The major goal of this application is to define viral interactions with host pathways that are essential for the assembly of infectious HIV-1 particles. We previously identified Rab11-FIP1C (FIP1C) as a key adaptor protein required for HIV-1 envelope protein (Env) trafficking and particle incorporation, a finding that established the model stating that host recycling pathways are required for particle incorporation. Recent work from our laboratory has extended this model, defining the role of KIF16B in Env trafficking and identifying discrete motifs within the Env cytoplasmic tail that are essential for recycling and particle incorporation. Preliminary data presented here now identifies the tubular recycling endosome (TRE) as a major new component of the recycling machinery involved in particle incorporation of Env. TRE components including MICAL-L1 and EHD1 were shown to be essential for Env trafficking and incorporation into particles. Remarkably, disruption of TRE trafficking severely depleted Env from particles, but only in those cells that have been shown to be nonpermissive for incorporation of cytoplasmic tail (CT)-depleted Env. Thus, this pathway holds the key to understanding the contribution of the Env CT to the recycling and particle incorporation of Env. Experiments in Aim 1 will define essential components of the TRE that contribute to Env trafficking, examining EHBP1, Rab10, Syndapin2, and MICAL-L1. In Aim 2, the critical role of EHD1-mediated scission from the TRE in Env trafficking to the PM site of assembly will be defined using cellular/biochemical and imaging approaches. A unique FAP-tagged Env construct will be employed to visualize and quantify Env trafficking from the TRE to the PM in live cells. Aim 3 will determine the mechanisms responsible for cell type- dependence of incorporation of Env, using a novel cell-cell fusion approach combined with identification of host factors that interact with the Env CT. Together, the work outlined here will expand upon a productive line of investigation that seeks to comprehensively define the mechanism of incorporation of Env into HIV-1 particles.