Fig 2: 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. nsp > 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. nsp > 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 log10 scale.

Fig 3: Treatment with I2 or 1564, or loss of KPNA2, partially prevents VEEV induced cell death. (A) HMC3 wells were pre-treated with 1564 (50 μM), I2 (50 μM) or DMSO for 1 h. The cells were then infected with VEEV TC-83 at MOI 1 for 1 h, after which they were post-treated with the respective treatment for 24 h. Cell viability was assessed using the CellTiter-Glo assay. Uninfected mock cells were included as a negative control. (B) 293 WT or KPNA2 KO cells on poly-L-lysine hydrobromide plates were transfected with either pcDNA3.1 or KPNA2 plasmid. At 24 h post transfection (hpt), the cells were infected with VEEV TC-83 for 24 h. Un-infected mock cells transfected with just pcDNA3.1 plasmid were included as controls. After 24 hpi, cell viability was determined using the CellTiter-Glo assay. (C) HMC3 wells were infected with VEEV TC-83 or VEEV Cm at an MOI 1 for 1 h, then fresh media was added. CellTiter-Glo assay was done after 24 h. Uninfected mock cells were included as a negative control. (D) 293 WT or KPNA2 KO cells on poly-L-lysine hydrobromide-coated plates were pre-treated with 1564 (50 μM), I2 (50 μM) or DMSO for 1 h. The cells were then infected with VEEV TC-83 at MOI 1 for 1 h, after which they were post-treated with the respective treatment for 26 h. Cell viability was assessed using CellTiter-Glo assay. nsp > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig 4: Loss of KPNA2 does not reverse capsid’s block of nucleocytoplasmic trafficking. (A) Schematic of the constructs of the two replicon systems (VEErep/C/4xTomato_3xNLS and VEErep/Cm/4xTomato_3xNLS) used for the nucleocytoplasmic trafficking assay. (B) 293 WT or KPNA2 KO cells were electroporated with either VEErep/C/4xTomato_3xNLS or VEErep/Cm/4xTomato_3xNLS. At 16 h post electroporation (hpe), cells were prepared for immunofluorescence microscopy. Blue indicates the nucleus (DAPI) and red indicates dTomato. (C) The nuclear-to-cytoplasmic ratio of dTomato was determined using the cellular analysis tool on Gen5 Image software. At least 40 cells per condition were utilized in the quantification. Y-axis is in log10 scale. nsp > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig 5: Loss of KPNA2 does not impact VEEV replication kinetics. 293 WT or KPNA2 KO cells on poly-L-lysine hydrobromide-coated plates were infected with VEEV TC83. Supernatants were collected at 3, 6, 9, 18, and 24 hpi, and viral titers were determined by plaque assay. nsp > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Data are represented as mean ± SD.