FIG. 4.

Complementation between different IN mutants. (A) Enzymatically defective trans-IN protein supports reverse transcription. Four micrograms of the S-IN, H12A, H16A, F185A, and Δ22 IN mutant proviral clones was transfected alone and separately cotransfected into 293T cells with 2 μg of the Vpr-IN^D116A or Vpr-IN expression vector. Forty-eight hours later, supernatant virions were prepared and used to infect HeLa-CD4 cells exactly as described in the legend to Fig. 1. The infected cells were washed 18 h later, and total DNA was extracted and treated with DpnI endonuclease. The late R-gag DNA product of reverse transcription was PCR amplified and analyzed as described above. The data are from a representative experiment that was repeated two times. (B) Complementation of proviral DNA integration. The D116A IN mutant was inserted into the SG3 hygromycin-resistant clone, generating Hy-SG3^D116A. The Hy-SG3^D116A mutant virus produces wild-type levels of viral DNA yet is integration defective. Four micrograms of Hy-SG3^D116A was transfected with 2 μg of the control vector (pLR2P) and individually cotransfected with 2 μg of the Vpr-IN, Vpr-IN^H12A, Vpr-IN^H16A, Vpr-IN^F185A, and Vpr-IN^Δ22 IN mutant expression vectors, respectively. Since the env region of Hy-SG3^D116A contains the hygromycin resistance marker, the virions were pseudotyped by including the pCMV-VSV-G env vector in the transfection reactions. Forty-eight hours after transfection, the culture supernatants were filtered through 0.45-μm-pore-size filters and analyzed for HIV-1 p24 antigen concentration by ELISA. Twenty-five nanograms (p24 antigen) of each pseudotyped virus stock was used to infect cultures of HeLa cells. The infected cells were maintained in hygromycin selection medium for 12 days and then stained to identify resistant colonies. These results were highly reproducible in three independent experiments. The data shown are from a single representative experiment.