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. 2017 May 26;91(12):e00286-17. doi: 10.1128/JVI.00286-17

FIG 1.

FIG 1

Design of chimeric HA/B constructs. (A) Monomer of the H5 HA structure (based on data reported under Protein Data Bank [PDB] accession number 2FK0 [52]). The head domain is shown in purple, and the stalk domain is shown in blue. The head domain is located between cysteines 52 and 277 (H3 numbering), which form a disulfide bond. The cysteines are indicated by yellow arrows and are highlighted in yellow on the structure. (B) Monomer of an influenza B virus HA (based on data reported under PDB accession number 4M44 [53]). In this case, the stalk domain is shown in light green, and the head domain is shown in dark green. The head domain is located between alanines 57 and 306 (B/Yamagata/16/88 numbering, starting with methionine), which are in positions on the structure similar to those of C52 and C277 on influenza A virus HAs. The alanines are indicated by orange arrows and are highlighted in orange on the structure. (C and D) The two cHA/B constructs that were generated with head domains from influenza A virus HA and stalk domains from influenza B virus. (C) The influenza A virus HA cysteines (now in positions C57/C306, labeled in purple, since they are derived from influenza A virus HA) were maintained as the demarcation line between the head (purple) and stalk (green), and the original influenza B virus alanines were deleted. (D) Similar construct where the alanines (labeled in green since they are derived from influenza B virus HA) were maintained and the cysteines were removed. *, the transmembrane domain (TM) (brown) and cytoplasmic tail domain (CTD) (blue) are derived from A/Puerto Rico/8/34 HA for all constructs for technical reasons. (E) Immunostaining of HEK 293T cells transfected with different cHA/B constructs, wild-type influenza B virus HA (WT B HA) (from B/Yamagata/16/88), or the empty vector (pDZ). cH5/Bcys contains the head domain of H5 HA combined with the stalk domain of B/Yamagata/16/88 HA, with “cys” indicating that the design shown in panel C was used. cH7/Bcys and cH8/Bcys were constructed in the same way, but the head domains are derived from H7 and H8, respectively. cH5/Bala, cH7/Bala, and cH8/Bala are based on the designs shown in panel D. Cells were stained with polyclonal anti-B/Yamagata/16/88 serum.