Exploitation of nuclear functions by human rhinovirus, a cytoplasmic RNA virus
Fig 7
SFPQ knockdown correlates with reduced HRV16 replication.
(A) Transfection of non-targeting siRNA (siNT) or SFPQ-targeting siRNA (siSFPQ) did not result in statistically significant differences in cell viability 96 h post-transfection, as assessed by trypan blue exclusion prior to infection (P > 0.05). Mean percent viable values are displayed with error bars representing one standard deviation. (B) HeLa cells transfected with siRNA for 96 h were infected with HRV16 (MOI 0.1) and cells and cell culture fluids were harvested at the indicated times. Virus titer was determined by plaque assay. Data represent the means of three biological replicate experiments with error bars indicating standard error of the means (SEM) (* P < 0.005, ** P < 0.0005). (C) Single cycle growth analysis of HRV16 from siRNA-transfected and HRV16-infected HeLa cells (MOI 10). As in panel B, data represent the means of three biological replicate experiments with error bars displaying SEM (* P <0.005, ** P <0.0005). (D) siRNA-transfected and HRV16-infected HeLa cell lysates corresponding to the time-points in panel B (MOI 0.1) were generated and subjected to Western blot analysis. Knockdown of SFPQ was confirmed and levels of 2C and precursor (2BC) represented viral protein production. GAPDH was used as a loading control. (E) Western blot analysis of lysates from siRNA-transfected and HRV16-infected HeLa cells corresponding to time points in panel C (MOI 10). Western blots in panels D and E are representative results from three biological replicate experiments. (F) RNA isolated from siRNA-transfected and HRV16-infected HeLa cells at time-points corresponding to panel B (MOI 0.1) was subjected to RT-PCR. Primers for PCR were specific for HRV16 RNA, SFPQ mRNA, or actin (ACTB) mRNA. PCR products were separated by agarose gel electrophoresis. (G) RT-PCR analysis of RNA isolated from siRNA-transfected and HRV16-infected HeLa cells at time-points corresponding to panel C (MOI 10). Data in panels F and G are representative results from biological duplicate experiments. hpi: hours post infection.