N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
Figures
Figure 1 with 4 supplements
HIV-1 RNA contains m6A modifications and YTHDF1–3 proteins bind to m6A-modified HIV-1 RNA.
(A–B) The distribution of m6A reads from m6A-seq mapped to HIV-1 genome (red line) in HIV-1 infected Jurkat cells (A) or primary CD4+ T-cells (B). Baseline signal from the RNA-seq of input samples …
Figure 1—figure supplement 1
HIV-1 RNA contains m6A modifications.
HEK293 T cells were transfected with a proviral DNA-containing plasmid (pNL4-3). Total RNA was extracted at 48 hr post-transfection and immunoprecipitated with an m6A-specific antibody. Enriched RNA …
Figure 1—figure supplement 2
Quantification of HIV-1 RNA m6A level using liquid chromatography-mass spectrometry.
HIV-1 RNA (250 ng) was isolated from highly purified HIV-1MN virions (total 600 μg of p24 capsid) and subjected to quantitative analysis of the m6A level using LC-MS/MS (n=3 of each sample). The …
Figure 1—figure supplement 3
Distribution of m6A in cellular RNAs and the frequency of m6A motifs in HIV-1-infected cells.
(A–B) Pie charts show the distribution of m6A peaks in the 5′ UTR, coding DNA sequence (CDS), 3′ UTR, and noncoding regions of transcripts from uninfected and HIV-1-infected Jurkat T-cells (A) or …
Figure 1—figure supplement 4
Gene ontology (GO) analysis of m6A-modified cellular genes in HIV-1 infected cells.
(A and B) GO terms specific to virus related pathways and corresponding p values, clustered from methylated genes detected in Jurkat cells (A) or primary CD4+ T cells (B) infected with HIV-1. (C and …
Figure 2
YTHDF1–3 proteins negatively regulate post-entry HIV-1 infection in HeLa cells.
(A–B) Overexpression of YTHDF1–3 proteins in HeLa cells significantly inhibits HIV-1 infection compared to vector control cells. (A) Overexpression of YTHDF1–3 proteins in HeLa cells was confirmed …
Figure 3
YTHDF1–3 proteins negatively regulate post-entry HIV-1 infection in CD4+ T-cells.
(A) Individual knockdown of endogenous YTHDF1–3 proteins in Jurkat CD4+ T cells was confirmed by immunoblotting. (B) Knockdown of YTHDF1–3 proteins does not affect proliferation of Jurkat cells. …
Figure 4 with 1 supplement
YTHDF1–3 proteins inhibit post-entry HIV-1 infection by blocking viral reverse transcription.
HeLa cells over-expressing or knocking-down (shRNA) individual YTHDF1–3 proteins were infected with HIV-1-Luc/VSV-G at an MOI of 0.5. (A, B and D) Genomic DNA was isolated from the cells 24 hr …
Figure 4—figure supplement 1
YTHDF1–3 proteins negatively regulate HIV-1 gag mRNA expression.
Specific shRNAs or scrambled shRNA vector-treated cells were infected with HIV-1 Luc/VSV-G at an MOI of 0.5. Total RNA was isolated from the cells 24 hr post-infection and HIV-1 gag mRNA levels were …
Figure 5
YTHDF1–3 proteins bind to HIV-1 gRNA in infected cells.
(A) Immunoblotting of YTHDF1–3 proteins in the input and immunoprecipitation (IP) samples from HIV-1-Luc/VSV-G infected HeLa cells. FLAG antibodies were used to immunoprecipitate FLAG-tagged …
Figure 6
The m6A writers and erasers affect HIV-1 Gag expression in virus-producing cells.
(A and B) Individual or combined knockdown of endogenous METTL3 and METTL14 inhibits HIV-1 Gag protein expression. HEK293T cells were transfected with indicated siRNA, and then with an HIV-1 …
Figure 7
Proposed mechanisms and dynamics of m6A modification of HIV-1 RNA in regulating viral infection in cells.
In the nucleus, the m6A writers (METTL3 and METTL14) add the m6A marker to HIV-1 genomic RNA (gRNA) or mRNA, and the m6A erasers (FTO and AlkBH5) remove the m6A modifications of HIV-1 RNA. The m6A …
Tables
Table 1
The shRNA sequences used in this study.
| shRNA | Sequences (5’-3’) |
|---|---|
| Non-specific (vector) control | CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT |
| YTHDF1 | CCGGCCCGAAAGAGTTTGAGTGGAACTCGAGTTCCACTCAAACTCTTTCGGGTTTTTG |
| YTHDF2 | CCGGCGGTCCATTAATAACTATAACCTCGAGGTTATAGTTATTAATGGACCGTTTTTG |
| YTHDF3 | CCGGGATAAGTGGAAGGGCAAATTTCTCGAGAAATTTGCCCTTCCACTTATCTTTTTG |
Table 2
The siRNA sequences used in this study.
| siRNA | Sequences (5’-3’) |
|---|---|
| METTL3 | 5’-CTGCAAGTATGTTCACTATGA-3’ 5’-AGGAGCCAGCCAAGAAATCAA-3’ |
| METTL14 | 5’-TGGTGCCGTGTTAAATAGCAA-3’ 5’-AAGGATGAGTTAATAGCTAAA-3’ |
| FTO | 5’-AAATAGCCGCTGCTTGTGAGA-3’ |
| AlkBH5 | 5’-AAACAAGTACTTCTTCGGCGA-3’ |
Table 3
Sequences of PCR primers and probes used in this study.
| Primers | Sequences (5’-3’) |
|---|---|
| HIV-1 gag forward | CTAGAACGATTCGCAGTTAATCCT |
| HIV-1 gag reverse | CTATCCTTTGATGCACACAATAGAG |
| Unspliced GAPDH forward | GGGAAGCTCAAGGGAGATAAAATTC |
| Unspliced GAPDH reverse | GTAGTTGAGGTCAATGAAGGGGTC |
| Spliced GAPDH forward | GGAAGGTGAAGGTCGGAGTCAACGG |
| Spliced GAPDH reverse | CTGTTGTCATACTTCTCATGGTTCAC |
| MH531 forward (for HIV-1 late reverse transcription (RT) products) | TGTGTGCCCGTCTGTTGTGT |
| BB reverse (for late RT products) | GGATTAACTGCGAATCGTTC |
| HIV-1 late RT product probe | TCGACGCAGGACTCGGCTTGCT |
| 2-LTR probe | AAGTAGTGTGTGCCCGTCTGTTGTGTGACTC |
| 2-LTR forward | GCCTGGGAGCTCTCTGGCTAA |
| 2-LTR reverse | GCCTTGTGTGTGGTAGATCCA |
| LW59 (forward, alternative for late RT detection in shRNA vector-transduced cells) | GACATAGCAGGAACTACTAGTACCC |
| LW60 (reverse, alternative for late RT detection in shRNA vector-transduced cells) | GGTCCTTGTCTTATGTCCAGAATGC |
