FIG 8.

Downregulation of endogenous SUN1 impairs nuclear entry of NL4-3luc but not that of the G89V mutant. HEK293A cells were transfected with a control siRNA (siCtrl) or siRNAs targeting different sites of SUN1. (A) Downregulation of endogenous SUN1 expression was confirmed by Western blotting. (B) Cells were infected with VSV_G-pseudotyped NL4-3luc or NL4-3luc-G89V. At 48 h postinfection, luciferase activity was measured. Fold inhibition was calculated as the luciferase activity in the control cells divided by that in the cells transfected with the SUN1-targeting siRNA and is indicated above the bars. (C to H) Cells were infected with VSV_G-pseudotyped NL4-3luc. At the indicated time points, Hirt-extracted DNA and total DNA were used for detection of the late RT product (C), 2-LTR DNA (D), and integrated viral DNA (E). The relative levels of DNA in the control cells were set to 100. At 48 h postinfection, the cells were harvested and analyzed for mRNA levels for Gag (F), Vif (G), and Nef-luc (H). Fold inhibition was calculated as the relative integrated viral DNA level in the control cells divided by that in the cells transfected with the SUN1-targeting siRNA and is indicated above the bars in panel E. (I to K) Cells were infected with VSV_G-pseudotyped NL4-3luc-G89V. At the indicated time points, Hirt-extracted DNA and total DNA were used for detection of the late RT product (I), 2-LTR DNA (J), and integrated viral DNA (K). The relative levels of DNA in the control cells were set to 100. Data presented are means and SD for three independent experiments. hpi, hours postinfection; ns, not significant (P > 0.05); *, P < 0.05; ***, P < 0.001.