LC/MS evaluation and deep sequencing reveal the correct RNA composition within the HIV-1 virion

Cell tradition, transfections and infections

The human CD4 + T-cell line MT4 (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. Douglas Richman) and the human embryonic kidney HEK293T cell line (American Sort Tradition Assortment, LGC Requirements, UK) have been cultured underneath commonplace circumstances at 37 °C underneath a humidified (>90%) ambiance of 5% CO2/95% air. The MT4 cell line was cultured in RPMI 1640 w/o L-Glutamine and 25 mM Hepes supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) (all Sigma-Aldrich). The HEK293T cell line was cultured in DMEM excessive glucose supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) (all Sigma-Aldrich).

Isolation of huge and small RNA fractions from the cell tradition

MT4 cells have been collected by centrifugation (225 × g, 5 min, and 20 °C), HEK293T cells have been collected by trypsinization and subsequent centrifugation as above. Cells pellets have been washed with PBS and cells have been lysed with RNAzol reagent (Sigma-Aldrich). Massive and small RNA fractions have been purified based on the RNAzol producer’s protocol. The RNA focus was decided on NanoDrop ONE (ThermoFisher Scientific) and the RNA pattern high quality management was carried out on a 4200 TapeStation System (Agilent).

An infection

MT4 cells have been initially contaminated with a cell-free HIV-1 pressure NL4-Three, which was generated by transient transfection of HEK293T cells with a pNL4-Three plasmid (obtained by way of NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. Malcolm Martin). The contaminated cultures have been subsequently expanded by co-cultivation. 48 h post-infection, cell tradition supernatants containing viral particles and contaminated cells have been added to uninfected MT-Four cells (5*105 cells per mL) at a ratio of 1:9. The co-culture was synchronized by three successive additions of contaminated tradition supernatant to uninfected MT4 cells (5*105 cells per mL, the ratio of 1:9, 27 h between infections).

Virus-containing supernatants have been harvested 40 h after the final synchronization step, cleared by centrifugation (225 × g, 5 min, 20 °C) and filtration (zero.45 µm pore dimension cellulose-acetate filter (VWR)), and saved at −80 °C.

Infectious titres have been decided as 50% tissue tradition infectious dose by endpoint titration utilizing serial 10-fold dilutions of the virus on TZM-bl cells47.

Viral particles purification and RNA isolation

Sucrose cushion isolation

Virus particles have been concentrated from cleared tradition medium by centrifugation by way of a cushion of 20% (wt./wt.) sucrose in phosphate-buffered saline (PBS) (90 000 × g, 90 min, Four °C). The pellet was resuspended in RNase/DNase buffer (Tris HCl 100 mM, MgCl2 25 mM, CaCl2 25 mM) with DNase I (10 U/mL, New England BioLabs – NEB), RNase I (200 U/mL) and RNase A (20 mg/mL, each ThermoFisher Scientific) and incubated 2 h at 37 °C. RNase/DNase remedy was stopped by including RNAzol (Sigma-Aldrich).

Mock medium was ready in the identical means from uninfected MT-Four cells.

OptiPrep gradient isolation

Virus particles have been concentrated and cleared by way of a sucrose cushion as described above. The virus pellet was resuspended in PBS, loaded to the highest of the iodixanol gradient (6% to 35% OptiPrep Density Gradient medium diluted in PBS, Sigma–Aldrich) and ultracentrifuged (90 000 × g, 90 min, Four °C). HIV-containing fractions have been detected by western blot evaluation utilizing the anti-HIV capsid protein antibody. Chosen fractions have been collected, diluted with PBS 5 occasions and the virus was pelleted by ultracentrifugation (90 000 × g, 45 min, Four °C). The virus-containing pellet was resuspended in RNase/DNase buffer and RNase/DNase remedy was carried out as described above.

Western blot evaluation

Samples from every fraction of the Optiprep gradient have been combined with loading buffer within the ratio of 5:1 and denatured for five min at 95 °C. Separation was carried out at 15% SDS PAGE at fixed voltage 150 V for 90 min. Separated proteins have been transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and blocked at casein (Blocker Casein, ThermoFisher Scientific). HIV-1 CA protein was detected with polyclonal anti HIV-1 CA antibody (sera of rabbit immunized with purified HIV-1 CA protein, produced at Institute of Physiology CAS; dilution 1:1000, 1 h, RT) and as secondary antibody was used Goat anti Rabbit IgG labelled with horseradish peroxidase (1:10000, 1 h, RT, Sigma-Aldrich). After washing, the membranes have been incubated with SuperSignal West Femto Most Sensitivity Substrate (ThermoFisher Scientific) and the depth of chemiluminescence was detected utilizing the CCD digicam (Las-3000, software program Picture Reader Las-3000, Fujifilm) (Supplementary Determine S7).

RNA from viral particles was purified by Zymo-Spin™ IIC Columns (Direct-zol™ RNA MiniPrep Plus, Zymo) based on the producer’s protocol. The standard of RNA samples was checked by HS RNA ScreenTape. (Supplementary Determine S4) RNA samples have been quantified by an RNA Excessive Sensitivity Assay (Quibit Four Fluorometer, ThermoFisher Scientific), and the presence of HIV gRNA was confirmed by RT-PCR (LightCycler, Roche) (Supplementary Determine S3).

RNA digestion and LC/MS evaluation

RNA samples (1–Four µg) have been totally digested by Nuclease P1 (1 U/µg of RNA, Sigma-Aldrich) in 40 µL of 50 mM ammonium acetate buffer (pH Four.5) for 1 h at 37 °C. After addition of Alkaline phosphatase (CIP, 1 U/µg of RNA, NEB) and CutSmart buffer (last focus 1 × , NEB), the samples have been incubated for an additional 1 h at 37 °C. Digested RNA samples have been diluted in 200 µL and purified over Microcon® – 10 kDa centrifugal filters (Merck). The flow-through was concentrated utilizing a SpeedVac system to the amount of 20 µL for LC-MS evaluation.

All samples have been measured in technical duplicates. The separation of the digested RNA samples (eight µL injection quantity) was carried out by an LC system (I-Class, Waters) on a C18 column (Acquity UPLC® BEH C18 1.7 µm, 15 cm, Waters) at 40 °C utilizing a gradient of water (A) and acetonitrile (B), every containing zero.1% (v/v) formic acid48. The gradient was zero–6 min, 100% A; 6–7.5 min, 100–99% A; 7.5–9.5 min, 99–94% A; 9.5–15 min, 94% A; 15–25 min, 94–50% A; 25–27 min, 50–20%; 27–29.5 min, 20% A; 29.5–30 min, 20–100% A; 30–40 min, 100% A. The stream charge was zero.05 mL/min. The autosampler cooled the samples to eight °C. The LC system was coupled on-line to a mass spectrometer (Synapt G2, Waters) to accumulate lots of nucleosides by electrospray ionisation. Ions have been scanned in a optimistic polarity mode over full-scan vary of m/z 100–1200. The supply parameters have been as follows: capillary voltage, Three kV; supply temperature, 150 °C; sampling cone, 40; extraction cone, 5; desolvation temperature, 450 °C; desolvation gasoline stream, 600 L/h.

All mass chromatograms have been analysed using the MassLynx V4.1 software program. Mixtures of nucleoside requirements (m1A, m6A, Am, A, t6A; Jena Bioscience, Sigma-Aldrich, CarboSynth) at three totally different quantities: 64, 320 and 1600 fmol every have been injected on a column to match the response of every nucleoside underneath outlined ionization circumstances. Mixtures have been measured within the technical triplicates. For every commonplace, an extracted ion chromatogram (EIC) was generated utilizing a serious fragment noticed in its full scan spectrum (fragmentation happens within the ion supply). The chromatographic peaks in EICs have been built-in. The usual peak space (space underneath the curve, AUC) was used to calculate the ionization effectivity ratio of the examined nucleosides on this focus vary (Supplementary Desk S3, Figures S1, S2).

The chromatographic peaks of the key fragments in EICs have been built-in. The AUC was used to calculate the share of the adenosine modifications (Fig. 1A,B).

Deep sequencing library preparation

Deep sequencing libraries have been ready utilizing a mixture of three protocols12,31,49.

Chemical fragmentation (metal-ion induced) was used to realize dimension distributions of the fragments from 50–200 nt. RNA samples (1–2 µg) have been incubated with 100 mM ZnCl2 in 100 mM Tris-HCl buffer at pH 7.Four at 75 °C for 1 min. The response was terminated by addition of EDTA at a last focus of 50 mM. Samples have been ethanol precipitated and the dimensions of the RNA fragments was verified by HS RNA Display Tape®.

One half of every pattern was incubated with alkaline buffer (50 mM Na2CO2, 2 mM EDTA, pH 10.Four) for 1 h at 60 °C to transform m1A into m6A through Dimroth rearrangement. Samples have been purified by RNA Clear & Concentrator columns (Zymo) and analysed on HS RNA Display Tape®.

After denaturation of the RNA samples at 90 °C for 30 s, adopted by cooling down on ice, dephosphorylation was carried out by zero.5 U of FastAP alkaline phosphatase (ThermoFisher Scientific) in dephosphorylation buffer (at a last focus 100 mM Tris-HCl, pH 7.Four, 20 mM MgCl2, zero.1 mg/mL BSA and 100 mM 2-mercaptoethanol) for 30 min at 37 °C. The entire process was repeated, adopted by last warmth deactivation of the enzyme at 75 °C for five min.

A pre-adenylated adaptor was ready as described beforehand49 (for the sequence see Supplementary Desk S6). Ligation on the Three′-end of RNA was carried out at Four °C for 72 h in dephosphorylation buffer, containing as well as, 15% DMSO, 5 µM adenylated Three′-RNA adaptor, zero.5 U/µL T4 RNA ligase (ThermoFisher Scientific) and 1 U/µL T4 RNA ligase 2 truncated (NEB). The response was stopped by heating to 75 °C for 15 min. Unligated Three′-adaptor was eliminated by 5′-Deadenylase (NEB) and Lambda exonuclease (ThermoFisher Scientific). The combination was ethanol precipitated.

Reverse transcription with SuperScript III (ThermoFisher Scientific, 10 U/µL) was carried out within the First-Strand Buffer, containing RT primer (5 µM), dNTP combine (zero.5 mM last conc.), DTT (5 mM), BSA (50 µg/µL) in 30 µL response combination for 1 h at 50 °C. RT primer extra was digested by a mixture of Lambda exonuclease, Exonuclease 1 and FastAP alkaline phosphatase (all ThermoFisher Scientific) after response. RNA was degraded by NaOH and samples have been ethanol precipitated.

Reverse transcription with TGIRT™-III (InGex, 1 µL, 500 nM) was carried out in 19 µL of response buffer (450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5), with DTT (5 mM) and RT primer (5 µM) for 30 min at room temperature. After 30 min dNTPs (1.25 mM every, last quantity 20 µL) have been added and the response was incubated at 60 °C for 50 min. The response was stopped by the addition of 1 µL of 5 M NaOH and was incubated for Three min at 95 °C. Samples have been cooled down at room temperature, neutralized with 1 µL of 5 mM HCl and ethanol precipitated.

Three′-Tailing of cDNA was carried out utilizing 1 U/µL of Deoxynucleotidyl transferase (TdT, ThermoFisher Scientific) in 1x TdT buffer containing 1.25 mM CTP for 30 min at 37 °C with warmth deactivation (75 °C, 10 min). Double stranded DNA anchor was ready as described beforehand49. Ligation of dsDNA adaptor (1.25 µM) was finished in 50 mM Tris-HCl buffer (pH 7.Four), containing 10 µM ATP and 20 mM MgCl2 with T4 DNA ligase (1.5 U/µL) at Four °C for 72 h. The enzyme was warmth deactivated at 65 °C for 10 min. Samples have been ethanol precipitated and dissolved in 12 µL of water (molecular biology grade).

PCR amplification was carried out with barcoded PCR primers (Supplementary Desk S6) in 38 cycles in ThermoPol response buffer 1 × (NEB), with 5 µM of every barcoded primer, zero.5 mM dNTPs (every) and zero.25 U of Taq DNA Polymerase (NEB) in 20 µL of whole response combination. Preliminary denaturation was carried out at 95 °C for 60 s, following by annealing for 60 s at 54 °C, elongation for 60 s at 68 °C and denaturation for 30 s at 95 °C. Closing extension was carried out at 68 °C for five min.

PCR response combination was loaded on 1.Three% agarose gel (140 V for two h). Fractions between 100–400 nt have been minimize and DNA was extracted from the gel by NucleoSpin® Gel and PCR Clear-up (Macherey –Nagel).


Libraries have been trimmed utilizing cutadapt50 and Atropos51. Clear sequences longer than 20 bp have been mapped to human genome and HIV-1 genome utilizing bwa aligner v0.7.10-r78952. All statistics and graphs have been generated by customized scripts (github:).

RNA detection by Northern blotting

Remoted RNA from HIV-1 particles and whole RNA remoted from contaminated MT4 cells have been every divided in two components. One half was chemically fragmented as described for the deep sequencing libraries. Denaturing acrylamide gel (20%, eight × 10 cm; 1 mm thickness) was ready from 19:1 acrylamide:bis-acrylamide in zero.5x MOPS buffer (10x MOPS buffer inventory containing zero.2 M MOPS pH, 50 mM NaOAc and 10 mM EDTA) and seven M urea. The polymerized gel was pre-run at 100 V for 30 min in zero.5x MOPS buffer. 15 µL of the RNA samples (400–600 ng of RNA/pattern, containing 7.Three% formaldehyde, 50% formamide, zero.5x MOPS buffer and zero.01% bromphenol blue) have been denatured for 15 min at 55 °C and loaded into the gel wells. The gel was initially run at 50 V for roughly 15 min to pay attention the samples in wells, after which at 150 V till the bromphenol blue reached 90% of the gel size. The gel was blotted onto a charged nylon membrane (Amersham Hybond-N + ; GE Healthcare) by capillary switch in 20x SSC buffer (Three M NaCl, zero.Three M tri-sodium citrate, pH adjusted to in a single day. The membrane was crosslinked twice on a default setting (120 mJ, 30 s) utilizing digital ultraviolet crosslinker (Ultralum). The crosslinked membrane was hybridized with 10 mL of Church buffer (70 mM NaH2PO4, 180 mM Na2HPO4, 7% SDS, 1% BSA, 1 mM EDTA, pH 7.2) at 45 °C for 1 h utilizing a ProBlot hybridization oven (Labnet). In the meantime, 5 μL of 100 μM probe (Sigma-Aldrich) was end-labelled utilizing 20 U of T4 polynucleotide kinase (NEB), 2 μL of γ-32P-ATP (Three.Three μM, 10 μCi/μL; Hartmann analytic) in 20 μL of supplemented kinase buffer. The labelling was carried out at 37 °C for 30 min. The enzyme was inactivated at 65 °C for five min and the probe was purified from unincorporated nucleotides utilizing Micro Bio-Spin P-30 columns (BioRad) based on the producer’s directions. The probe was added to the membrane in 10 mL of recent Church buffer and hybridized at 45 °C in a single day. The probe was washed twice for 10 min every with low stringency buffer (2x SSC + zero.1% SDS) and as soon as with excessive stringency buffer (zero.1x SSC + zero.1% SDS), all at 45 °C. The membrane was sealed in foil, incubated with phosphor imaging plate (GE healthcare) and browse utilizing Storm FLA 9500 (GE Healthcare).

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