PMC

Generation of promoter PCR (pPCR) amplicons. (A) Schematic presentation of the pPCR procedure. The CMV promoter and the HIV-1 rev/env cassette were amplified separately. The CMV promoter was then added to the HIV-1 rev/env gene at the 5′ end by overlapping PCR. (B) PCR and pPCR amplification of the CMV promoter and the env gene from a functional env control clone TRO.11 visualized on a 0.7% agarose gel.

Dose dependent infectivity of pPCR pseudovirions. The pPCR products were 1:2 serially diluted (800 ng − 12.5 ng) and cotransfected with pSG3Δenv into 293T cells. The infectivity was determined by measuring luciferase activity. The data is shown as the mean ± standard error (n = 6 independent assays).

Infectivity of pseudovirions derived from pPCR DNA or corresponding functional env clones. The pPCR product (1000 ng) or corresponding env plasmid (1000 ng) was cotransfected with pSG3Δenv into 293T cells in a T25 flask. Supernatants were harvested 48 hrs after transfection and were used to infect TZM-bl cells. The luciferase activity was measured two days after infection. The data is shown as the mean ± standard error (n = 24 independent assays).

Western blot analysis of HIV-1 protein expression. The 293T cells transfected with pPCR products and pSG3Δenv were lysed 48 hrs after transfection. The viral proteins were separated on a 4–12% gradient reducing gel, transferred to nitrocellulose, and were reacted with an HIV-1 infected patient serum and a mouse mAb 13D5 to the Env protein. Viral proteins were visualized with secondary antibodies IRDye800 conjugated goat anti-human and Alexa-Fluor680 goat anti-mouse using a LiCor Odyssey Infrared Imaging system.

Infectivity of pseudovirions derived from pPCR from patient 05ZM373. (A) PCR and pPCR amplification of env genes from patient 05ZM373 plasma. Five HIV-1 rev/env cassettes were obtained using the single genome amplification (SGA) method. The CMV promoter was amplified and then added to all five env genes at the 5′ end by pPCR. (B) The pPCR DNA was cotransfected with pSG3Δenv into 293T cells. Pseudovirions were harvested 48 hrs after transfection and were used to infect TZM-bl cells. The luciferase activity was measured two days after infection. The data is shown as the mean ± standard error (n = 4 independent assays).