Background: Efforts to understand the mechanism of CD4⁺ T-cell depletion has led to the extensive investigation of the molecular mechanism of cell death during infection. However, the mechanism of viral cytopathicity, including viral components involved, still remains undefined due to many conflicting reports. In this study, a variety of HIV-1 mutants was systematically examined for their ability to cause cytopathicity in CD4⁺ T-cells in order to identify a viral component involved in the direct T-cell killing.

Methods: Various combinations of knockout mutations were created by site-directed mutagenesis in the HIV-1 genome of NL4-3. The viral stocks of various mutants were prepared in the presence of a helper plasmid and used to infect a Jurkat T-cell line. The cytopathicity of mutant viral strains lacking the expression of one or more HIV-1 genes was investigated in highly infected Jurkat cells, using flow cytometry.

Results: An env and nef deficient strain of HIV-1 kills Jurkats in 48 hrs after infection. The loss of gag and pol expression in addition to env and nef decreases the rate of cytopathicity by 1– 2 days; however, it does not attenuate T-cell death per se. A knockout mutation in one accessory gene of either vpr, vpu, or vif, in addition to those in gag and pol, further decreases the rate of cytopathicity by 3 days. The loss of an additional accessory protein, e.g., any combinations of Vpr, Vpu, and Vif, reduces the kill rate by another 2 days, thus manifesting the gradual loss of infected Jurkats over 7 days. In contrast, Jurkat cells infected with the mutant HIV-1 strain lacking the Gag, Pol, Vpr, Vpu, and Vif shows a maximum of 10%–40% lethality, and the majority of highly infected Jurkats are still viable a month after infection.

Conclusions: The mutant strain deficient in gag, pol, vpr, vpu, and vif expression showed attenuated viral cytopathicity, indicating that Tat and Rev are dispensable for direct T-cell killing by HIV-1. Moreover, the number of genes eliminated from HIV-1 was inversely correlated with viral cytopathicity, raising the possibility that the number of viral genes expressed in cells, rather than a particular protein, may determine viral cytopathicity.