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 (WE790-791 and WW796-797) 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. IMPORTANCEThe HIV-1 envelope glycoprotein (Env) is an essential component of the virus, and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 to the incorporation of Env into particles, and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.