HIV Nef, paxillin, and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases.

The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNFα converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNFα and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNFα. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment.

HIV Nef- and Notch1-dependent Endocytosis of ADAM17 Induces Vesicular TNF Secretion in Chronic HIV Infection.

Tumor necrosis factor (TNF) is a key cytokine in HIV replication and pathogenesis. For reasons that are not entirely clear, the cytokine remains upregulated despite anti-retroviral therapy (ART). Here we demonstrate that HIV Nef induces an alternative TNF secretion mechanism that remains active in chronic infection. Ingestion of Nef-containing plasma extracellular vesicles (pEV) from ART patients by primary immune cells, but also Nef expression, induced intracellular proTNF cleavage and secretion of vesicular TNF endosomes. Key event was the Nef-mediated routing of the TNF-converting enzyme ADAM17 into Rab4+ early endosomes and the Rab27+ secretory pathway. Analysis of lymph-node tissue by multi-epitope-ligand-cartography (MELC) confirmed a vesicular TNF secretion phenotype that co-localized with persistent Nef expression, and implicated Notch1 as an essential co-factor. Surprisingly Notch1 had no transcriptional effect but was required for the endosomal trafficking of ADAM17. We conclude that Nef expression and Nef-containing pEV mobilize TNF from endosomal compartments in acute and chronic infection.

HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection.

Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvß3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c(+) CD14(+) cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity.