Fig. 5: Effect of KIAA1456 overexpression and depletion on mcm5U and mcm5s2U modification levels and on CHIKV infection. | Nature Communications

Fig. 5: Effect of KIAA1456 overexpression and depletion on mcm5U and mcm5s2U modification levels and on CHIKV infection.

From: CHIKV infection reprograms codon optimality to favor viral RNA translation by altering the tRNA epitranscriptome

Fig. 5

a Western blot showing KIAA1456 protein upregulation upon CHIKV infection. Values denote relative KIAA1456 expression normalized by tubulin expression calculated from 3 independent replicates and expressed as mean ± SEM. b Western blot showing KIAA1456 protein levels under plasmid-driven overexpression (KIAA1456-OE) and CHIKV infection conditions. Values represent relative KIAA1456 expression normalized by vinculin expression. A representative example from two independent replicates is shown. Note, that to be able to compare expression levels, 1:5 dilutions of the KIAA1456–OE overexpression samples were loaded. c Relative mcm5U and mcm5s2U modification levels in mock-infected cells, CHIKV-infected cells and in cells overexpressing KIAA1456 (KIAA1456-OE) determined by mass LC-MS-MS; bars represent the mean value ± SEM of 4 (in the case of mcm5 after KIAA1456-OE) respectively 5 (in the remaining cases) independent replicates. p-values: mcm5 - mock vs CHIKV: 0.011, mcm5 – mock vs KIAA-OE: 0.016. d Relative amount of CHIKV RNA levels (p-value 0.0046) and (e) of CHIKV capsid protein levels in CHIKV-infected cells transfected with an empty plasmid or with a plasmid overexpressing KIAA1456 (KIAA1456-OE) determined by qPCR; bars represent the mean value ± SEM of 5 (empty) and 4 (KIAA-OE) independent replicates. CHIKV RNA and capsid protein expression levels were normalized by TUBB6 RNA and tubulin, respectively. f Western blot showing KIAA1456 protein levels upon transfecting siRNA control (siCTL) and siKIAA1456. Values denote relative KIAA1456 protein expression normalized by vinculin expression calculated from at least 3 independent samples and expressed as mean ± SEM. g Relative levels of mcm5U and mcm5s2U modification (p-value 0.007), h CHIKV RNA levels (p-value 0.047) and i CHIKV capsid protein levels in CHIKV-infected cells, transfected with siCTL or siKIAA1456 determined as in (c, d and e). Bars represent the mean value ± SEM of 3 independent replicates. All CHIKV infections were carried out at an MOI of 4 and samples were collected 12 hpi. All statistical analyses were performed using the two-tailed t-test. *p < 0.05.

Back to article page