Figure 2.

Analysis of viral functions from the viral molecular clones. (A) Ability of the molecular clones to encode functional Tat protein. The 8 molecular clones were individually cotransfected into HEK293 cells along with a reporter construct expressing EGFP under the control of HIV-1 subtype C LTR. Subtype C reference molecular clone, Indie-C1, serves as a positive control and the parental cloning vector as a negative control. GFP+ cells were documented 48 h following the transfection. (B) Virus production from the molecular clones. Spent media from the above cultures were monitored for p24 using a commercial antigen-capture ELISA. Note that only 4 out of 8 clones express p24 indicating virus production. (C) The ability of virus produced by the molecular clones to propagate through the PBMC in culture. Mitogen-activated PBMC from two different healthy donors (striped and filled bars) were infected with the virus produced from the 4 molecular clones that secreted p24 in 'B' above. Viral stocks equivalent of 10 ng of p24 were used for the infection. Following 4 h of incubation, the residual virus was removed by washing, the cells were cultured and production of p24 into the culture medium was monitored using an antigen-capture assay. Data for day-7 have been presented. Note that only two molecular clones, D17 and D24, were found to be infectious. (D) Syncytium formation by the infectious molecular clones in MT2 cells. Both D17 and D24 induced syncytium formation (arrows) in MT2 cells within a couple of days, same as NL4-3. (E) Analysis of the viral antigens by western blotting. HEK293T cells were transiently transfected with D17, D24 or Indie-C1 molecular clones. Cell-free culture supernatants were collected 72 h post-transfection and subjected to high-speed centrifugation to pellet the virus. Viral pellets were directly suspended in lysis buffer and the viral antigens were separated on a 6 to 15% gradient SDS-PAGE gel. Proteins were transferred to PVDF membrane and analyzed by immunoblotting using pooled sera derived from individuals infected with HIV-1 subtype C. Predicted viral antigens are indicated.