Determinants in nsP2 promote impairment of MHC-I antigen presentation by CHIKV-infected cells.

(A) Schematic depicting the location of nsP2 V-loop mutations (QMS; blue) compared with wildtype nsP2 (ATL; red). (B) WT and Ifnar1^-/- murine embryonic fibroblasts (MEFs) were inoculated with CHIKV-VENKL or CHIKV^QMS-VENKL at an MOI of 0.1 FFU/cell. At 0 (input), 1, 24, 48, and 72 hpi, the amount of infectious virus present in culture supernatants was quantified by focus formation assay. (C-H) EVA cells were mock-inoculated or inoculated with CHIKV-VENKL or CHIKV^QMS-VENKL. At 24 hpi, cells were assessed for cell surface expression of H2-K^b, and SIINFEKL-loaded H2-K^b (H2-K^b-SIINFEKL) by flow cytometry. (C) Representative flow cytometry plots depicting H2-K^b-SIINFEKL and H2-K^b cell surface expression on live, CD45^- and live, CD45^-, VENUS⁺ cells from mock-, CHIKV-VENKL-, and CHIKV^QMS-VENKL-inoculated EVA cells. (D) Percentage of VENUS⁺ cells among live, CD45^-, EVA cells. (E) CD29 cell surface expression on live, CD45^-, VENUS⁺ EVA cells. (F) Quantification of H2-D^b and H2-K^b surface expression on live, CD45^-, VENUS⁺ EVA cells. (G) Percentage of double-positive (H2-K^b+H2-K^b-SIINFEKL⁺) cells on live, CD45^-, VENUS⁺ EVA cells. (H) pMHC-I presentation efficiency on live, CD45^-, VENUS⁺ EVA cells. Data are representative of 4 experiments (n = 12). P values were determined by unpaired student’s t-test (D-H). **, P<0.01; ***, P<0.001; ****, P<0.0001.