Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: Probing and correction of HIV-1 base modifications called by nanopore dRNA-seq. ( A ) Nanopore modification-calling results for HIV-1 viral RNA extracted from Jurkat cell cultures treated with STM2457, a drug that inhibits METTL3 m 6 A modification activity. The position of each high frequency m 6 A nucleotide in the HIV-1 genome is indicated on the x -axis. ( B ) Comparison of modifications at DRACH motif sites where m 6 A was called at high frequency. The nucleotide position of the m 6 A modification within the DRACH motif is indicated on the x -axis. Upper plot: comparison of Jurkat cell samples infected with HIV-1 without (back row) or with (front row) 30 μM STM2457 treatment. Lower graph: comparison of Jurkat cell sample infected with HIV-1 before (back row) and after (front row) baseline correction. Comparison of modification calling between NL4-3 from Jurkat cells ( C ) and two synthetic HIV-1 RNA fragments, one unmodified ( D ) and one bearing m 6 A ( E) at two DRACH motifs. The nucleotide position corresponding to the NL4-3 genome is indicated on the x -axis. All modifications called in panel (D) are considered incorrect while modifications called in panel (E), besides m 6 A at position 8975 and 8989, are considered incorrect.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Activity Assay, Comparison, Infection

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: Nanopore-based modification calling of the most reliable, baseline-subtracted HIV-1 RNA modifications on viral RNA from Jurkat cells. The HIV-1 genome architecture is illustrated above. Modifications are shown if, after baseline correction, the average modification frequency was at least 10%. ( A ) m 6 A (blue), ( B ) m 5 C (green), ( C ) pseudouridine (psi) (yellow), ( D ) inosine (purple), and ( E ) 2′- O -methylation (orange). Inset in panel (A) is a close-up of the 3′ end of the NL4-3 HIV-1 genome where m 6 A is most densely called. Results are the average of three separate biological replicates. Error bars are standard deviation.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Methylation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: Differential modification frequencies in HIV-1 RNA splice isoforms. Comparison of modification frequency in each splice isoform as compared to the full-length (unspliced) isoform. X -axis represents individual nucleotides in the HIV-1 genome. Y -axis represents percentage of reads that contained the mutation of interest for that nucleotide, relative to the unspliced form. Bar color indicates modification type: m 6 A (blue), m 5 C (green), pseudouridine (yellow), inosine (purple), and 2′- O -methyl (orange). Only modifications with a difference of 10% from the unspliced RNA in at least one isoform are shown. Shaded background shows portion of the full transcript retained in the spliced isoform. Splice isoforms are subsets of direct-RNA sequencing sample 7C. The A6 splice site is exclusive to the HXB2 genome and therefore not indicated here.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Comparison, Mutagenesis, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: A nanopore-based HIV-1 reference epitranscriptome

doi: 10.1093/nar/gkag220

Figure Lengend Snippet: A preliminary HIV-1 antisense epitranscriptome, the effect of cART and cell type on HIV-1 modification calling, and conservation of m 6 A in HIV-1 genomes from PLWH. (A) Nanopore modification-calling for HIV-1 antisense RNA from Jurkat cells. m 6 A (blue), m 5 C (green), pseudouridine (yellow), inosine (purple), and 2′- O -methylation (orange). Inset shows a close-up of the asp gene. (B) Nanopore modification-calling results for HIV-1 RNA taken from Jurkat cells treated without (darker color) or with (lighter color) cART treatment. Error bars represent the standard deviation from three separate biological replicates. (C) Nanopore modification-calling results for HIV-1 RNA taken from Jurkat cells, primary CD4+ T cells infected in vitro , supernatant of primary CD4+ T cells infected in vitro , and CD4+ T cells from PLWH samples. In each nucleotide position cluster, the first bar is Jurkat cell samples infected with HIV-1, the second bar is CD4+ T cells from healthy donors and infected with HIV-1, the third bar is the supernatant from the CD4+ T cells from healthy donors, and the fourth, fifth, and sixth bars are samples taken from CD4+ T cells from PLWH donors. (D) Top: comparison of m 6 A modifications called from HIV-1 RNA from Jurkat cells against preservation of the DRACH motifs for these m 6 A modifications sequenced from three PLWH samples. Bottom: analysis of conservation of known m 6 A modification sites in a larger dataset of HIV-1 mutations. Positions containing m 6 A between nucleotide positions 8000 and 9171 were identified and the average and median Shannon entropy for these positions are reported.

Article Snippet: For replicative NL4-3, we concentrated the supernatants over a 20% sucrose cushion at 5600 × g overnight in 15 ml tubes. p24 ELISA was performed to quantify the viral preparations (R&D Systems, HIV-1 Gag p24 Quantikine ELISA Kit).

Techniques: Modification, Methylation, Standard Deviation, Infection, In Vitro, Comparison, Preserving