Additional file 1 of YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity
Creators: name: N’Da Konan, Sarah
name: Ségéral, Emmanuel
name: Bejjani, Fabienne
name: Bendoumou, Maryam
name: Ait Said, Mélissa
name: Gallois-Montbrun, Sarah
name: Emiliani, Stéphane
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology
subject: Virology
subject: Computational Biology

description: Additional file 1: Figure S1. Schematic representation of HIV-1 RNAs detected by RT-qPCR in this study. Organization of the NL4-3 genome and positions of SD and SA sites are indicated. Introns and exons nomenclature are according to [34]. Thick lines represent retained exon and thin lines excised introns. RNA species were named as indicated on the left side according to [34]. Forward (-f) and reverse (-r) primers used in this study are indicated by arrows. Of note, Vpr3 primers designed to detect the Vrp3 IS isoform also amplifies the Vrp1 MS isoform (not shown). However, Vpr3 represents more than 80% of its Vpr transcripts family compare to less than 7% for Vpr1 [34]. Figure S2. YTHDF2 depletion increases HIV-1 mRNA transcripts abundance in HeLa infected cells (A) HeLa cells were transfected with a control (Ctrl) or YTHDF1, YTHDF2 and YTHDF3 targeting siRNAs, as indicated. 3 days later, the respective mRNA abundance of each of the 3 readers was measured by RT-qPCR. Data are presented as mean ± S.D. (n = 5 for siYTHDF1 and n = 3 for siYTHDF2 and siYTHDF3). (B) Cellular proteins knockdown was confirmed by western blot analysis of cell lysates transfected with indicated siRNAs. Note that YTHDF1 was detected on a membrane already probed with the YTHDF2 antibody (°, YTHDF2; *, nonspecific band). (C) 3 days after siRNA transfection, cells were infected with a single round VSVg-pseudotyped HIV-1 virus. 24 h.p.i., the relative abundance of US, IS and MS viral RNAs was monitored by RT-qPCR. Data are presented as mean ± S.D. (n = 3). Results are expressed in fold change over the control siRNA (Ctrl). Data are presented as mean ± S.D. (n = 3). P values were calculated using one-sample t-test (*, p &lt; 0.05, **, p &lt; 0.001). Figure S3. YTHDF2 binds preferentially US HIV-1 RNA. (A) HeLa cells were infected with VSVg-pseudotyped HIV-1 and 24 h later YTHDF2 was immunoprecipitated using an antibody against the endogenous protein. IgGs were used as control immunoprecipitation. The specificity of the YTHDF2 immunoprecipitation was confirmed by western blotting and RNA was extracted from input and immunoprecipitated samples. (B) Levels of YTHDF2-associated US, IS and MS viral RNAs were quantified by RT-qPCR. Data are presented as mean ± S.D. (n = 3). P values were calculated using paired t-test (*, p &lt; 0.05). The p-value for MS RNAs is not significant (p = 0.0821). Figure S4. YTHDC1 depletion affects Vpu and Env expression in HeLa and HEK293T producing cells. (A) HeLa cells were transfected with control (Ctrl) or a siRNA targeting YHTDC1 (DC1-1). The following day, cells were transfected with pNL4-3 and 48 h. later cells harvested and cell lysates were analyzed by western blotting for HIV-1 viral proteins Env, Gag and Vpu and Nef. (B) HIV-1 proteins band intensities were quantified by phosphoimaging and normalized to GAPDH (n = 3). Fold change of protein levels upon YTHDC1 knockdown over the control are plotted. Data are presented as mean ± S.D. (n = 3). P values were calculated using one sample t-test (**, p &lt; 0.01).