N-acetyltransferase 10 promotes mRNA stability of immune response factors to modulate Zika virus infection

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that poses serious risks to human health. In previous work, we used RNA immunoprecipitation and mass spectrometry to show that more than 30 distinct RNA modifications, or chemical moieties, were present on the RNA genome of ZIKV. Among these, N4-acetylcytosine (ac4C) was one of the most abundant modifications. In this study, we investigated the role of N-acetyltransferase 10 (NAT10), the writer enzyme that acetylates cytidine, in ZIKV gene expression. Using NAT10 knockout cell lines, RNA interference (RNAi), and overexpression rescue strategies, we found that loss of NAT10 led to increased levels of ZIKV protein and RNA. However, the production of infectious virus particles was not significantly affected. Interestingly, in NAT10-deficient cells compared to wild-type (WT) cells, ZIKV protein and RNA were detectable earlier during infection, suggesting that the loss of NAT10 facilitated increased viral replication. Despite this increase, ZIKV RNA was more rapidly degraded, although the accumulation of small flaviviral RNAs was not significantly altered by the absence of NAT10. Further analysis of key components of the innate immune response revealed that, in the absence of NAT10 and during early infection, STAT1, IFIT1, and MX1 mRNA transcripts were rapidly degraded, leading to reduced expression of the respective innate immune proteins. Taken together, our findings demonstrate that NAT10, the ac4C writer enzyme, modulates the stability of specific innate immune mRNAs and thereby plays a regulatory role in ZIKV infection dynamics. IMPORTANCERNA modifications are chemical groups that are deposited post-transcriptionally on RNA. In recent years, RNA modifications in viral RNAs have been shown to have profound effects on viral gene expression and hence viral function. Indeed, numerous studies have investigated the role of N6-methyladenosine and 5-methylcytosine RNA modifications and the respective enzymes that deposit (writer), remove (eraser) and facilitate (reader) function on viral infection. In this study we show how N4-acetyltransferase 10 (NAT10), a writer enzyme that acetylates cytidine to form N4-acetylcytidine (ac4C), affects Zika virus gene expression. Specifically, we found that NAT10 regulates viral infection kinetics by affecting the stability of select mRNAs involved in the innate immune response pathway. Our findings highlight another mode by which the innate immune response is robustly regulated in response to ZIKV infection.