Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication

IFNs stimulate HCMV major IE expression. HFF cells were either mock infected (M) or infected with HCMV (C) for different times as indicated above the lanes (hpi). The cells were treated with IFN-α or IFN-γ for 2 h before infection (α+C), 4 h after infection (C+α or C+γ), or left untreated (C). Northern blot assays were performed to measure HCMV IE1 expression. Fold activation of IE1 is indicated at the bottom of the upper panel. The cellular 7SK gene was used as an internal control (lower panel).

Deletion analysis of two VRS1 elements in the HCMV major IE promoter. (A) The two VRS1 elements within a 131-bp fragment of wild-type AD169_BAC (W) were replaced by a kan/lacZ cassette to generate a VRS1 deletion mutant, AD169VRS-Δ (Δ). The names, locations, and orientations of PCR primers for verifying the correct clones are indicated by arrows. (B) Using PCR, the deletion clone (Δ) was compared with the wild-type clone (W). The results showed that the VRS fragment was not present in the mutant clone (lane 2) but was present in the wild type (lane 1). The kan/lacZ cassette was inserted in the correct genomic position, as evidenced by the presence of the right (lane 8) and left (lane 6) junctions in the deletion clone but not in the wild-type clone (lanes 7 and 5). UL4 was used as a control (lanes 3 and 4). (C) The wild-type (W) and deletion (Δ) BAC DNAs were digested by EcoRI and SalI, and the deletion of VRS elements was confirmed further by Southern blotting using the VRS fragment as a probe. The VRS fragment is only present in the wild type (W) and not in the deletion (Δ) clones. (D) A multistep growth curve analysis was performed to compare the wild type (W; black circle) and VRS deletion mutant (Δ; black triangle) at an MOI of 0.1. The peak titer of mutant virus was 100- to 1,000-fold lower than that of the wild-type virus.

Analyses of point mutations in the VRS elements. (A) The VRS1 wild-type and point mutation reporter plasmids, pElu-VRS-W and pElu-VRS-P, were constructed. A 131-bp MIEP/E fragment containing either wild-type or mutated VRS1 (as shown in boxes) was inserted into the isg54K promoter (Pisg54K) in pElu-NRS (47). In pElu-VRS-P, one VRS1 was converted into an XhoI site and the other was converted into an XbaI site as indicated, resulting in three point mutations in the GAS consensus sequences, as indicated by underlines. The GAS consensus sequence is also indicated. Two previously constructed VRS wild-type and mutant clones, pElu-GAS and pElu-GAS^m, also are illustrated. The intervening sequence between the two VRS1 elements in MIEP/E was removed in these two plasmids. (B) pElu-GAS and pElu-GAS^m were transfected into HFF cells. Ten days posttransfection, the cells were either mock infected (mock) or infected with UV-inactivated HCMV (UV-HCMV) at an MOI of 1 for 8 h, and luciferase assays were performed. The relative luciferase activities from the average of triplicate points are shown. (C) The DNA-binding activities of the wild-type and mutated VRS1-containing fragments were analyzed using EMSA. The 131-bp wild-type (W) and point mutation (P) fragments were amplified from pElu-VRS-W and pElu-VRS-P by PCR. These fragments were labeled as probes (PB) and incubated with or without HCMV-infected cell nuclear extracts (NE). A specific interaction was observed when the wild-type (lane 2), but not the mutant (lane 4), probe was used in the reaction.

Generation of the VRS1 point mutation and rescue viruses. (A, diagram 1) pGEM-lox-zeo was constructed by inserting a loxP/zeo^r cassette into pGEM-T. The 131-bp MIEP/E fragment containing the VRS1 mutations then was cloned between the SphI and BamHI sites of pGEM-lox-zeo to create pGEM-lox-zeo-VRS-P. The two VRS1 sites were converted into XhoI and XbaI, as shown in Fig. 4A. (Diagram 2) The VRS-P/zeo fragment was amplified by PCR. (Diagram 3) This PCR fragment was electroporated into DY380 carrying the AD169VRS-Δ BAC clone (VRS-Δ). (Diagram 4) The kan/lacZ fragment was replaced by the VRS-P/zeo to produce an HCMV clone containing mutated VRS1 elements with zeo^r (VRS-P-zeo). (Diagram 5) The VRS-P-zeo DNA was cotransfected with a CRE-expressing plasmid into HFF cells to generate the VRS1 point mutant (VRS-P), in which zeo^r was removed from the genome and one loxP site was left in the recombinant virus genome. (Diagram 6) A rescue virus (VRS-R) was generated using the same method, with the wild-type VRS1 replacing the mutated VRS1 in the MIEP/E. (B, blot 1) The VRS1 point mutation and rescue clones were confirmed by Southern blotting. The AD169VRS_BAC (W), AD169VRS-Δ (Δ), AD169VRS-R (R), and AD169VRS-P (P) BAC DNA samples were digested with EcoRI and SalI, and Southern blot analysis was performed using a 131-bp probe containing the VRS sites. (Blot 2) The AD169VRS-P clone was confirmed by PCR. The 1.2-kb PCR product was either uncut (UC), cut by XhoI into 700 and 500 bp, or cut by XbaI into 620 and 580 bp. (Blot 3) The BAC DNAs were transfected into HFF cells to produce viruses. The correct recombinant viruses were verified by PCR. Glycoprotein B (gB), zeocin^r (zeo), and VRS-containing fragments were amplified from AD169VRS-Δ (ΔV), AD169VRS-P (PV), and AD169VRS-R (RV) viruses and AD169VRS-R BAC DNA (RB). (Blot 4) The 1.2-kb VRS fragment amplified by PCR from AD169VRS-R (RV) and AD169VRS-P (PV) viruses were either uncut (UC) or cut by XhoI or XbaI as indicated. (C) Growth curve analyses at an MOI of 1 (left panel) and an MOI of 0.1 (right panel) of AD169VRS-P (P) and AD169VRS-R (R) viruses. Each point was obtained from an average of triplicates.

The expression of IE1 and IE2 is reduced in AD169VRS-P infection. (A) HFF cells were mock infected (M) or infected with AD169VRS-P (P) or AD169VRS-R (R) virus at an MOI of 1 for 2 h. The infected cell lysates (30 and 10 μl) were analyzed by Western blotting using a monoclonal antibody against HCMV tegument protein pp65. Actin was used as an internal control. (B) Expression of HCMV IE1 and IE2 in the VRS1 mutant and rescue viruses was measured by Northern blotting and Western blotting. HFF cells were either mock infected (M) or infected with AD169VRS-P (P) or AD169VRS-R (R) virus at an MOI of 1 or 0.1. Total RNA and protein were prepared at the indicated hpi. The IE1 RNA level was measured by Northern blotting (row 1). A cellular 7 SK RNA was used as an internal control (row 2). The IE1 and IE2 protein levels were detected by Western blotting using anti-IE1 antibody, 1B12 (row 3), or anti-IE1/2 antibody mAB810 (row 4). Actin was used as an internal control (row 5).

IFNs failed to stimulate IE1 expression in the VRS1 mutant virus. HFF cells were either mock infected (M) or infected with the VRS1 mutant, AD169VRS-P (P), or the rescue virus, AD169VRS-R (R). The infected cells were then either untreated (lanes 2 and 6) or treated with IFN-α (lanes 3 and 7) or IFN-γ (lanes 4 and 8) at 2 hpi. HCMV IE1 protein was measured at 8 hpi by Western blotting using an anti-IE1 antibody, 1B12. Actin was used as an internal control.