Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Insertion of V5 tag at the N-terminus of capsid enables successful detection of the capsid. (A) Sequence alignment between a region of VEEV TC-83 and VEEV Cm showing key mutation sites [created in BioRender. Kehn-hall, K. (2026), https://BioRender.com/d8zhltb ]. (B,C) Schematic of VEEV and VEEV Cm with V5 tag inserted at the N-terminus of the capsid protein [created in BioRender. Kehn-hall, K. (2026), https://BioRender.com/nk52hc0 ]. (D,E) Graphs depicting the replication kinetics of V5-C and V5-Cm relative to their respective parental viruses. Vero cells were infected at an MOI 1, and viral titers were determined by plaque assay at 4, 6, 8, 12, and 24 hpi. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Data are represented as mean ± SD. N = 3. (F–I) Western blots for V5-C and V5-Cm expression at various time points (MOI 5) in HMC3 and Vero cells. E1-V5 sample was used as a positive control. Mock-infected cells were used as a negative control, and actin was probed as a loading control.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Sequencing, Mutagenesis, Infection, Plaque Assay, Western Blot, Expressing, Positive Control, Negative Control, Control

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Compounds 1564 and I2 reduce capsid-importin α1 interaction. (A) Co-immunoprecipitation was performed using HMC3 cells infected with VEEV V5-C at an MOI of 1. Mock uninfected cells were included as controls. Cells were collected at 9 hpi, protein lysate was quantified and normalized, and subjected to co-immunoprecipitation assay. Pull-down of V5-tagged capsid was done using V5 tag antibody, then western blot was done by probing for importin α1 (KPNA2), CRM1, or V5. A hemagglutinin (HA) antibody pulldown was included as a co-immunoprecipitation control. (B) HMC3 cells were pre-treated with respective inhibitor (50 μM) or DMSO for 1 h, then transfected with capsid-V5 plasmid for 6 h, and post-treated with the inhibitor or DMSO for 18 h. Following protein lysates collection and normalization, capsid was pulled down using V5 antibody, then probed for KPNA2 or V5. Pulldown with HA was included as a co-immunoprecipitation control.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Immunoprecipitation, Infection, Co-Immunoprecipitation Assay, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Loss of KPNA2 reduces capsid nuclear localization. (A) 293 WT cells on PLL-coated coverslips were infected with VEEV V5-C or VEEV V5-Cm at an MOI of 10 for 9 h, then prepared for immunofluorescence imaging. Images were acquired using confocal microscope. (B) The capsid nuclear-to-cytoplasmic ratio in 293 WT cells infected with either VEEV V5-C or V5-Cm was determined using the cellular analysis tool on Gen5 image software. At least 20 cells per condition were utilized in the quantification. Statistics were determined using an unpaired t -test. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (C) KPNA2 KO or 293 WT cells on PLL-coated coverslips were transfected with either pcDNA3.1 or KPNA2 plasmid for 24 h, then infected with VEEV V5-C at MOI 10 for 9 h and processed for immunofluorescence microscopy. (D) Western blot confirmation for successful transfections of KO cells with pcDNA3.1 or KPNA2 plasmid. (E) The capsid nuclear-to-cytoplasmic ratio was determined using the cellular analysis tool on Gen5 mage software. At least 60 cells per condition were utilized in the quantification. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Slides were stained with DAPI and probed for V5 tag. Blue indicates the nucleus (DAPI) and red indicates V5 tagged capsid protein. Both A, E are presented in log 10 scale.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Infection, Immunofluorescence, Imaging, Microscopy, Software, Transfection, Plasmid Preparation, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Compounds 1564 and I2 significantly reduce nuclear import of VEEV capsid. (A) HMC3 cells on PLL-coated coverslips were pre-treated with 1564 (50 μM), I2 (50 μM), or DMSO, then infected with V5-C at MOI 1, and post-treated with 1564, I2, or DMSO. The slides were then probed, stained, and prepared for immunofluorescence microscopy. Images were acquired using confocal microscope. Blue indicates the nucleus (DAPI) and red indicates V5-tagged capsid protein. (B) The capsid nuclear-to-cytoplasmic ratio was determined using the cellular analysis tool on Gen5 Image software. At least 60 cells per condition were utilized in the quantification. Y-axis is in log 10 scale. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Infection, Staining, Immunofluorescence, Microscopy, Software

Journal: Frontiers in Microbiology

Article Title: Importin α1 contributes to Venezuelan equine encephalitis virus-induced cell death, but is not required for the capsid-mediated blockage of nucleocytoplasmic trafficking

doi: 10.3389/fmicb.2026.1784611

Figure Lengend Snippet: Capsid interacts with other importin α family members. Following infection with VEEV V5-C (MOI 1), HMC3 cells were collected, protein lysates were quantified, and subjected to co-immunoprecipitation assays. A hemagglutinin (HA) antibody pulldown was included as a co-immunoprecipitation control. (A) Immunoprecipitation of V5-tagged capsid was done using a V5 tag antibody and the blot was probed with either a KPNA3 (importin α4) antibody or V5 antibody. (B) Immunoprecipitation of V5-tagged capsid was done using a V5 tag antibody and the blot was probed with either a KPNA4 (importin α3) or V5 antibody.

Article Snippet: Blots were probed with mouse anti-V5 primary antibody (Bio-Rad, MCA1360) and goat anti-mouse secondary antibody (Invitrogen, 32430).

Techniques: Infection, Immunoprecipitation, Control