HIV-1 Nef modulates the protein content of T cells extracellular vesicles with the depletion of antiviral proteins IFITMs

Author(s)

Mara Elisama da Silva Januário

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-08-31

Examining board members

Luis Lamberti Pinto da Silva; Luciana Jesus da Costa; Larissa Dias da Cunha; Ana Cláudia Trócoli Torrecilhas

• Advisor: Luis Lamberti Pinto da Silva

Abstract

Nef is a multifunctional HIV protein that contributes to increased viremia and disease progression. Nef exerts these functions by modifying the expression levels of various antiviral proteins in different compartments of the host cell, such as the plasma membrane, Golgi apparatus, and extracellular vesicles (EVs). In this work, a quantitative proteomic analysis of EVs released by lymphocytes was performed to characterize the global changes in their protein content promoted by Nef. The hypothesis tested was that Nef would have unidentified targets with altered levels in the EVs. Among the various proteins with modified levels, the results showed that Nef is able to reduce the levels of the antiviral protein IFITM2 in EVs. The IFITMs (Interferon-Induced Transmembrane) family of proteins act against various viruses, including influenza, dengue, zika, and HIV. Interferon stimulation regulates the expression of IFITMs, and at least three different IFITMs exhibit antiviral activity (IFITM1, IFITM2, and IFITM3). In the context of HIV infection, it was previously shown that IFITMs prevent the fusion of the virus with the plasma membrane, favoring the targeting of the virus to a lysosomal degradation pathway. To date, there is no evidence of viral proteins that antagonize IFITMs. The discovery that Nef reduces IFITM2 levels in EVs motivated the investigation of the possible role of Nef in antagonizing IFITMs in lymphocytes. Western blot assays demonstrated that Nef reduces IFITM1, IFITM2, and IFITM3 protein levels in T cell EVs but does not affect total intracellular levels. Biotinylation and lipid raft enrichment assays showed that Nef decreases the association of IFITM1, IFITM2, and IFITM3 with the plasma membrane and lipid raft domains, respectively, which is the leading site of EV biogenesis in lymphocytes. Analysis of the subcellular distribution of IFITM3 in the presence of Nef showed that the protein has reduced levels on the cell surface and is redistributed from the cytosol to the perinuclear region. Protein transfer assays using EVs allowed us to observe that IFITM3 can be efficiently delivered to acceptor cells. Finally, analyzes of the expression of IFITMs genes in response to interferon revealed that Nef strongly blocks its expression by blocking the type I IFN signaling pathway. The results allow us to conclude that Nef antagonizes IFITMs in transcription and post-translation, blocking their interferon-induced expression and removing these proteins from the lipid raft domains in the plasma membrane. Such action leads to a sustained exclusion of IFITMs from EV budding sites. (AU)

• FAPESP's process: 17/18477-9 - Study of molecular mechanisms associated to the modulation of HIV pathogenesis mediated by extracellular vesicles
• Grantee: Mara Elisama da Silva Januário
• Support Opportunities: Scholarships in Brazil - Doctorate