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. 2019 May 11;15:489–501. doi: 10.1016/j.isci.2019.05.010

Figure 4.

Figure 4

LGTV NS3 E117A Mutant Virus Is Attenuated In Vitro

(A) Representative images of confocal microscopy of HEK293 cells transfected with either LGTV or LGTV_E117A in vitro-transcribed genomic RNA. Cells were processed at 72 h post-transfection and immunostained with anti-NS3 (red) and anti-E (green) antibodies. Nuclei were stained with DAPI (blue); scale bar (white solid line), 50 μM.

(B) Measurement of viral foci in Vero cells infected with recovered LGTV, LGTV NS3_D19A, or LGTV NS3_E117A. The area (mm2) of individual foci formed by each virus was calculated using ImageJ. Representative foci of each virus are presented below the column graph. Data are average of three independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; **p < 0.01; ***p < 0.001.

(C) Representative images of confocal microscopy of HEK293 cells infected with LGTV, LGTV NS3_D19A, or LGTV NS3_E117A. HEK293 cells were infected at an MOI of 0.1 and were processed at 24 hpi. Cells were immunostained with anti-E (green) and anti-NS3 (red) antibodies. Nuclei were stained with DAPI (blue); scale bar (white solid line), 200 μM.

(D) Titration of infectious particles in the supernatant from HEK293 cells infected with LGTV, LGTV NS3_D19A, or LGTV NS3_E117A (MOI of 0.1) for indicated times (h). Viral titers were determined by immunofocus assay. Data are representative of three or more independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; ***p < 0.001; ****p < 0.0001.

(E) Immunoblot analysis of whole-cell extracts from HEK293 cells infected with LGTV, LGTV NS3_D19A, or LGTV NS3_E117A (MOI 0.1) for indicated times (h). Blots were probed with antibodies to LGTV NS3, LGTV E, TRAF6, and β-actin. Results representative of three or more independent experiments.

(F) Titration of infectious particles in the supernatant from WT or TRAF6−/− MEFs infected with LGTV or LGTV NS3_E117A (MOI of 0.1) at 24 hpi. Viral titers were determined by plaque assay. Data are representative of three independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; ***p < 0.001.