Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

Abstract

Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N⁴ position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. 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. 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. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. 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.

Keywords: HIV-1; N-acetyltransferase 10; N4-acetylcytidine; NAT10; RNA modification; ac4C; epitranscriptomic; gene regulation; human immunodeficiency virus 1; retrovirus; virus.

Fig 1.. NAT10-dependent ac4C is deposited at multiple locations on HIV-1 RNAs

(A) ac4C sites were mapped by PA-ac4C-seq on the poly(A)⁺ fraction (mRNA) of mock or HIV-1 infected CEM-SS T cells, along with virion RNA produced by infected Sup T1 and CEM-SS cells. See also Fig. S1. (B) Schematic of HIV-1 genome organization drawn to scale (C) PA-ac4C-seq was performed on HIV-1 virion RNA produced in control (Ctrl) or ΔNAT10 CEM cells. NAT10 knock-down is validated in Fig. 3A. (D) PAR-CLIP was performed on CEM cells stably expressing FLAG-NAT10 or FLAG-GFP to identify NAT10 binding sites on HIV-1 RNA. Sequence reads were aligned to the NL4–3 genome. Consistent (across mRNA and virion) ac4C sites are highlighted in yellow and numbered above. 4SU-based CLIP methods result in T>C conversions where protein is cross-linked to 4SU residues, here shown as red-blue bars. All sequencing read pileups shown in counts per million (CPM), where read counts were normalized to the total read count of reads >15nt long with a FASTQ quality score >Q33.

Fig 2.. NAT10 enhances the rate of HIV-1 spread in culture.

(A-C) CEM cells in which the NAT10 gene had been edited using CRISPR/Cas (ΔNAT10, see also Fig. S2), along with control CEM cells expressing Cas9 and a non-targeting guide RNA (Ctrl), were assayed for NAT10-associated viral replication phenotypes. (A) WT HIV-1 replication levels, in a spreading or mock infection of Ctrl and ΔNAT10 cells at 3 dpi, were analyzed by Western blot for the HIV-1 capsid protein p24 (B) Ctrl and ΔNAT10 cells were infected with the NL4–3NLuc reporter virus and NLuc activity determined at 3 dpi (C) HIV-1 RNA levels in the samples shown in panel A were determined using qRT-PCR. Three different ΔNAT10 single cell clones were used in panels B & C, with Ctrl cells set at 1. n=4 to 7, error bars=SD. ** 2-tailed T-test, p<0.01. (D) CEM, Ctrl, and ΔNAT10 cells were treated with the NAT10-inhibitor Remodelin. Infected CEM cells were counted at 1 dpi to determine Remodelin toxicity (shown in gray), infected Ctrl (dark blue) and ΔNAT10 (light blue) cells were harvested at 2 dpi and viral RNA levels assayed by qRT-PCR. n=3, error bars=SD. 2-tailed T-test on Ctrl cells for each condition compared to the 0 μM level, **p<0.01, *p<0.05. (E) Schematic of NAT10 functional domains and point mutations. (F) 293T cells were transfected with a plasmid expressing WT NAT10 (WT), or the K290A or G641E NAT10 mutant, or empty vector. Cells were co-transfected with plasmids expressing CD4 and firefly luciferase (FLuc). 3 days later, transfected cells were infected with the NL4-NLuc reporter virus. NLuc expression levels, assayed at 2 dpi, are shown normalized to the FLuc internal control. n=3 to 6, error bars=SD. ** 2-tailed T-test, p<0.01. (G) Western blot showing NAT10 over-expression, NAT10 probed with both FLAG and NAT10 antibodies and GAPDH probed as a loading control.

Fig 3.. NAT10 depletion destabilizes HIV-1 transcripts.

Ctrl (dark blue) and ΔNAT10 (light blue) cells were infected with HIV-1, treated with the reverse transcriptase inhibitor Nevirapine (NVP) at ~16 hpi and harvested at ~48 hpi for the following single cycle infection assays. (A) Viral Gag levels assayed on Ctrl and 3 single cell clones (#9, #3, #7) of ΔNAT10 cells by Western blot. HIV-1 Gag band intensities (p24 + p55) were quantified, normalized to Ctrl levels (set to 1), and are shown in the right hand panel (Ctrl n=3, ΔNAT10 n=7). (B) Aliquots of the samples visualized in panel A were assayed for viral RNA levels by qRT-PCR. (C) Viral DNA levels in infected Ctrl and ΔNAT10 CEMs, quantified by qPCR using an LTR U3 primer set, n=4. (D) Subcellular fractionation of infected Ctrl & ΔNAT10 cells. Viral RNA in each fraction was quantified by qRT-PCR and is shown as the percentage of cytoplasmic RNA over total (cytoplasmic + nuclear) RNA, n=3. Fractionation validated by Western blot in the right panel, with Lamin A/C as the nuclear marker and GAPDH as the cytosolic marker. Statistical analyses shown in (A-D) used the two-tailed Student’s T test, error bars=SD, **p<0.01. (E) Viral RNA stability analyzed by 4SU-metabolic labeling, followed by isolation of 4SU⁺ nascent RNA at 0, 2 and 4hrs post-4SU-labeling, n=5. Slopes of regression lines compared by ANCOVA, **p=0.0008. See also Fig. S3.

Fig 4.. Silent mutagenesis of ac4C sites in env diminishes viral Gag expression.

(A) ac4C sites #4–8 in the HIV-1 env CDS were mutated to remove as many ac4C sites as possible without changing the encoded amino acid. Example of silent mutations in ac4C site #7 in the lower panel. See also Fig. S4. (B) 293T cells were transfected with WT pNL4–3 HIV-1 (WT), or the mutant viral plasmid (mut.) with ac4C sites in the env gene mutated, and Gag expression determined by Western blot. (C) Virions released into the supernatant media of 293T cells transfected with WT or mut HIV-1 expression plasmids were quantified by p24 ELISA. n=4, **p=0.002 (D) WT or ac4C mut virus were normalized using the p24 levels from panel C, and used to infect Ctrl & ΔNAT10 CEM cells. Viral Gag expression from single round (NVP-treated) infections were assayed at 48 hpi by Western blot. (E) The Gag protein bands (p55+p24) from 6 repeats of panel D were quantified and the WT/mut ratio from Ctrl & ΔNAT10 CEMs plotted. (F) Similar to panel F, WT/mut. ratios of the p24 bands only. Significance determined using paired two-tailed Student’s T test, n=6, error bars=SD.