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.

Efficient SIVcpz replication in human lymphoid tissue requires viral matrix protein adaptation

“…This lack of antitetherin activity must explain, at least in part, why SIVcpz constructs containing the human Gag30 adaptation still replicate less efficiently than HIV-1 strains in HLT cultures. In fact, previous studies have shown that a defective vpu gene decreased replication of HIV-1 in ex vivo-infected human tonsillary tissue by about 3-fold, although it was not determined whether this reduction was due to lack of antitetherin activity or other Vpu functions, such as degradation of CD4 (40). Nonetheless, the results suggest that effective tetherin antagonism may be required for efficient replication of SIVcpz in HLTs.…”

“…Human tonsils removed during routine tonsillectomies were dissected into 2-to 3-mm 3 blocks, cultured, and infected with viral stocks normalized based on p24 content as described previously (20,21,40). Productive HIV-1 and SIVcpz infection were evaluated by measuring the amount of p24 core antigen released into the medium using the InnoTest HIV Antigen mAb ELISA (Innotest HIV Antigen mAb; Innogenetics).…”

Human Immunodeficiency Virus Type 1 Inhibits DNA Damage-Triggered Apoptosis by a Nef-Independent Mechanism

“…Bicistronic cytomegalovirus-based pCG expression vector coexpressing the green fluorescent protein (GFP) and the NL4-3, NA7, or SIVmac239 nef allele and the NL4-3 proviral constructs carrying either functional (nef ϩ ) or defective (nef-defective) nef reading frames followed by an internal ribosome entry site (IRES) and the GFP gene have been previously described (17,42). To generate proviral IRES-GFP vectors containing defective vpu or vpr genes, the intergenic region of NL4-3 mutants containing deletions in these genes (41) was cloned into the original proviral reporter constructs using the unique PflMI and StuI restriction sites.…”

Proline 35 of Human Immunodeficiency Virus Type 1 (HIV-1) Vpr Regulates the Integrity of the N-Terminal Helix and the Incorporation of Vpr into Virus Particles and Supports the Replication of R5-Tropic HIV-1 in Human Lymphoid Tissue Ex Vivo

“…1B to D). HLT was chosen, as it represents a relevant physiological system to study accessory proteins such as Vpr that exercise their functions particularly in primary cells (10,19,20).…”

“…1B. Since the replication of HIV-1 in HLT generally exhibits donor-dependent differences (9), an average of replication profiles in tissues derived from several donors revealed that productive infection of both R5-tropic and X4-tropic HIV-1 variants resulted in the typical Vpr-dependent replication kinetics (19,20): the inactivation of the vpr gene reduced the replication of X4-tropic HIV-1 NL4-3 to 50% Ϯ 8.8% (mean Ϯ standard error of the mean [SEM]) (number of HLT donors [n] ϭ 9; P Ͻ 0.0001), and the replication of R5-tropic viruses decreased to 37% Ϯ 8.2% (n ϭ 10; P Ͻ 0.0001) (Fig. 1B to D).…”