Fig. 5: ORF11 and BUD23 are required for the efficient translation of key late lytic KSHV mRNAs which contain uORFs.

a Changes in translational efficiency of viral genes calculated for libraries originating from TREx BCBL1-Rta cells expressing a Scr shRNA compared to shRNA 1 targeting BUD23 at 36 h post KSHV lytic reactivation. A false discovery rate was set at <0.005 and fold change threshold set at ±0.45 (log2). Volcano plot representation of all KSHV gene translational efficiency changes (n = 2 biologically independent samples). b Table of all KSHV genes with significantly changed translational efficiencies >± 0.45 (log₂). c Schematic of luciferase reporter assays with upstream KSHV mRNA 5' UTRs containing (1) uORFs or (2) with the uORF start codon mutated in HEK 293T cells. d Normalised luciferase intensity was quantified from luciferase reported plasmids containing uORFs or with the start codon of the uORF mutated (n = 3 biologically independent samples). Significance was calculated by using two-tailed unpaired t-tests (p = 0.0208, 0.0060 and 0.0177). e–g Normalised luciferase intensity of KSHV uORF containing reporter plasmids was quantified in HEK 293T cells stably expressing a non-targeting scrambled (Scr) shRNA or shRNAs 1 or 2 targeting BUD23 or ORF11-FLAG exogenous expression (n = 3 biologically independent samples). Data are presented as mean ± SD. Significance was calculated by one-way ANOVA with a Newman–Keuls multiple comparison post-test. Asterisks denote a significant difference between the specified groups (*p ≤ 0.05, **p < 0.01 and ***p < 0.001). NS Not significant.