Fig 1.
YTHDF3-deficient T cells are more susceptible to HIV infection.
(A) A3R5-Rev-GFP NTCg1 and A3R5-Rev-GFP YTHDF3g1 T cells were generated by nucleofection of in vitro assembled CRISPR/Cas9 ribonucleoproteins. A representative western blot is shown. NTCg1: non targeting guide RNA 1; YTH3g1: YTHDF3 gRNA1. (B) Infection of A3R5-Rev-GFP NTCg1 and A3R5-Rev-GFP YTHDF3g1 with HIV NL4-3. The number of infected cells (GFP+) was measured by FACS at day 4 post infection. Infections were performed in duplicates. The number of infected A3R5-Rev-GFP YTHDF3g1 cells (GFP+) is shown relative to the infected A3R5-Rev-GFP NTCg1 cells. * denotes p ≤ 0.05 as determined by an unpaired, two-tailed student’s T test. Three independent experiments were performed. (C) YTHDF3 knockdown in primary human CD4+ T cells nucleofected with CRISPR/Cas9 ribonucleoproteins. A representative Western blot is shown. (D) Infection of primary CD4+ T cells modified as shown in Fig 3C with HIV-NL4-3 (HIV NL4-3 X4 Renilla Luciferase). Luciferase expression was quantified four days post infection. Infection was calculated relative to NTCg1-targeted T cells. Error bars denote SEM. **** denotes p ≤ 0.0001, as determined by an unpaired, two-tailed student’s T test. Data shown for two different donors (donor 1 and 2) with infection being done in, at least, triplicates.
Fig 2.
YTHDF3 are incorporated into virions in a nucleocapsid dependent manner.
(A, B) Concentrated viral supernatants (A) and cell lysates (B) of HEK293T cells co-transfected with HIV NL4-3 and individual plasmids expressing FLAG-tagged YTHDF3 (Y3) or control (C) were analyzed by Western blotting. The antibodies used are denoted. A representative Western blot of five individual experiments is shown. (C) Schematic representation of the four HIV NL4-3-derived Gag plasmids used is shown. (D, E) Concentrated viral like particles (VLP, D) and cell lysates (E) of HEK293T cells co-transfected with Gag WT or Gag mutant expression plasmids and FLAG-tagged YTHDF3 (Y3) or control (C) plasmids were analyzed by Western blotting. The antibodies used are denoted. A representative Western blot of five individual experiments is shown. * Please note that β-actin control for ΔGH overlaps with GagΔGH.
Fig 3.
YTHDF3 incorporation in HIV particles negatively affects virus infection.
(A) HEK293T cells were co-transfected with HIV NL4-3 and plasmids expressing FLAG-tagged YTHDF3 or control. P24 levels in the viral supernatant were measured to quantify virus production. Error bars denote standard error of the mean (SEM). *** denotes p<0.001 and **** denotes p<0.0001 as determined by student’s two-tailed T test. The average of two experiments is shown with each experiment comprising three biological replicates. (B) Viral infectivity was determined by infection of TZM-bl cells with 1ng p24 equivalent of each virus produced as shown in Fig 3A. Error bars denote standard error of the mean (SEM). ** denotes p<0.01 and **** denotes p<0.0001 as determined by a student’s two-tailed T test. The average of two experiments is shown with each experiment comprising three biological replicates. (C) Experimental approach to directly test the impact of virion-incorporated YTHDF3 on the next round of infection (panels 2C, 2D, 2E). Please note that HEK293TΔYTH3 cells are used for these experiments. (D) Single cycle HIV stocks were produced by co-transfecting HEK293TΔYTH3 cells with NL4-3 lacking a functional envelope (NL4-3 ΔEnv), VSV-G envelope, and FLAG-YTHDF3 or control. Culture supernatants were concentrated and analyzed by Western blotting. The viral stocks are referred to as HIV+control (blue) versus HIV+YTHDF3 (green). (E) A3R5-Rev-GFP T cells were infected with different doses of single cycle viral stocks (HIV+YTHDF3 versus HIV+control). Quantification of GFP+ expressing T cells was performed by FACS in technical triplicate 3–4 days post infection. One representative experiment with technical triplicates is shown (total number of experiments = 3). (F) Normalization of the data shown in panel 3E. Fold change relative to control was calculated. Each row was analyzed individually, without assuming a consistent standard deviation. ** denotes p<0.01 and *** denotes p<0.001 as determined using the Holm-Sidak method, with alpha = 0.05. Data from three individual experiments were included in the graph. (G) Infection of HEK293T-ΔYTH3 with HIV+YTHDF3 or HIV+control single cycle viruses in the presence or absence of the non-nucleoside reverse transcriptase inhibitor Nevirapine (NVP, 1mM). Quantification of early RT products was performed using DNA from infected cells harvested at 5, 23, 29, and 53 hours post-infection. Two biological replicates were measured in duplicate or triplicate. Data shown is representative of two independent experiments. Corresponding legends are shown in Fig 3H. (H) Same infection as shown in panel 3G only that the quantification of late RT products is shown. Two biological replicates were measured in duplicate or triplicate. * denotes p<0.05 and ** denotes p<0.01 as determined by two-tailed student’s T-test. Data shown is representative of two independent experiments.
Fig 4.
HIV Protease cleaves YTHDF3 in the virus.
(A) Endogenous YTHDF3 is incorporated into HIV. Western blot of concentrated HIV NL4-3 (left) and A3R5-Rev-GFP NTCg1 or A3R5-Rev-GFP YTHDF3g1 T cell lysates (right) are shown. HIV NL4-3 viral stocks were produced by spreading infection of A3R5-Rev-GFP NTCg1 and A3R5-Rev-GFP YTHDF3g1 T cells in duplicate. Mock represents concentrated culture supernatants collected from mock infected A3R5-Rev-GFP NTCg1 or A3R5-Rev-GFP YTHDF3g1 T cells. Western blots were probed with anti-YTHDF3 ab103328, anti-Gag-p24, and anti-beta-actin. (B) HIV NL4-3Δenv virus was produced by co-transfecting HEK293TΔYTH3 cells with FLAG-YTHDF3 in the presence and absence of the protease inhibitor Indinavir (IDV 5 μM). Concentrated virus and cells were lysed and analyzed by Western blotting. Membranes were probed with anti-YTHDF3 ab103328 and anti-Gag-p24. Data is representative of five independent experiments. (C) The same samples as shown in Fig 4B were probed with the FLAG epitope antibody used in previous experiments. (D, E) NL4-3Δenv virus was produced by co-transfecting HEK293TΔYTH3 cells with FLAG-YTHDF3 in the presence of increasing concentrations of protease inhibitors Indinavir (IDV, range 0–5μM, panel D) or amprenavir (AMP, range 0–5μM, panel E). Western blots of two biological replicates are shown.
Fig 5.
Cleavage ofYTHDF3 in protease-resistant HIV mutant strains.
(A) Western blot of concentrated virus lysates produced in HEK293TΔYTH3 cells co-transfected with FLAG-YTHDF3 and HIV NL4-3Δenv WT, NL4-3Δenv-MDR1 (Indinavir resistance associated mutations I84V & L90M), NL4-3Δenv-MDR2 (L90M), or control plasmid. Increasing doses of Indinavir or DMSO-only control was added to cells one day after transfection. Membranes were probed with anti-YTHDF3 ab103328 and anti-Gag p24. Data shown is representative of three individual experiments. (B) Quantification of virion incorporated full-length YTHDF3 protein (64 kD, YTHDF364) and the YTH3 fragment (44 kD, YTHDF344) as determined by Western blot (e.g., as shown in Fig 5A). Percent YTHDF364 was calculated relative to the total YTHDF364 and YTHDF344 in each lane. Data shown for two independent experiments are shown. The DMSO-only control is artificially placed at 0.0016 μM for the purpose of graphical representation.
Table 1.
Overview of the drug resistance associated mutations found in the protease and RT regions of HIV NL4-3-MDR1 and HIV NL4-3-MDR2.
The molecular backbone is isogenic (HIV NL4-3 Δenv/eGFP). The protease mutations associated with reduced susceptibility to Indinavir and Amprenavir are in bold.
Fig 6.
Detection and mapping of YTHDF3 fragments by LC/MS/MS.
(A) Western blot of concentrated viruses produced by co-transfection of HEK293TΔYTHDF3 with HIV NL4-3 and FLAG-YTHDF3 in the presence or absence of Indinavir (IDV, 5μM). (B) Silver stain SDS PAGE gel of the viral stocks shown in Fig 6A. The region of interest was cut out and sent for LC/MS/MS. (C) The most N-terminal peptide identified by mass spectrometry is shown (peptide sequence is shown above the spectrum). (D) The most C-terminal peptide identified by mass spectrometry is shown (peptide sequence is shown above the spectrum). (E) Graphical representation of the different protein domains within YTHDF3, the unique tryptic peptides identified by mass spectrometry (MS/MS peptides, yellow), the predicted protease cleavage sites (P, black), and the location of the YTHDF3 antibody ab103328 (“αYTH3 binds” box) epitope are shown.