FIG. 4.

Repression of viral spread in U937 cells infected with pAC3-GFP vector carrying the 142-3pT sequence. (A) Replication kinetics of pAC3-GFP, pAC3-GFP-142-3pT, and pAC3-GFP-142-3pT4X vectors in U937 cells. Cells were infected with each vector at an MOI of 2 on day 0 and passaged at the indicated time points. The percentage of GFP-positive cells was determined by flow cytometry, with proper gating to exclude GFP-negative cells. The replication kinetics of each vector was obtained by plotting the percentage of GFP-positive cells versus time. (B) Stability of pAC3-GFP, pAC3-GFP-142-3pT, and pAC3-GFP-142-3pT4X proviral DNA in U937 cells. DNA molecular marker (1 Kb Plus marker) was included in the first lane of the gel. The numbers above each lane indicate the days postinfection. The arrowheads indicate the size of the PCR product expected for the undeleted IRES-GFP region (1445 bp for pAC3-GFP, 1492 bp for pAC3-GFP-142-3pT, and 1575 bp for pAC3-GFP-142-3pT4X vectors). NC, naive cells, negative control. The 17-day lane for pAC3-GFP-1423pT appears under-amplified for unknown reasons, but shows a full-length band. (C) Vector copy number of proviral DNA in U937 cells infected with pAC3-GFP, pAC3-GFP-142-3pT, and pAC3-GFP-142-3pT4X vector. (D) Normalized expression level of cellular viral RNA in U937 cells infected with pAC3-GFP, pAC3-GFP-142-3pT, and pAC3-GFP-142-3pT4X vector. Expression levels are presented relative to the parental vector, which is set to 1. (E) Viral titers produced by infected U937 cells. At the end of infection, cells were seeded at 1×10⁶ in 5 ml of culture medium. At 48 hr postseeding, viral supernatant from each sample was collected for titration in PC-3 cells by qPCR. (F) Viral proteins produced by U937 cells infected with pAC3-GFP, pAC3-GFP-142-3pT, and pAC3-GFP-142-3pT4X vectors. At the end of infection, cells were seeded at 1×10⁶ in 5 ml of culture medium. At 48 hr postseeding, cells were harvested and lysed for immunoblotting. Twenty micrograms of cell lysate was loaded onto each lane as indicated by the loading control, GAPDH. Lanes 1–4: noninfected cells and cells infected with pAC3-GFP, pAC3-GFP-142-3pT, or pAC3-GFP-142-3pT4X vector, respectively. Asterisk (*) indicates deletion of the IRES-GFP cassette in vectors carrying the 142-3pT sequences.