Despite some degree of variability in signal intensity, we were able to identify ~11 conserved sites of ac4C addition on HIV-1 transcripts that were detected across these three replicates and on additional replicates performed in HIV-1 infected CEM cells and primary CD4 + T cells ( Figure S1C), but not in mock-infected cells. In Figure 1A, we report the PA-ac4C-seq analysis of HIV-1 gRNA, isolated from HIV-1 virions generated in the CEM-SS or SupT1 T cell line, or on intracellular viral RNAs, isolated from CEM-SS cells. ) that ac4C residues on cellular mRNAs are primarily localized to coding sequences (CDS), although ac4C residues were also detected in the 3′ UTRs, but not 5′ UTRs, of cellular mRNAs ( Figure S1B). Lastly, we show that the previously described NAT10 inhibitor remodelin ( While HIV-1 gene expression was also reduced when silent mutagenesis was used to remove a subset of viral ac4C sites, this effect was strongly attenuated in the NAT10-depleted CEM T cells. The reduction in HIV-1 gene expression in NAT10-depleted CEM T cells could be fully explained by a decrease in viral RNA stability. CRISPR-mediated depletion of NAT10 in the CEM T cell line resulted in a decrease in both viral RNA acetylation and gene expression, whereas overexpression of wild-type (WT), but not inactive mutant forms, of NAT10 increased viral gene expression. ![]() We now report that HIV-1 transcripts indeed bear ac4C residues at multiple discrete sites and that the addition of ac4C to viral transcripts is mediated by cellular N-acetyltransferase 10 (NAT10). We therefore hypothesized that addition of ac4C residues to HIV-1 transcripts might also serve to boost HIV-1 gene expression and replication. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development. ![]() ![]() Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. Moreover, acetylation of the N 4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function.
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