Background: Standard HIV-1 stocks have been shown to contain cell-derived microvesicles and secreted HIV proteins. HIV virions can be isolated away from these contaminants using a velocity gradient method of purification. We investigated whether the contaminating factors in standard viral preparations influence the infectivity or cytopathicity of HIV-1.

Methods: Standard viral stocks were prepared by transfecting 293T-cells with HIV-1 proviral constructs. One-half of each standard viral prep was purified using the OptiPrep velocity gradient protocol. First, the supernatant was filtered through a 20% OptiPrep solution and pelleted. The viral pellets were then suspended in PBS and centrifuged in an OptiPrep gradient for 1.5 hr at 38,000 rpm. The infectious titers of the standard and purified viral preps were quantitated by TCID₅₀ assay. Primary Human CD4⁺ T-cells were infected with purified or standard virus at 0.01 moi. Released p24 was quantitated by p24 ELISA, apoptosis was detected by Annexin V staining, and infected cells were detected by intracellular p24 staining.

Results: CD4⁺ T-cell cultures infected with a purified X4 strain of HIV-1, LAI, released greater amounts of soluble p24 into the supernatant than cultures infected with standard LAI stocks. Conversely, a higher percentage of infected cells was detected in cultures infected with the standard virus. In addition, more apoptotic cells were detected at earlier time points following infection with the standard virus. Slightly higher percentages of infected cells were detected in cultures infected with the standard prep of the R5 strain, YU-2, although the levels of released p24 and percentages of apoptotic cells were similar with both standard and purified YU-2 preps.

Conclusions: Highly purified X4 HIV-1 is less cytopathic to CD4⁺ T-cells than non-purified X4 virus. Although more infected cells are detected in cultures infected with standard viral preps, cells die quickly and release less progeny virus than cells infected with purified virus. These data demonstrate that contaminants in standard viral preparations affect the infectivity of HIV-1 and can induce cytopathic effects on the target cell population, which may erroneously be associated with the virus itself.