Titration of lentiviral vectors onto restricting cells. Two-fold serial dilutions of HIV-eGFP were titrated onto 10⁵ OMK cells (A), LLC-MK2 (▴) and FRhK4 (●) cells from rhesus macaque (B). (C) Two-fold serial dilutions of SIV-LacZ were titrated onto OMK (●), FRhK4 (▴), and LLC-MK2 (■) cells. Two-fold serial dilutions of HIV-eGFP (●) or SIV-eGFP (▴) were titrated onto African green monkey CV1 cells (D) or rabbit SIRC cells (E). Results are representative of two independent experiments. Lines are guides for slopes of 1 and 2.

Abrogation of restriction of SIV-eGFP by SIV-LacZ and HIV-puro in African green monkey CV1 cells. TE671 (□), LLC-MK2 (●), OMK (×), or CV1 cells (▴) were exposed to 5 × 10⁶ TE671 i.u. of SIV-LacZ or 5 × 10⁶ TE671 i.u HIV-puro (♦) (CV1 only) for 4 h, washed, and exposed to 5 × 10⁴ i.u. of SIV-eGFP. Percentage infection was measured by analysis of eGFP expression 48 h later by FACS. Fold abrogation is calculated by dividing the increased percentage infection after abrogation by unabrogated control HIV-eGFP infection. Approximately 1% of unabrogated monkey controls and 10% of human TE671 controls typically were infected. Data are representative of two independent experiments.

Abrogation by reverse transcriptase mutant and time course of abrogation decay. OMK (●) or FRhK4 (▴) cells (10⁵) were exposed to serial dilutions of reverse transcriptase-defective HIV-eGFP D185E (A) or wild-type HIV-puro (B) for 4 h. Cells were washed and exposed to 10⁵ TE671 i.u. of HIV-eGFP. Percentage infection was measured by analysis of eGFP expression 48 h later by FACS. Fold abrogation is calculated by dividing the increased percentage infection after abrogation by unabrogated control HIV-eGFP infection. About 1% of unabrogated controls typically were infected. p24 concentrations of wild-type and mutant supernatants were similar with around 500 ng/ml corresponding to a titer of 10⁷ TE671 i.u. wild-type HIV-puro. OMK cells (10⁵) were exposed to 10⁷ TE671 i.u. of HIV-puro for 4 h, washed, and exposed to 10⁵ TE671 i.u. of HIV-eGFP at 4-h intervals (C). Percentage infection and fold abrogation were determined as above. Data are representative of two independent experiments.

(A) Abrogation of HIV-1 restriction in owl monkey cells. FACS plots of side scatter versus GFP fluorescence are shown. Owl monkey cells are uninfected (i), infected with 10⁵ TE671 i.u. of HIV-eGFP (ii), or exposed to 10⁷ TE671 i.u. of HIV-puro for 4 h, washed, and then infected with 10⁵ TE671 i.u. of HIV-eGFP. Region of GFP-positive cells (R1) and percentages of positive cells are indicated. (B) Abrogation of HIV restriction in monkey and rabbit cells. Cells (10⁵) were exposed to 5 × 10⁶ TE671 i.u. of either HIV-puro (■) or SIV-LacZ (□), incubated for 4 h, washed, and then exposed to 10⁴ (TE671), 10⁵ (OMK, FRhK4), 5 × 10⁴ (LLC-MK2, CV1), and 5 × 10⁶ (SIRC) TE671 i.u. of HIV-eGFP. Percentage infection was measured by analysis of eGFP expression 48 h later by FACS. Fold abrogation is calculated by dividing the increased percentage infection after abrogation by unabrogated control HIV-eGFP infection. Typically 1–5% of unabrogated controls were infected. Errors are standard error of the mean of three independent experiments.

Quantitative PCR of restricted and unrestricted infection. FRHK4, OMK, CV1, or TE671 cells (10⁵) were infected preexposed to 10⁷ TE671 i.u. of HIV-puro for 4 h (■) or left unexposed (□), washed, and then infected with 5 × 10⁴ TE671 i.u. of HIV-eGFP. For SIV on CV1 cells 10⁶ TE671 i.u. of HIV-puro (■) or 10⁶ TE671 i.u. of SIV-LacZ (striped bar) was used to abrogate and then 5 × 10⁴ i.u. of SIV-eGFP was used to infect cells. Four hours after the second round of infection, total DNA was extracted and 100 ng was subjected to quantitative PCR (see Materials and Methods). Viral template copy number per 100 ng of total DNA was calculated by reference to a standard curve. Approximately 1–10% of unabrogated monkey control cells were infected, and 35% of TE671 controls. Data are representative of two independent experiments with duplicate PCR. Errors are standard error of the mean. Parallel samples were analyzed by FACS, and levels of restriction were similar to those in Figs. 3 and 4.