Fig. 3: NAT10 lactylation at Lys290 promotes viral mRNAs translation and virion production of KSHV.
A Schematic representation of DUF1726 domain (marked in blue), RNA helicase domain (marked in yellow), N-acetyltransferase domain (marked in green) and RNA-binding domain (marked in purple) of NAT10. The identified lactylation lysine site (K) within the RNA helicase domain was marked in red, which was mutated to arginine (R) and marked in dark blue. B The iSLK-KSHV cells transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h were subjected to the anti-Flag immunoprecipitation, and then examined by Western blot with the anti-Pan Kla antibody. C The sequences around NAT10 Lys290 from different species were aligned. Conserved Lys290 was marked in red. D The total RNAs from cells shown as in (B) were subjected to dot blot assay with anti-ac4C antibody. Methylene blue staining was used as the internal control. E The iSLK-KSHV cells with THUMPD1 overexpression (THUMPD1-Myc) were transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h. Cells were subjected to the anti-Flag immunoprecipitation and analyzed by Western blot using anti-Myc antibody. F The total RNAs from iSLK-KSHV cells transduced with the wild type (NAT10) and the mutant (K290R) NAT10 for 48 h were subjected to Northern blot with probes to tRNASer-CGA-1-1, and U6 as the control. G The expression levels of vIRF1 and K-bZIP in cells treated as in (B) were examined by Western blot with the corresponding antibodies. H The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in (B). *P < 0.05, **P < 0.01 and ***P < 0.001 by Student’s t-test. I The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with the wild type (NAT10-WT) or the mutant (NAT10-K290R) NAT10 overexpression, which were complemented with the wild type (tRNASer-CGA-1-1-WT) or the mutant (tRNASer-CGA-1-1-Mut) tRNASer-CGA-1-1 for 48 h. *P < 0.05, **, P < 0.01 and ***P < 0.001 by Student’s t-test. J By the assessment of ORF26, real-time DNA-PCR for cells treated as in (B) was performed to examine viral copy number after doxycycline stimulation for 72 h. ***P < 0.001 by Student’s t-test.
