Tryptophan-based motifs in the LLP3 Region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation

The HIV-1 envelope glycoprotein complex (Env) is incorporated into developing particles at the plasma membrane (PM). The cytoplasmic tail (CT) of Env is known to play an essential role in particle incorporation, while the exact mechanisms underlying this function of the CT remain uncertain. Upon reaching the PM, trafficking signals in the CT interact with host cell endocytic machinery, directing Env into endosomal compartments within the cell. Prior studies have suggested that Env must traffic through the endosomal recycling compartment (ERC) in order for Env to return to the plasma membrane (PM) site of particle assembly. Expression of a truncated form of the ERC-resident trafficking adaptor Rab11-Family Interacting Proteins C (FIP1C) resulted in CT-dependent sequestration of Env in the condensed ERC, preventing recycling of Env to the PM. In this work, the motifs within the CT responsible for ERC localization of Env were systematically mapped. A small deletion encompassing the N-terminal portion of LLP3 eliminated ERC localization. Site-directed mutagenesis identified two tryptophan-based motifs (WE(790–791) and WW(796–797)) within the N-terminus of LLP3 that were essential for ERC localization of Env. Mutant viruses bearing substitutions in these motifs were deficient in Env incorporation, with a corresponding loss of particle infectivity and a significant defect in replication in a spreading infection assay. These results identify two tryptophan-based motifs at the N-terminal portion of LLP3 that mediate ERC localization and Env incorporation, providing additional supporting evidence for the importance of cellular recycling pathways in HIV-1 particle assembly.