Contribution of endosomal proteins EHD1 and EHD4 to the HIV-1 replication cycle

Regulators of membrane trafficking, particularly within the endocytic pathway, are critical for both virus fitness and evasion of the host immune defenses. Here, we investigate the role of two key regulators of endosomal trafficking, Eps15 Homology Domain-containing proteins 1 and 4 (EHD1 and EHD4), in the HIV-1 replication cycle. Our results reveal that both proteins are required in target cells to facilitate infection and in producer cells for the production of infectious virions. Mechanistically, we show that EHD1 regulates the cell surface expression of HIV-1 receptor CD4, thereby contributing to HIV-1 entry. We also uncover a functional interplay between the EHD proteins and Interferon-Inducible Transmembrane Proteins 1, 2, and 3 (IFITM1/2/3), innate immune restriction factors that inhibit diverse viruses by blocking entry and reducing infectivity. Specifically, we show that EHD1/4 proteins interact with IFITMs and control their subcellular distribution and incorporation into extracellular vesicles and virions. Moreover, this interplay modulates viral infection via the endocytosis pathway. Overall, our findings reveal that EHD1 and EHD4 orchestrate multiple steps in HIV-1 replication by coordinating endosomal trafficking, viral entry, and infectivity, in close connection with immune factors such as CD4 and IFITMs.