E2 and VENUS are equally detectable in CHIKV-infected EVA cells at 24 hpi.

EVA cells were inoculated with 106 PFU of CHIKV-VENKL for 24hpi, harvested, fixed, permeabilized, and stained with anti-E2 CHIKV (CHK-11) at decreasing (2-fold) serial dilutions. Cells were analyzed for E2 and VENUS expression by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of cell frequency for E2 and VENUS positive cells (red = infected, black = mock). (EPS)</p

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 CHIKVQMS-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 CHIKVQMS-VENKL. At 24 hpi, cells were assessed for cell surface expression of H2-Kb, and SIINFEKL-loaded H2-Kb (H2-Kb-SIINFEKL) by flow cytometry. (C) Representative flow cytometry plots depicting H2-Kb-SIINFEKL and H2-Kb cell surface expression on live, CD45- and live, CD45-, VENUS+ cells from mock-, CHIKV-VENKL-, and CHIKVQMS-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-Db and H2-Kb surface expression on live, CD45-, VENUS+ EVA cells. (G) Percentage of double-positive (H2-Kb+H2-Kb-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). **, PPP<0.0001.</p

CD8⁺ T cells are activated by CHIKV-infected cells when peptide processing and ER transport are bypassed.

(A-B) EVA cells were inoculated with 106 PFU CHIKV-VENKL or CHIKV-SIINFEKLβ2m. At 24 hpi, EVA cells were cocultured with a 1:1 mixture of 106 bystander and 106 OT-I SIINFEKL-specific CD8+ T cells for 6 h. Bystander CD8+ T cells were distinguished from OT-I CD8+ T cells by gating on CD8+, KbOVA257-264 tetramer positive or negative cells, and both populations were assessed for the expression of CD69, CD25, IRF4 and CD8 by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of CD25, CD69, IRF4 (intracellular), and CD8 cell surface expression (upper graphs) and frequency (lower graphs) for OT-I cells above bystander CD8+ T cells. Data are representative of two independent experiments (n = 6). P-values were determined by unpaired student’s t-test. *, PPPP<0.0001.</p

Frequency of VENUS⁺ cells after transfection of EVA cell cultures.

EVA cells were co-transfected with the designated WT or mutant pCMV-nsP2 plasmids or an identical control plasmid lacking the methionine start codon for nsP2 (vector) and pCMV-VENKL. At 24 h post-transfection, cells were evaluated by flow cytometry for expression of VENUS. (A) Representative flow cytometry plots for vector (black, circle), nsP2 (red, circle) and nsP2QMS (blue, circle) and frequency of VENUS+ cells among live, CD45- cells. (B) Representative flow cytometry plots for vector (black, circle), nsP2 (red, circle), nsP2K192A (red, triangle), nsP2C478S (red, square) and nsP2QMS (blue, circle) and frequency of VENUS+ cells among live, CD45- cells. (EPS)</p

Gating strategy for CHIKV-VENKL- vs CHIKV^QMS-VENKL-infected EVA cells.

Representative flow cytometry plots demonstrating gating strategy for CD45-, CD29+, VENUS+ fibroblasts from EVA cell cultures. Cell surface expression of CD29 was assessed within singlets, CD45-, VENUS+ or VENUS- gates. (EPS)</p

CHIKV-infected primary joint tissue fibroblasts display decreased MHC-I cell surface expression.

(A) Schematic of the recombinant CHIKV-VENKL. The coding sequence for the VENUS-SIINFEKL chimeric protein was inserted in-frame into the structural ORF of the CHIKV genome with known cleavage motifs flanking SIINFEKL. (B-E) EVA cells were inoculated with PBS (mock) or 106 PFU CHIKV-VENKL. (B) At 24 hpi, the percentages of CD45- (open circles) and CD45+ (open squares) cells among mock-inoculated cells and VENUS+ cells were determined. (C) The magnitude and frequency of expression of CD29 on CD45- cells was determined by flow cytometry. (D) Representative flow cytometry plots of H2-Kb and VENUS expressing cells from mock (black) and CHIKV-VENKL-infected cell populations (VENUS+, red; VENUS-, grey). (E) Quantification of surface expression and frequency of H2-Kb and H2-Db from mock and CHIKV-VENKL-infected EVA cells. Data are pooled (B), or representative (C, E) from 3–4 independent experiments. P values were determined by unpaired student’s t-test (B) or one-way ANOVA with Tukey’s test for multiple comparisons (C,E). *, PPPP<0.0001.</p

WT CHIKV-infected cells inefficiently activate CD8⁺ T cells <i>in vitro</i>.

(A-D) EVA cells were mock-inoculated or inoculated with 106 PFU of CHIKV-VENKL or CHIKVQMS-VENKL. At 24 hpi, EVA cells were cocultured with a 1:1 mixture of 106 bystander CD8+ T cells and 106 OT-I SIINFEKL-specific CD8+ T cells for 6 h. Bystander CD8+ T cells were distinguished from OT-I CD8+ T cells by gating on CD8+, KbOVA257-264 tetramer positive or negative cells, and both populations were assessed for the dual expression of CD69 and CD25 by flow cytometry. (A) Representative flow cytometry plots. (B) Quantification of CD25+CD69+ double positive cell frequencies for bystander (black) and OT-I (colored) CD8+ T cells. (C) Cell surface expression of CD69 (gMFI). (D) Cell surface expression of CD25 (gMFI). Data are representative of two independent experiments (n = 6). P values were determined by paired student’s t-test. ***, PP<0.0001.</p

Bypassing peptide processing and ER transport restores MHC-I antigen presentation by CHIKV-infected cells.

(A) Schematic of the recombinant CHIKV-SIINFEKLβ2m viral genome. The coding sequence for the β2mSIINFEKL chimeric protein was inserted into the CHIKV genome in-frame in the viral structural ORF similar to the VENKL coding sequence. (B-C) EVA cells were mock-inoculated or inoculated with CHIKV-VENKL, CHIKVQMS-VENKL, or CHIKV-SIINFEKLβ2m. At 24 hpi, live, CD45-, E2 (CHIKV)+ cells were assessed for cell surface expression of H2-Kb and SIINFEKL-loaded H2-Kb (H2-Kb-SIINFEKL) by flow cytometry. (B) Representative flow cytometry plots depicting the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells among live, CD45-, CHIKV+ cells. (C) Quantification of the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells (left) and pMHC-I presentation efficiency (right). (D-E) At 24 hpi, ankle cells from C57BL/6 mice mock-inoculated or inoculated with 103 PFU CHIKV-VENKL, CHIKVQMS-VENKL, or CHIKV-SIINFEKLβ2m were assessed by flow cytometry. (D) Representative flow cytometry plots depicting the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells among live, CD45-, CHIKV+ cells. (E) Quantification of the frequency of double-positive (H2-Kb+H2-Kb-SIINFEKL+) cells (left) and pMHC-I presentation efficiency (right) among live, CD45-, CHIKV+ cells. Data are representative of 2 independent experiments (n = 8). P values were determined by one-way ANOVA with Tukey’s multiple comparison test. **, PPP<0.0001.</p

OT-I CD8⁺ T cell enrichment efficiency, gating scheme, and baseline activation.

CD8+ T cells from naïve OT-I x CD45.1 mice were negatively selected for CD8+ cells by magnetic separation enrichment. (A) Frequency of CD8+ T cells before and after enrichment. (B) Gating scheme to differentiate bystander (KbOVA257-264 tetramer-) from OT-I (KbOVA257-264 tetramer+) CD8+ T cells. (C) Baseline activation of bystander (light grey; upper plot) and OT-I (black; lower plot) CD8+ T cells without coculture with EVA cells. (EPS)</p

Gating strategy for CHIKV-VENKL vs CHIKV^QMS-VENKL infected ex vivo joint-associated fibroblasts.

Representative flow cytometry plots illustrating the gating strategy for CD45-, CD29+, VENUS+ fibroblasts from ex vivo joint-associated tissues. Cell surface expression of CD29, CD44 were assessed within live, singlet, CD45-, VENUS+ or VENUS- gates. (EPS)</p