HIV - 1 Gag Associates with Specific Uropod - Directed Microdomains in 1 a Manner Dependent on its MA Highly Basic Region

29 In polarized T cells, HIV-1 Gag localizes to a rear end protrusion known as the uropod in 30 a multimerization-dependent manner. Gag-laden uropods participate in formation of virological 31 synapses, an intercellular contact structure that plays a key role in cell-to-cell HIV-1 32 transmission. Our previous observations suggest that Gag associates with uropod-directed 33 microdomains (UDMs) that eventually co-migrate with Gag to the uropod over the cell surface. 34 However, the nature of Gag multimerization required for this movement, the composition of the 35 UDMs, and the molecular determinants for Gag association with these microdomains remain 36 unknown. In this study, we found that Gag multimerization prior to budding but beyond 37 dimerization is necessary for Gag localization to the uropods, indicating that uropod localization 38 occurs early in the assembly process. We also found that prior to membrane curvature, Gag 39 multimers associate with a specific subset of UDMs containing PSGL-1, CD43 and CD44, but 40 not ICAM-1, ICAM-3 or CD59. Notably, upon association, Gag excludes ICAM-3 from this 41 subset of UDMs, revealing an active and selective reorganization of these microdomains by Gag. 42 This specific association between Gag and UDMs is dependent on the highly basic region (HBR) 43 in the Gag MA domain. The overall positive charge of the HBR was needed for the interaction 44 with the specific UDM subset, while the exact HBR sequence was not, unlike for MA binding to 45 the plasma membrane phospholipid PI(4,5)P 2. Altogether, these findings revealed that HIV-1 46 Gag associates with specific microdomains present in polarized T cells in an MA-dependent 47