Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression

Abstract

Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.

Figure 1. m⁶A site discovery in HIV-1 isolate NL4-3

(A) Overview of the general PAR-CLIP experimental design. (B) A schematic of the PA-m⁶A-seq and PAR-CLIP site discovery strategy is depicted. A typical transcript containing an m⁶A editing site is shown with an incorporated adjacent 4SU molecule (orange star). Upon binding, the m⁶A specific antibody or a host YTHDF reader protein is crosslinked to the 4SU. T>C transitions are generated from crosslinked 4SU during reverse transcription/cDNA synthesis. (C) PA-m⁶A-seq and PAR-CLIP were performed 64 h after infection with VSV-G pseudotyped HIV-1 strain NL4-3. Shown are the entire genome coverage tracks for PA-m⁶A-seq in CEM-SS cells, and then the FLAG-GFP control and YTHDF1, 2 and 3 tracks in 293T cells. (D) Expanded view of the 3'UTR region of HIV-1 containing the detected m⁶A editing sites. This ~1.4 kb region extends from the second coding exon of Rev to the end of the R region. Red/Blue bars indicate sites of T>C conversions. Reads are aligned to an HIV-1 genome that begins with the U5 region and ends with U3-R to avoid repeat alignments. The PA-m⁶A-seq has a Y axis of 0-200 reads, and all others are depicted with Y axes of 0-900 reads. For related data, see Fig. S1.

Figure 2. m⁶A site discovery using primary HIV-1 isolates BaL and JR-CSF

(A) YTHDF1 or YTHDF2 PAR-CLIP binding clusters were mapped for HIV-1 isolates NL4-3, BaL and JR-CSF for the 3’ region of the HIV-1 genome from the second exon of Rev to the end of the R region, as indicated. The three novel YTHDF protein binding clusters discovered for these two viruses are annotated below the relevant track. The Y axes for these alignments are NL4-3: 0-900 reads, BaL: 0-2000 reads, and JR-CSF: 0-1500 reads. (B) Alignment of two segments from the NL4-3 and BaL genome, with a putative novel methyl receptor adenosine present in BaL shown in red. (C) Similar to panel B, except aligning two regions of NL4-3 and JR-CSF, with the two novel methyl acceptor adenosines present in JR-CSF indicated. For related data, see Figs. S1 and S4.

Figure 3. Consensus m⁶A editing sites mapped to the NL4-3 genome

Shown in (A-D) are the 4 mapped YTHDF PAR-CLIP clusters present in NL4-3 with consensus m⁶A sites indicated. Adjacent T to C conversions, that result from 4SU photo-crosslinking (T=blue, C=red), are indicated. Below are the potential viral m⁶A editing sites shown in red, with a black line indicating the nucleotide position in the YTHDF binding cluster relative to the mutated T residue. This figure identifies all sites with a minimal (5’-RAC-3’) m⁶A consensus but this does not demonstrate that all of these A residues are actually modified. For related data, see Fig. S4.

Figure 4. 3'UTR m⁶A sites boost mRNA abundance and protein expression

Dual luciferase indicators were constructed in which the 3'UTR of RLuc in psiCheck2 was replaced by HIV-1 3'UTR sequences in either a wildtype form or with the m⁶A sites listed in Fig. 3 replaced by G residues. The “HIV 3’ UTR” construct contains the entire ~1.4 kb 3'UTR region of HIV-1, encompassing all four m⁶A clusters, extending from the second coding exon of Rev through the viral poly(A) addition site. The U3/NF-kB/TAR indicator, which contains the viral 3’ UTR from 5’ of the LTR NF-kB repeats again through the viral poly(A) addition site, retains only the U3/NF-kB and TAR m⁶A sites. (A) The indicators were transfected into 293T cells and RLuc and internal control FLuc levels assayed at 48 h post-transfection. (B) This transfection was performed in 293T cells, as described in (A). Steady state transcript abundance was measured by qRT-PCR for both the internal control FLuc and the m⁶A cluster-containing RLuc mRNAs. RLuc mRNA abundance is shown normalized first to endogenous GAPDH mRNA and then to the control FLuc mRNA. (C) Similar to (A), except these luciferase assays were performed in transfected CEM-SS T-cells. (D) Similar to (B) except that this qRT-PCR analysis of FLuc and RLuc mRNA expression levels was performed in transfected CEM-SS T cells. (E) Cellular YTHDF PAR-CLIP clusters with 1, 2, 5, or 6 predicted m⁶A editing sites were compared using the same RLuc indicator assay as described in (A) and (C). These clusters were cloned into the 3'UTR of RLuc in a wildtype or mutant form, lacking m⁶A editing sites and RLuc activity determined. (F) YTHDF fusion proteins were constructed where the carboxy-terminal m⁶A binding domain was replaced with the MS2 coat protein, and these were compared to a negative control GFP-MS2 fusion after co-transfection into 293T cells along with a psiCHECK2 dual luciferase vector with and without MS2 binding sites inserted into the RLuc 3'UTR. (A through F). Average of from three to six independent experiments with SD indicated. For related data, see Fig. S2.

Figure 5. Overexpression of YTHDF m⁶A reader proteins boosts HIV-1 protein and RNA expression

(A and B) qRT-PCR was used to quantify the expression level of the dominant spliced HIV-1 mRNA isoforms encoding Rev, Tat or Nef as well as the unspliced genomic RNA (gRNA). Assays were performed at 24 h (A) or 48 h (B) post-infection (hpi) using 293T cells stably overexpressing GFP (Neg) or one of the three YTHDF proteins (Y1 is YTHDF1 etc). Data were normalized to endogenous GAPDH mRNA. (C and D) Shown are representative Western blots from HIV-1 infection experiments similar to those described in (A and B). Infected 293T cells over-expressing GFP (Neg) or one of the YTHDF proteins were lysed at 24 hpi or 48 hpi then probed with an antibody specific for the HIV-1 p24 capsid protein, Nef, the FLAG tag on the overexpressed YTHDF protein or endogenous β-actin. Shown below the respective bands are actin-normalized quantifications. p55 represents uncleaved HIV-1 Gag polyprotein while p24 is the mature viral capsid (E and F). Shown are quantifications of band intensities from three independent Western experiments, similar to those shown in (C and D), performed at 24 hpi (E) or 48 hpi (F), with SD indicated.

Figure 6. Recruitment of YTHDF2 to viral m⁶A editing sites boosts viral replication in CD4+ T cells

(A) A representative growth curve for HIV-1 NL4-3 in control CEM-SS cells, in a CEM-SS sub-clone lacking a functional YTHDF2 gene (Y2-KO) or in a CEM-SS sub-clone overexpressing YTHDF2 (Y2-OE). HIV-1 replication was monitored by p24 ELISA. (B) This graph shows the total level of protein recovered from the cell pellets harvested at the indicated time points from the cultures analyzed in (A). (C) This bar graph shows the average of 3 independent replicate p24 ELISA growth curve experiments at 96 hpi, with significance of differences indicated. (D) A representative Western blot of samples treated as in (A) at 72 hpi. This Western analyzes the level of intracellular expression of HIV-1 p24, Nef and YTHDF2, with endogenous β-actin used as a loading control. Equal quantities of protein, as determined by BCA analysis, were loaded in each lane. Mock: mock infected culture. For related data, see Fig. S3.