FIG 5.

HCMV infection or IE1 expression inhibits STAT3 tyrosine phosphorylation and promotes nuclear accumulation of unphosphorylated STAT3 in NPCs. (A) Inhibition of STAT3 tyrosine (Y705) phosphorylation by HCMV infection. NPCs were mock infected (M) or infected with TNWT at an MOI of 3 (V) and collected at the indicated times postinfection. The protein levels of IE1, pSTAT3, and total STAT3 were determined by Western blotting. Actin served as a loading control. (B) Nuclear trapping of STAT3 by HCMV infection. NPCs were mock infected (M) or -infected with TNWT at an MOI of 1 (V). (Left) For indirect immunofluorescence analysis, NPCs on coverslips collected at 8 hpi were stained with antibodies against STAT3 (green) or IE1/IE2 (red), and nuclei were counterstained with Hoechst 33342 (blue). Infected (IE1/IE2-positive) cells are indicated by arrows. Scale bar, 10 μm. (Right) For cellular-fractionation analysis, fractions enriched in cytoplasmic (Cyt) or nuclear (Nuc) proteins were prepared from cells collected at 4 or 8 hpi. Protein levels of pSTAT3 and total STAT3 in each fraction were determined by Western blotting. GAPDH and lamin B1 served as controls for the Cyt and Nuc fractions, respectively. (C to G) Inhibition of tyrosine phosphorylation and nuclear sequestration of unphosphorylated STAT3 by IE1. Fractions enriched in cytosolic or nuclear proteins or total cell extracts were prepared. (C) For transient-transfection analysis, NPCs were nucleofected with pcDNA3-IE1 or empty vector (Ctrl) and harvested at 48 h postnucleofection. Protein levels of IE1, pSTAT3, and total STAT3 were determined by Western blotting. GAPDH and lamin B1 served as controls for the Cyt and Nuc fractions, respectively. (D) To examine dose-dependent effects of IE1 on pSTAT3 levels, NPCs transfected with the indicated amounts of pcDNA-IE1 and empty vector (pcDNA3.0) were harvested 48 h postnucleofection. The protein levels of IE1, STAT3, pSTAT3, and SOX2 were determined by Western blotting. *, P ≤ 0.05. (E to G) For HCMV infection analysis, NPCs were mock infected (M) or infected with TNWT (WT), TNdlIE1 (dlIE1), or TNrvIE1 (rvIE1) viruses at an MOI of 10. The levels of the indicated viral and cellular proteins in whole-cell extracts at 72 hpi (E) and 24 hpi (F) or in the Cyt and Nuc fractions at 24 hpi (G) were determined by Western blotting. Actin, GAPDH, and lamin B1 served as controls for total extracts or Cyt and Nuc fractions, respectively. *, P ≤ 0.05; **, P ≤ 0.01.