FIG 5.
Effects of the loss of SPCS1 function on virus entry into cells, genome RNA replication, and protein processing during JEV infection. (A) WT and SPCS1 KO HEK-293 cells were infected with JEV single-round infectious RRPs at an MOI of 0.5. At 48 hpi, cell nuclei were stained with Hoechst reagent. Cells positive for GFP fluorescence indicated infection with RRPs. Data from one experiment of three are shown. (B) Infectivity analyzed by using the HCS system. The data are pooled from three experiments in duplicate. Statistical significance was determined by Student's t test (*, P < 0.05). (C) Real-time qRT-PCR analysis of JEV during the early stages of infection. The data are pooled from three experiments in duplicate. Statistical significance was determined by Student's t test (*, P < 0.05). (D to F) WT and SPCS1 KO HEK-293 cells were infected with JEV at an MOI of 1.0. At 4, 6, 8, 10, and 12 hpi, infected cells were harvested, and JEV genome RNAs were analyzed by qRT-PCR. To examine the expression of the JEV E, prM, and NS1 proteins in infected cells, WT and SPCS1 KO HEK-293 cells were infected with JEV at an MOI of 20. At 48 hpi, cell lysates were blotted with anti-JEV E protein (D), anti-JEV prM protein (E), or anti-JEV NS1 protein (F) monoclonal antibodies. Higher-molecular-mass bands (Ehi, Mhi, and NS1hi) reacting with the respective monoclonal antibodies are indicated. Data from one experiment of two are shown.