Fig. 4: Recognition of MHCII is mediated by the head domain of the hVIC/11 HA.

a hVIC/11 HA trimer model made using PyMOL based on previously published crystal structure (PDB: 4WE8⁵⁶). Sialic acid RBS is highlighted in pink while residues 138, 186, and 193 (H3 numbering) are shown in red. HEK-293T cells transfected with HLA-DR- (b) or SLA-DR-expressing plasmids (c) and then desialylated with 100 mU/mL of neuraminidase. Subsequently, cells were infected at an MOI of 0.01 with hVIC/11-A138S, hVIC/11-V186G, or hVIC/11-F193Y. At 24 hpi cells were stained for MHCII (APC) and FLUAV (Alexa488). d FLUAV-positive cells among MHCII-expressing HEK-293T cells was determined by gating live, MHCII⁺ cells only. Percentages were obtained by flow cytometry. Data is presented as the mean ± SEM of n = 3 independent experiments. e Total amount of FLUAV-infected HEK-293T cells was determined by multi-color flow cytometry. Data are presented as the mean ± SEM of three independent experiments. To evaluate virus replication in A549 cells, shRNA-scrambled (red), shRNA-CMAS cells transfected with an empty plasmid (blue), shRNA-CMAS cells transfected with HLA-DR-encoding plasmids (black), shRNA-CMAS cells transfected with SLA-DR-expressing plasmids (yellow), and shRNA-CMAS cells transfected with a shRNA-resistant CMAS cDNA (white) were infected at an MOI of 0.01 with either hVIC/11-A138S (f), hVIC/11-V186G (g), or hVIC/11-F193Y (h). Viral titers in the supernatant were quantified at 0, 12, 24, 48, and 72 hpi by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. L.D. limit of detection.