Figure 1.
See text for details. SREBP, sterol regulatory element binding protein; SCAP, SREBP cleavage activation protein; Insig1, insulin inducible gene 1.
Figure 2.
PERK expression is highly increased in HCMV-infected cells and critical for HCMV growth.
(A) PERK protein levels in HCMV infection. Whole cell extracts from mock- or HCMV-infected cells at indicated times post infection were analyzed to determine the levels of PERK by Western. (B) Efficiency of shRNA treatment in depleting the levels of PERK protein in HF cells. Lentiviral vectors were used to introduce a control shRNA, shGFP, and two independent shRNAs targeted to PERK mRNA (shPERK, #1 and #2). Whole cell extracts were collected for Western analysis three days after shRNA treatment. (C) HCMV viral growth curves in the presence (shGFP) and absence of PERK (shPERK, #1 and #2) in HF cells. (D) HCMV viral protein expression in the presence (shGFP) and absence of PERK (shPERK #1) in HF cells.
Figure 3.
Depletion of PERK inhibits the induction of lipid synthesis in HCMV infection.
(A) shLuc and shPERK treated HF cells, grown on fibronectin coated coverslips, were infected with HCMV and stained with lipophilic dye BODIPY 493/503 at 48 hpi. (B) and (C) Total lipid synthesis was assayed in HF cells. HF cells were treated with shGFP or shPERK, and then mock- or HCMV-infected for 48 hours, then the cells were labeled with 14C-acetate for 3 hours; total lipids were extracted and counted by scintillation counter.
Figure 4.
Depletion of PERK inhibits lipogenic gene expression in HCMV-infected HF cells.
mRNA levels of lipogenic enzymes were determined by quantitative RT-PCR using total RNA extracted from mock- and HCMV-infected cells that had been treated with shGFP or shPERK at 72 hpi. ACC1, acetyl CoA carboxylase 1; ACL, ATP-citrate lyase; FAS, fatty acid synthetase; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase.
Figure 5.
Depletion of PERK inhibits maturation of SREBP1.
(A) and (B) Levels of SREBP2 in mock- and HCMV-infected cells. Whole cell extracts from mock- and HCMV-infected HF cells at the indicated times were analyzed to determine levels of SREBP2 precursor and mature form by Western. P, precursor SREBP2; Short, short exposure; Long, long exposure. (C) Whole cell extracts were prepared from HF cells treated with shGFP or shSREBP2 for three days and serum-starved for one day prior to mock- or HCMV-infection. Western analysis was performed by anti-SREBP2 antibody to determine the levels of the precursor and mature forms of SREBP2. M, mock infection; V, HCMV infection; P, precursor SREBP2; the arrow indicates the mature form of SREBP2. (D) The cleavage of SREBP1 and SREBP2 in PERK-depleted HF cells. HF cells were treated with shGFP or shPERK for 3 days, then mock- or HCMV-infected. Whole cell extracts were prepared at 48 hpi and analyzed by Western to determine the cleavage of SREBP1 and SREBP2. P, precursor; M, mature form; *. nonspecific protein.
Figure 6.
The cleavage of SREBP1 and 2 in the presence of sterol in HCMV-infected cells.
Serum-starved HF cells were infected with HCMV or mock infected. At 2 hpi, the cells were either refed with serum-free medium alone or medium supplemented with 10 µg/ml cholesterol and 1 µg/ml 25-hydroxycholesterol. At 72 hpi, protein samples were harvested and tested for the effects of sterols on SREBP1 and 2 cleavage.
Figure 7.
The cleavage of SREBP1 and SREBP2 in cells depleted of either SREBP1 or SREBP2, respectively.
(A). HF cells were treated with shGFP or shSREBP1 for 3 days, then mock- or HCMV-infected. Whole cell extracts were prepared at 48 hpi and analyzed by Western to determine the levels of precursor and mature SREBP1 and SREBP2. (B) HF cells were treated with shGFP or shSREBP2 for 3 days, then mock- or HCMV-infected. Whole cell extracts were prepared at 48 hpi and analyzed by Western to determine the levels of precursor and mature SREBP1 and SREBP2. P, precursor; M, mature form.
Figure 8.
Activation of SREBP1 is sufficient to induce lipid synthesis.
(A) Total lipid synthesis was measured in mock- and HCMV infected cells at 48 hpi that had been treated with shGFP, shSREBP1 or shSREBP2. Assays were performed as described in Figure 3B and 3C. (B) HCMV growth at 72 and 96 hpi was measured in HFs treated with shGFP, shSREBP1 or shSREBP2 as described in Materials and Methods.
Figure 9.
Restoration of HCMV induced lipid synthesis by expression of SREBP1a(N) in PERK-depleted HF cells.
(A) Expression of Flag-tagged SREBP1a(N) in HF cells by retroviral transduction. Retro-GFP, retrovirus expressing GFP; Retro-SREBP1a(N), retrovirus expressing 2×FLAG-SREBP1a(N). (B) Total lipid synthesis in HF cells expressing SREBP1a(N). See text for details. * P<0.006; ** P<0.002; *** P<0.0005; P values were determined by Student's t-test.
Figure 10.
Introduction of Insig1-Myc inhibits HCMV growth and lipid synthesis in HCMV-infected cells.
(A) The effect of PERK depletion on Insig1 levels and eIF2α phosphorylation. Human fibroblasts stably transfected with pCMV-Insig1-Myc were treated with shGFP or shPERK for three days, followed by one day serum starvation and then either mock- or HCMV-infected. Whole cell extracts were prepared for Western analysis at 48 hpi. Extracts were probed with anti-PERK, anti-Myc, anti-phospho-eIF2α, anti-total eIF2α and anti-actin. Two exposures of the Insig1-myc analysis are provided. S, short exposure; L, long exposure. (B) Introduction of exogenous Insig1-Myc inhibits HCMV growth. HF cells were treated with shGFP or shPERK for two days subsequent to transient transfection with pCMV-Insig1-Myc or the vector control (pCDNA3), then the cells were serum-starved for two hours and infected with HCMV. Viruses were harvested for titration at 72 hpi. See text for details. (C) Lipid synthesis in Insig1-Myc expressing HF cells. HF cells were treated as described in (B) prior to mock or HCMV infection. Total lipid synthesis was measured at 48 hpi.