Figure 5

HK2 knockdown attenuates human rhinovirus infection that is rescued by forced expression of cytoplasm-restricted hnRNP A1. (a) HeLa T4+ cells were transfected with HK2-targeting (siHK2) or nontargeting control (siCTRL) siRNA for 48 h and subsequently infected with HRV for 10 h. The expression of viral capsid proteins was detected by immunofluorescence and nuclei were stained with Hoechst. Representative immunofluorescence confocal images are shown. (b) Quantification of the number of infected cells (identified as those that stained positive for viral capsid protein relative to the total number of cells identified by Hoechst) from (a), where ∼350 cells per condition were counted; n=3, P=0.0002. (c) Levels of HRV RNA in HK2-targeting (siHK2) or nontargeting control (siCTRL) siRNA-transfected HeLa T4+ cells infected for 4 or 6 h were determined by RT-qPCR. Absolute amount of HRV RNA normalized to GAPDH mRNA levels is shown; n=3, P=0001. (d) Kinetic analysis of caspase 3/7 activity in HRV-infected HeLa T4+ cells previously transfected with HK2-targeting (siHK2) or nontargeting control (siCTRL) siRNA. The y axis represents the number of fluorescent cells per image normalized to cell confluency; n=3, P<0.0001 for siHK2 versus siCTRL HRV-infected cells. (e) HeLa T4+ cells were transfected with HK2-targeting (siHK2) or nontargeting control (siCTRL) siRNA, subsequently transfected with plasmids expressing FLAG-hnRNP A1 F2 or FLAG-hnRNP A1ΔM9 for 24 h and then infected with HRV. The number of infected cells was determined as in (a), where ∼350 cells per condition were counted; P=0.0124