Journal: bioRxiv

Article Title: The cellular poly(rC)-binding protein 2 prevents the early steps of hepatitis C virus virion assembly

doi: 10.1101/2022.04.12.488029

Figure Lengend Snippet: (A) Schematic representation of the experimental approach for 2’CMA experiments: two days post-siRNA transfection, Huh-7.5 cells were infected with JFH-1 T (MOI = 0.05). Three days post-infection, cells were treated with 2 ’ CMA or DMSO (control). Total RNA, intracellular and extracellular virus was collected at t = 0, 6 and 12 h post-treatment. (B) Quantitative RT-PCR analysis after 2’CMA treatment. (C) Intracellular viral titers and (D ) intracellular virus accumulation rate calculated by linear regression. (E) Extracellular viral titers and (F) virus secretion rates calculated by linear regression. (G) Model of PCBP2’s role in the HCV life cycle: PCBP2 normally inhibits the first stage of virion assembly, where the viral NS5A protein transfers the viral RNA to the core protein. All data are representative of three independent replicates and error bars in (B), (C) and (E) represent the standard deviation of the mean. Error bars in (D) and (F) represent the slopes of the linear regressions ± standard error. P-values were calculated by two-way ANOVA (ns, not significant; ** p < 0.01; **** p < 0.0001).

Article Snippet: Three days post-infection, the media on these cells was changed for complete Huh-7.5 media with 5 μM 2’CMA (2’C-methyladenosine, Carbosynth), an HCV NS5B polymerase inhibitor, or DMSO (vehicle control) ( ).

Techniques: Transfection, Infection, Control, Virus, Quantitative RT-PCR, Standard Deviation

Journal: PLoS Pathogens

Article Title: Host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis C virus replicase complex formation

doi: 10.1371/journal.ppat.1007284

Figure Lengend Snippet: Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. Seven days after transduction samples of the cells were collected for Western-blot analysis using antibodies against lipin1 and lipin2 and actin as loading control. Parallel cultures were subjected to an MTT assay to determine their viability as described in the methods section. (A) Representative Western-Blot showing cellular lipin1 and lipin2 protein expression levels and a loading control (actin). (B) Average expression values for lipin1 and lipin2 as determined by Western-Blot and cell viability as determined by an MTT assay. Data are shown as average and SD of six independent transduction experiments (n = 6). (C) Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. At day 3 post-transduction, cells were infected at MOI 0.01 with HCV D183 virus. Samples of cell supernatants were collected at days 3 and 5 post-infection to determine the extracellular infectivity titer. Average and SD of the infectivity titers at day 3 and 5 of two independent infections performed in triplicate (n = 6). (D) Huh-7.5 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. Silenced cells were infected 7 days after transduction at MOI 0.05 with genotype 1a HCV (TNcc) in the presence or absence of 2mAde (10μM; shControl+DAA). Intracellular HCV RNA was determined by RT-qPCR 72 hours post-infection. Data are shown as average and SD of three experiments performed in triplicate (n = 9). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

Article Snippet: HCV antiviral compounds 2´-c-methyladenosine (2mAde), sofosbuvir and daclatasvir were obtained from Boc Sciences (NY, USA), Selleckchem (Texas, USA) and Medchem Express (New Jersey, USA) respectively and dissolved in DMSO to obtain 10mM stock solutions.

Techniques: Transduction, Expressing, Control, Western Blot, MTT Assay, Infection, Virus, Quantitative RT-PCR

Journal: iScience

Article Title: Experimental verification of strain-dependent relationship between mycovirus and its fungal host

doi: 10.1016/j.isci.2023.107337

Figure Lengend Snippet:

Article Snippet: 7-deaza-2′-C-methyladenosine , Biosynth Ltd. , ND08351.

Techniques: Recombinant, Multiplex Assay, Isolation, Virus, Sequencing, Software