The Vif and Vpr accessory proteins independently cause HIV-1-induced T cell cytopathicity and cell cycle arrest

Abstract

HIV type I (HIV-1) can cause G(2) cell cycle arrest and death of CD4(+) T lymphocytes in vitro and inexorable depletion of these cells in vivo. However, the molecular mechanism of viral cytopathicity has not been satisfactorily elucidated. Previously, we showed that HIV-1 kills T cells by a necrotic form of cell death that requires high level expression of an integrated provirus but not the env or nef genes. To determine which viral protein(s) are required for cell death, we systematically mutated, alone and in combination, the ORFs of the NL4-3 strain of HIV-1. We found that the elimination of the viral functions encoded by gag-pol and vpu, tat, and rev did not mitigate cytopathicity. However, elimination of the vif and vpr accessory genes together, but not individually, renders the virus incapable of causing cell death and G(2) cell cycle blockade. We thus identify vif and vpr as necessary for T cell cytopathic effects induced by HIV-1. These findings may provide an important insight into the molecular mechanism of viral pathogenesis in AIDS.

Fig. 1.

Locations of mutations in HIV-1_NL4-3 and corresponding loss of protein expression due to mutations. (A) NL4-3_HSA strains contained various individual or multiple mutations in the ORFs that alter the indicated amino acids shown in the standard single letter code. The mutations either prevent (ATG mutations) or prematurely terminate (stop codons) protein translation. Mouse heat stable antigen (HSA/CD24) replaced the nef gene coding sequence (21). To verify the expected loss of protein expression, lysates from 293T cells transfected with mutant NL4-3_HSA strains were analyzed by Western blotting. Mock transfected 293T cells served as a negative control, and the expression of β-actin was used as a loading control. (B and C) The Western blot was probed with HIV-1-infected patient sera to examine Gag and Pol expression (B) or antibodies specifically directed against Vif or Vpr (C).

Fig. 2.

The NL4-3_HSA strain of HIV-1 causes cytopathicity in Jurkat cells. (A) Jurkat cells were mock infected (a) or infected with NL-EGFP (b) or NL4-3_HSA Env⁻ (c) at a MOI of 5. The viability of infected cells was analyzed by flow cytometry by using forward light scatter (FSC; x axis) and staining with PI (y axis). Each panel represents 10,000 total events on day 4 after the start of infection. The polygonal gate indicates the percent of viable, PI-negative cells. (B) Jurkat cells were infected with the NL4-3_HSA Env⁻ strain at various MOI, and viability was monitored by flow cytometry over time. Mock-infected Jurkat cells were at least 95% viable on day 4 based on PI staining. Viability of infected (HSA-positive) cells over time was plotted on the graph.

Fig. 3.

Cytopathicity of mutant NL4-3_HSA strains in the absence of Gag and Pol. Mutant virus stocks were generated in the presence of ΔR8.2ΔVpr helper plasmid in 293T producer cells and used to infect Jurkat cells at a MOI of 5. Cytopathicity of the total population (A) and fractions of infected cells (B) were monitored by flow cytometry. Cell viability was analyzed as a function of time after the initiation of infection by propidium iodide exclusion staining as shown in Fig. 2A. The fraction of infected cells was monitored by HSA staining. Viability associated with virus expression is shown on the y axis, and time in days is shown for each row of samples.

Fig. 4.

Cytopathicity of mutant HIV-1_NL4-3 strains. Jurkat T cells were mock-infected or infected with various strains of NL4-3_HSA at a MOI of 3 (A) and 5 (B). The viability of the total population was measured as described in Fig. 2A. The unpaired Student’s t test for mutant vs. the parental NL4-3_HSA Env⁻ strain on day 7 indicated P < 0.05 for the viability of cells infected with Vif⁻Vpr⁻ and Vif⁻Vpr⁻Vpu⁻ strains.

Fig. 5.

Time course measurements of the viability of CD4⁺ T cell lines infected with NL4-3_HSAEnv⁺ strains carrying mutations in accessory genes. (A) Peripheral CD4⁺ T cells after 3 days of stimulation and infection with VSV-G pseudotyped viruses at a MOI of 30. The extent of infection and viability were monitored by flow cytometry by staining for the HSA marker and with PI staining, respectively, as described in Fig. 2. The percentage of PI-negative infected cells is shown in the graph. (B and C) MOLT-4 (B) and SupT1 (C) T cell lines were infected with mutant strains of NL4-3_HSA, and the viability of infected cells was monitored by flow cytometry.

Fig. 6.

Cell cycle profiles of Jurkat cells infected with various mutant NL4-3_HSA strains. Infected Jurkat cells were examined for DNA content (x axis) vs. cell number (y axis) 2 days after infection to assess their position in the cell cycle.