Elucidation of the mechanisms and cellular factors involved in the intracellular redistribution of SERINC5 promoted by HIV-1 Nef

Author(s)

Cristina Santos da Costa

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-06-11

Examining board members

Luis Lamberti Pinto da Silva; Gonzalo Antonio Mardones Cofré; Luciana Jesus da Costa; Mariana Kiomy Osako

• Advisor: Luis Lamberti Pinto da Silva

Abstract

SERINC5 is a restriction factor that significantly reduces the infectivity of HIV-1. Expressed on the plasma membrane, SERINC5 is incorporated into newly synthesized viral particles, compromising these viruses\' ability to infect a new target cell. In contrast, HIV-1 encodes the multifunctional protein Nef, capable of preventing the incorporation of SERINC5 into viruses by manipulating the intracellular protein trafficking machinery. Specifically, Nef accelerates the endocytosis of SERINC5 from the plasma membrane, mediated by the clathrin adaptor complex 2 (AP-2), removing SERINC5 from the assembly and budding site of HIV-1. However, the cellular components and molecular mechanism involved are not fully understood. The present study aimed to elucidate the mechanisms by which HIV-1 Nef alters the subcellular trafficking of SERINC5. We initiated our study by identifying the intracellular compartment to which Nef redistributes SERINC5. Through immunofluorescence and confocal microscopy, we observed that Nef induces a perinuclear accumulation of SERINC5, which colocalized with a Golgi marker in HeLa cells. Since the AP-1 complex - a well-described Nef\'s interaction partner - selects and controls the trafficking of protein-cargo between endosomes and the Golgi complex, we investigated whether AP-1 plays a role in this effect of Nef. To achieve this goal, we used wildtype (WT) or AP-1µ1a knockout (KO) HeLa cells to perform immunofluorescence and confocal microscopy, flow cytometry, viral particle ultracentrifugation, and western blot analysis. The data showed that in the absence of AP-1, Nef retains its ability to remove SERINC from the plasma membrane, presumably by AP-2. However, instead of inducing protein accumulation in the Golgi, SERINC5 was observed in a vesicular pattern associated with compartments positive for LAMP-1, a marker of the late endocytic pathway. A similar phenotype was found using antibody uptake assays, suggesting that AP-1 plays an essential role in the acceleration of SERINC5 retrograde trafficking promoted by Nef. Our investigation also indicated that Nef interferes with the exit of newly synthesized SERINC5 from the Golgi, which may contribute to the observed intracellular retention of SERINC5. Our preliminary virus production and western blot results indicated that in the absence of AP-1, the ability of Nef to reduce SERINC5 incorporation of into viral particles is not compromised. However, AP-1 depletion reduced viral production and also resulted in decreased incorporation of SERINC5 into HIV-1, independently of Nef. Indeed, our subsequent analyses showed that the localization of SERINC5 on the plasma membrane, as well as its stability, are altered in the absence of AP-1. Together the data presented here revealed that Nef alters both the retrograde and anterograde trafficking of SERINC5, leading to its accumulation in the Golgi complex in an AP-1-dependent manner. We also demonstrated that AP-1 acts in the regular trafficking of SERINC5, possibly playing a relevant role in the incorporation of this protein into viruses. (AU)

• FAPESP's process: 19/27725-1 - Study of the mechanisms by which HIV-1 Nef promotes viral infectivity neutralising host cell restriction factors
• Grantee: Cristina Santos da Costa
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)