Journal: Virology Journal

Article Title: 2-Deoxy-D-glucose inhibits lymphocytic choriomeningitis virus propagation by targeting glycoprotein N-glycosylation

doi: 10.1186/s12985-023-02082-3

Figure Lengend Snippet: LCMV infection induces changes in the expression of genes involved in glucose uptake and utilization. MRC-5 cells were mock- or LCMV-infected at an MOI of 3 and the mRNA levels of GLUT1 ( A ), GLUT3 ( B ), HK1 ( C ), HK2 ( D ), PDHA1 ( E ), and LDHA ( F ) were quantified by RT-qPCR at indicated times after infection. Gene expression levels normalized to β-actin ( ACTB ) were determined according to the 2 –∆∆Ct method. Data were log 2 transformed and expression values from four independent experiments are shown as a box plot with Tukey’s whiskers. The median value and outliers are depicted by the horizontal line and solid dots, respectively. Statistical differences between groups were analyzed using Welch’s ANOVA with Tamhane’s T2 post hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns not significant, hpi hours post-infection

Article Snippet: Anti-β-actin antibody (#3700) was purchased from Cell Signaling Technology, anti-α-tubulin antibody (ab7291) was purchased from Abcam, anti-GP2 antibody (83.6) was a kind gift from Prof. Stefan Kunz (Institute of Microbiology of the University of Lausanne, Lausanne, Switzerland), anti-NP (M87) and anti-Z (MJ-3) are in-house made monoclonal antibodies [ ].

Techniques: Infection, Expressing, Quantitative RT-PCR, Transformation Assay

Journal: Virology Journal

Article Title: 2-Deoxy-D-glucose inhibits lymphocytic choriomeningitis virus propagation by targeting glycoprotein N-glycosylation

doi: 10.1186/s12985-023-02082-3

Figure Lengend Snippet: Addition of mannose can reverse the negative impact of 2-DG on the LCMV life cycle. MRC-5 cells were infected with LCMV at an MOI of 3. After adsorption, the inoculum was removed and replaced with fresh media according to experimental design, followed by 24-h incubation. A , B Whole-cell extracts were harvested and used for the immunoblot analysis of viral protein expression levels using anti-GP2 ( A ), anti-NP, and anti-Z antibodies ( B ). The signals obtained with anti-α-tubulin or anti-β-actin antibodies were used as loading controls. The ratios of GP-C/tubulin, GP2/tubulin, NP/actin, and Z/actin were determined based on the densitometric analysis of protein abundance. Final values are expressed relative to control, which was set to 1. C LCMV titers in the supernatants were analyzed by FFA. Data from six independent experiments are shown as a scatter of individual values. Mean and SD are shown as column and error bars, respectively. D Total cellular RNA was isolated and LCMV RNA was detected by RT-qPCR. Copy numbers of viral NP were calculated using a standard curve. Data from three independent experiments are shown as a scatter of individual values. Mean and SD are shown as column and error bars, respectively. Statistical differences between groups were analyzed using Welch’s ANOVA with Tamhane’s T2 post hoc test ( C ) or one-way ANOVA with Tukey’s post hoc test ( D ). Different letters above the columns indicate significant differences ( P ≤ 0.05), while identical letters indicate no significant difference. E Whole-cell extracts were subjected to either mock or PNGase F treatment and used for the immunoblot analysis of viral glycoprotein levels using anti-GP2 antibody. The signal obtained with anti-β-actin antibody was used as a loading control. NP, nucleoprotein; Z, Z protein; glyco/unglyco GP-C, glycosylated/unglycosylated glycoprotein precursor complex; GP2, glycoprotein 2; glyco/unglyco GP2, glycosylated/unglycosylated glycoprotein 2; FFU/ml, focus-forming units per milliliter

Article Snippet: Anti-β-actin antibody (#3700) was purchased from Cell Signaling Technology, anti-α-tubulin antibody (ab7291) was purchased from Abcam, anti-GP2 antibody (83.6) was a kind gift from Prof. Stefan Kunz (Institute of Microbiology of the University of Lausanne, Lausanne, Switzerland), anti-NP (M87) and anti-Z (MJ-3) are in-house made monoclonal antibodies [ ].

Techniques: Infection, Adsorption, Incubation, Western Blot, Expressing, Isolation, Quantitative RT-PCR