High throughput functional analysis of HIV-1 env genes without cloning

Abstract

Functional human immunodeficiency virus type 1 (HIV-1) env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3 Delta env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple env genes from HIV-1 infected individuals.

Figure 1

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.

Figure 2

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).

Figure 3

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).

Figure 4

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.

Figure 5

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).