Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo

Abstract

The relevance of the accessory vpr, vpu, and nef genes for human immunodeficiency virus type 1 (HIV-1) replication in human lymphoid tissue (HLT), the major site of viral replication in vivo, is largely unknown. Here, we show that an individual deletion of nef, vpr, or vpu significantly decreases HIV-1 replication and prevents CD4+ T-cell depletion in ex vivo HLT. However, only combined defects in all three accessory genes entirely disrupt the replicative capacity of HIV-1. Our results demonstrate that nef, vpr, and vpu are all essential for efficient viral spread in HLT, suggesting an important role in AIDS pathogenesis.

FIG. 1.

Replication of HIV-1 variants in human lymphoid tissue ex vivo. For each of the indicated HIV-1 variants, 27 tissue blocks were inoculated with 100 ng of p24 and medium was collected every 3 days. (A) Representative replication kinetics of wild-type NL4-3 and deletion mutants. (B) Average production of virus. Matched tissues from 13 donors were inoculated with the wild-type virus or with accessory gene-deleted mutants as indicated, and for each condition cumulative production of p24 by 27 tissue blocks over 15 days was measured. Presented are means ± standard errors of the means of these values expressed as percentages of those measured in cultures infected with the wild-type virus.

FIG. 2.

CD4⁺-T-cell depletion in human lymphoid tissue infected ex vivo with HIV-1 variants. (A) Percentages of infected cells; (B) loss of CD4⁺ T cells in human lymphoid tissue infected ex vivo with HIV-1. Productively infected CD4⁺ T cells were defined as CD3⁺ CD8⁻ p24⁺, as described in the text. To evaluate CD4⁺-T-cell depletion, cells were mechanically isolated from control and infected matched tissues (27 pooled blocks for each variant) on day 12 postinfection, stained for CD3, CD4, CD8, and p24, and analyzed with flow cytometry. Depletion is expressed as 100% minus the percentage of CD4⁺ T cells that remained in the tissue after 12 days of infection, evaluated as described earlier (23, 29). Presented are average depletion values ± standard errors of the means for tissues from 4 to 12 donors. (C) Correlation between depletion and virus infection of CD4⁺ T cells in ex vivo-infected human lymphoid cultures. Accessory gene deletions are indicated in the following order: Vpr, Vpu, Nef.

FIG. 2.

CD4⁺-T-cell depletion in human lymphoid tissue infected ex vivo with HIV-1 variants. (A) Percentages of infected cells; (B) loss of CD4⁺ T cells in human lymphoid tissue infected ex vivo with HIV-1. Productively infected CD4⁺ T cells were defined as CD3⁺ CD8⁻ p24⁺, as described in the text. To evaluate CD4⁺-T-cell depletion, cells were mechanically isolated from control and infected matched tissues (27 pooled blocks for each variant) on day 12 postinfection, stained for CD3, CD4, CD8, and p24, and analyzed with flow cytometry. Depletion is expressed as 100% minus the percentage of CD4⁺ T cells that remained in the tissue after 12 days of infection, evaluated as described earlier (23, 29). Presented are average depletion values ± standard errors of the means for tissues from 4 to 12 donors. (C) Correlation between depletion and virus infection of CD4⁺ T cells in ex vivo-infected human lymphoid cultures. Accessory gene deletions are indicated in the following order: Vpr, Vpu, Nef.

FIG. 3.

Effect of exogenous IL-2 on replication of HIV-1 variants in human lymphoid tissue ex vivo. Matched infected tissues were inoculated with HIV-1 variants and cultured without or with IL-2 (50 U/ml). For each condition and each donor tissue, 27 tissue blocks were inoculated. (A) Representative time course of p24 production in unstimulated and IL-2-stimulated tissues inoculated with wild-type virus or HIV-1 variants with accessory gene deletions. (B) Cumulative viral production over 12 days of infection by wild-type virus or HIV-1 variants with accessory gene deletions. Presented are means ± standard errors of the means of the fold increases of p24 production (relative to the replication of the wild-type virus in the absence of IL-2) in tissues from seven to nine donors inoculated ex vivo with the indicated HIV-1 variants. The numbers above each bar give the fold increase of virus production in the presence of IL-2 relative to production of the respective HIV-1 mutants in the absence of IL-2.