HIV-1 gag recruits PACSIN2 to promote virus spreading

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Late assembly (L) domains in viral Gag proteins (RSV p2b, HIV-1 p6) are essential for virus release by interacting with the ESCRT machinery.
• PACSIN2 is a BAR domain protein involved in membrane remodeling and actin cytoskeleton dynamics.

Purpose of the Study:

• To investigate the role of PACSIN2 in virus assembly and release, particularly its interaction with viral Gag proteins and the ESCRT pathway.
• To determine the contribution of PACSIN2 to HIV-1 replication and cell-to-cell spreading.

Main Methods:

• Recruitment assays using virus-like particles (VLPs) to assess PACSIN2 binding to RSV and HIV-1 Gag.
• Analysis of PACSIN2 interaction with ESCRT components (TSG101, ALIX) and ubiquitin ligases (ITCH, NEDD4-2s).
• Functional studies involving PACSIN2 depletion in T cell lines and primary cells to evaluate HIV-1 replication and spreading, including rescue experiments with wild-type and mutant PACSIN2.

Main Results:

• RSV L domain and HIV-1 p6 recruit PACSIN2 into VLPs, independent of ESCRT factors TSG101 and ALIX.
• PACSIN2 is robustly recruited by NEDD4-2s, correlating with Gag-ubiquitin conjugate formation, suggesting ubiquitin binding.
• PACSIN2 depletion severely impairs HIV-1 cell-to-cell spreading but not single-cycle replication; this defect is rescued by wild-type PACSIN2 but not an SH3 domain mutant.

Conclusions:

• PACSIN2 acts as a bridge, connecting HIV-1 Gag to the actin cytoskeleton via WASP/N-WASP, thereby promoting viral cell-to-cell transmission.
• PACSIN2's role in HIV-1 spreading is critical and mediated through its interaction with the actin cytoskeleton, independent of its direct role in ESCRT-mediated budding.
• These findings reveal a novel mechanism for viral spread involving host actin regulatory proteins.