Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes

Abstract

The influenza virus hemagglutinin molecule possesses a globular head domain that mediates receptor binding and a stalk domain at the membrane-proximal region. We generated functional influenza viruses expressing chimeric hemagglutinins encompassing a variety of globular head and stalk combinations, not only from different hemagglutinin subtypes but also from different hemagglutinin phylogenetic groups. These chimeric recombinant viruses possess growth properties similar to those of wild-type influenza viruses and can be used as reagents to measure domain-specific antibodies in virological and immunological assays.

Fig 1

Schematic of chimeric hemagglutinins and sequence comparisons. (A) Diagrams of wild-type and cH1/1 viruses. The chimeric HA was constructed by swapping the globular head domain located between Cys52 and Cys277 of PR8 (H1) HA (dark green) with that of the A/California/4/09 (H1) HA (light green). The resulting chimeric HA has the stalk region of the A/PR8/34 (H1) HA with the globular head domain of the A/California/4/09 (H1) HA and is designated cH1/1. (B) Theoretical schematics of the folded structures of the different wild-type and chimeric HAs (from left to right, wild-type PR8 HA, the chimeric cH1/1 HA, the chimeric cH5/1 HA, the wild-type Perth HA, the chimeric cH7/3 HA, and the chimeric cH5/3 HA). The different HAs are color coded as follows: dark green, PR8 HA; light green, Cal/09 HA; red, VN H5 HA; blue, Perth/09 HA; cyan, H7 HA. The following full-length HA structures were downloaded from the Protein Database (PDB): PR8 HA (PDB identification code [ID] 1RU7) and Perth/09 HA (represented by A/duck/Ukraine/63 [H3N8]) HA (PDB ID 1MQN). Final images were generated with PyMol (Delano Scientific). In order to represent the cHAs, only the colors of the HA head domains from the wild-type structures were changed. (C) Tables comparing amino acid identities between the H1, H3, H5, and H7 HAs used in this study. The percentages of amino acid identity were calculated using ClustalW (excluding the signal peptide) and were compared for full-length HA, the globular head domain, and the stalk domain. Shaded cells indicate 100% identity.

Fig 2

Surface expression and functional analysis of chimeric HA constructs. (A) The surface expression of chimeric HA constructs was evaluated in transiently transfected or infected cells. At 48 h posttransfection, 293T cells were trypsinized, and the expression of chimeric HA proteins on the cell surface was analyzed by flow cytometry. (Top) Mock-transfected 293T cells (shaded histograms) are compared to cells transfected with either PR8, cH1/1, or cH5/1 (open histograms). (Middle) Mock-transfected 293T cells (shaded histograms) are compared to cells transfected with either Perth/09, cH7/3, or cH5/3 (open histograms). (Bottom) MDCK cells were infected with recombinant viruses expressing either Perth/09, cH7/3, or cH5/3. At 12 h postinfection, the expression of the different HAs on the cell surface was analyzed using flow cytometry. (B) Luciferase-encoding pseudoparticles expressing chimeric HAs were used to transduce MDCK cells. The relative light units (RLU) generated in the luciferase assay indicate that pseudoparticles expressing chimeric HAs are able to enter cells.

Fig 3

Generation of recombinant viruses bearing chimeric hemagglutinins. (A) Western blot analysis of cells infected with recombinant cHA-expressing viruses. Extracts from MDCK cells that were either mock infected or infected with the indicated viruses at an MOI of 2 were prepared and were probed with the following antibodies at 16 hpi: anti-A/PR8 HA (H1) (PY102), anti-A/Cal/09 HA (H1) (29E3), anti-A/VN HA (H5) (MAb 8), anti-H3 HA (12D1), anti-H7 HA (NR-3152), and anti-A/NP (HT103). Anti-GAPDH was used as a loading control. (B) Immunofluorescence analysis of MDCK cells infected with recombinant viruses using anti-A/NP (HT103), anti-A/H1 HA (6F12), anti-A/PR8 HA (PY102), anti-A/Cal/09 HA (29E3), anti-A/VN HA (MAb 8), anti-H3 HA (12D1), and anti-A/H7 (NR-3152) antibodies.

Fig 4

Growth kinetics and plaque phenotypes of wild-type and recombinant viruses. (A) Ten-day-old embryonated chicken eggs were infected with 100 PFU per egg of wild-type or recombinant virus, and viral growth was monitored for 72 h postinfection. Data points are averages and standard deviations for experimental replicates. (B) The plaque phenotypes of recombinant viruses were assessed by plaque assays. MDCK cells were infected with either a wild-type or a recombinant virus. Cells were fixed 48 h postinfection and were immunostained to reveal plaque phenotypes by using the antibody against A/NP (HT103).

Fig 5

A stalk-specific monoclonal antibody neutralizes cHA-expressing viruses and pseudotype particles. The ability of a MAb (KB2) to neutralize cHA-expressing viruses or pseudotype particles was assessed by a plaque reduction assay or a pseudotype particle inhibition assay. MDCK cells were infected or transduced with cHA-expressing viruses or pseudotype particles either in the presence of the indicated amounts (in micrograms per milliliter) of the MAb or without an antibody. Plaque formation or luciferase activity was used as a readout to determine the degree of inhibition by the MAb. (A) MAb KB2 neutralizes the replication of the cH1/1 (squares) and cH5/1 (triangles) viruses in a dose-dependent manner, with 100% inhibition at concentrations above 100 μg/ml. Data points are averages and standard deviations for experimental replicates. (B) MAb KB2 also inhibits the entry of cH1/1 and cH5/1 pseudotype particles in a dose-dependent manner, with complete inhibition above 4 μg/ml. Data points are averages and standard deviations for experimental replicates. The pseudotype inhibition assays were processed independently.