Background: Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4⁺ T-cell depletion in HIV-1 infected individuals remains unknown. The Vpr accessory protein of HIV-1 has been reported to cause cell death, as well as enhance transcriptional activity of the proviral long terminal repeat (LTR), target the pre-integration complex to the nucleus, and arrest cells in the G2/M phase. Although several Vpr functions have been associated with cell killing, it is not clear which, if any, of the known Vpr activities causes death. We investigated the molecular mechanism by which Vpr contributes to direct viral cytopathicity in CD4⁺ T-cells in the context of HIV-1 infection as well as in its virion-associated form.

Methods: Vpr cytopathicity resulting from either HIV-1 infection with NL4-3 or virion-associated Vpr was measured by flow cytometry and compared amongst wild-type Vpr and cell cycle arrest mutants of Vpr. Vpr-induced cell cycle blockade was detected by propidium iodide staining for DNA content. T-cell killing by virion-delivered Vpr was assessed for apoptosis by Annexin V binding.

Results: C-terminal truncation of Vpr was found to completely abrogate virion-associated Vpr cell death in Jurkat T-cells. Furthermore, virions containing these Vpr mutants were unable to induce cell cycle arrest in G2/M, as measured by hyperdiploid DNA content, suggesting that virion Vpr cytotoxicity is associated with the arrest-inducing activity of Vpr. In addition, Vpr-mediated killing does not appear apoptotic, as cells dying from virion-associated Vpr did not flux phosphatidylserine. The cytopathic role of the C-terminus of Vpr was further supported by chronic infection of T-cells infected with NL4-3 containing Vpr C-terminal deletions without cytopathic manifestations characteristic of infection with wild-type HIV-1.

Conclusions: Vpr was found to exert a cytopathic effect on T-cells when delivered into cells by HIV-1 virions and when expressed during HIV-1 infection in vitro. We have mapped a cytopathic determinant of Vpr to the C-terminus, which is also associated with mediating the G2/M cell cycle arrest. Further understanding of the mechanism of Vpr toxicity may provide valuable insight for strategies to prevent CD4⁺ T-cell loss by viral cytopathicity.