Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Screening of neurotransmitter receptor modulators reveals novel inhibitors of influenza virus replication

doi: 10.3389/fcimb.2025.1562650

Figure Lengend Snippet: Selected hit compounds inhibit internalization of the influenza virus. (A) Virus Attachment Assay: Cells were precooled at 4°C for 30 minutes and then infected with PR8 (MOI = 10) in the absence or presence of each candidate compound at the indicated concentrations for 1 hour at 4°C. The cells were then fixed with 4% paraformaldehyde (PFA). The viral NP was fluorescently stained green, and the nuclei were stained blue with DAPI. Images were captured using a Leica SP8 confocal microscope. Scale bar: 50 µm. (B) The total fluorescence intensities per field in A were quantified using ImageJ software; the result is one representative IFA data set. (C) Indirect Immunofluorescence Assay (IFA): Following viral attachment (MOI = 25) at 4°C for 1 hour, the cells were transferred to 37°C for 1 hour to allow for internalization in the absence or presence of each candidate compound at the specified concentrations. The level of virus internalization was measured using IFA. Scale bar: 25 µm. (D) The quantitative endocytosis in (C) was determined by counting the number of NP fluorescent foci in the cytoplasm per cell. Values are expressed as the means ± SEM of two independent experiments. (E) Internalization of influenza virus determined by acid treatment and Western botting Analysis: After viral attachment (MOI = 5) at 4°C for 1 hour, cells were washed twice with cold PBS (pH 7.2). One group underwent three washes with cold PBS-HCl (pH 1.3) to remove unattached virions, followed by cell lysis and Western blotting, which served as a control to indicate the removal efficiency of attached virions. Other groups of cells were transferred to 37°C for 30 minutes to allow for internalization, either in the absence of the drug or in the presence of each candidate drug. Afterward, the cells were lysed, and the NP protein and GAPDH levels were assessed through Western blotting. (F) The quantitative analysis of the intensity ratio of NP to GAPDH of two Western blots using ImageJ software; The relative value of the DMSO group was set as 1. (G) The inhibition of influenza A virus (IAV) entry by each candidate compound is associated with the distribution of clathrin. After viral attachment (MOI = 25) at 4°C for 1 hour, cells were treated with either the candidate compound at the specified concentrations or a control. The temperature was then raised to 37°C for 1 hour to allow for internalization. Following this, the cells were fixed, permeabilized, and immunostained for nucleoprotein (NP) in green and clathrin heavy chain in red. Scale bar: 8 µm.

Article Snippet: Mouse monoclonal antibodies against influenza A virus nucleoprotein (NP) were purchased from Sino Biological (Beijing, China).

Techniques: Virus, Infection, Staining, Microscopy, Fluorescence, Software, Immunofluorescence, Western Blot, Lysis, Control, Inhibition