Function of HIV-1 Env cytoplasmic tail domain - How HIV-1 glycoprotein Env traffics through infected cells to sites of viral budding and viral synapse formation is largely unknown. This enigma presents an exciting opportunity to discover new targets for antiviral drug development, which remains a critical need as the AIDS pandemic enters its fifth decade. Env trafficking is complex, with newly synthesized glycoprotein transported to the plasma membrane only for it to be rapidly endocytosed and reintroduced to viral budding sites. However, Env transport in the cell appears more nuanced, as additional pathways for cell-surface Env expression have been recently reported. Adding to the complexity are cell-type dependent differences in the incorporation of Envs with truncated CTDs. Most lymphocyte-derived cells cannot package Envs with CTD truncations between 19 and 140 amino acids and, therefore, do not support HIV-1 replication. By contrast, nonhematopoietic cell lines have no such restriction. This proposal takes advantage of an inhibitor-resistant Env variant that emerged in cultures of HIV-1- infected lymphocyte-derived CEM cells. The new env gene contains a frameshift mutation approximately two- thirds into the CTD coding region leading to a new stop site three codons downstream. The resulting glycoprotein has a 47 amino-acid truncation (D47) plus three new amino acids at its C-terminus: Lys-Cys-Cys. Inhibitor profiling suggests that this new Env variant achieves high-level resistance by switching the primary HIV-1 replication route from virus-cell infection to cell-to-cell viral transmission. The CTD changes have apparently altered Env trafficking, not only to permit incorporation of this truncated Env onto virus, but also to enhance viral synapse formation. As di-Cys motifs are often modified with lipids (especially at the C-terminus), and protein lipidation often impacts intracellular trafficking, we are led to the central hypothesis of this proposal: the truncated cytoplasmic tail of 5-Helix-resistant Env is lipidated on its C-terminal di-Cys motif, and this specific post-translational modification enables the glycoprotein to traffic to sites of viral budding and viral synapse formation, enabling HIV-1 spreading infection in restrictive cells like CEMs. To test our hypothesis, we will create Env variants with point mutations in the C-terminal Lys-Cys-Cys residues and use them to explore the impact on viral replication (Aim 1.A) and cell-to-cell viral transmission (Aim 1.B) in restrictive cells (CEM and Jurkat). We will also directly interrogate the lipidation status of the di- Cys motif using two complementary assays employing mass spectrometry and metabolic bio-orthogonal labeling (Aim 1.C). Finally, we will develop a proximity-dependent biotinylation (PDB)-mass spectrometry (MS) method and employ quantitative proteomics to identify cytoplasmic interaction partners critical for Env packaging into HIV-1 in restrictive cells (Aim 2). The results from these proposed experiments will refine our knowledge of Env trafficking and provide data for an expanded grant application on the cell and structural biology of Env CTD interactions and pharmacological interventions that could disrupt them.
