sites have undergone antigenic drift to a different extent in nature implies that the mechanisms responsible for antigenic drift act selectively on distinct structures of the HA molecule.

MATERIALS AND METHODSCells, Virus, and Virus Infection. HeLa, Vero, BHK21, CV-1, HEp-2, and human astrocyte cells were maintained in Eagle's minimal essential medium containing 10% fetal calf serum, 1% glutamine, and antibiotics. These cells were cultured in T75 flasks (Falcon) at 370C in 5% CO2 and passed twice weekly. Astrocytes derived from a human brain biopsy sample were positive for glial fibrillary acidic protein by immunofluorescent staining.

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this ®nding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.

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