Role of clic/geec pathway in VCC formation and HIV-1 transmission from macrophages

The National Institute of Allergy and Infectious Diseases awarded Cincinnati Children's Hospital Medical Center (doing business as Children's Home Healthcare) $703,409 on September 1, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to investigate the role of the CLIC/GEEC endocytic pathway in virus-containing compartment (VCC) formation and HIV-1 transmission from macrophages.

The research defines mechanisms by which myeloid cells internalize HIV-1 particles and form the intracellular VCC, a compartment that facilitates trans-infection of target T cells. Aim 1 experiments will characterize the role of CD98 heavy chain and its binding partners in VCC formation using novel chimeric approaches in induced pluripotent stem cell-derived macrophages. Aim 2 will evaluate clathrin-independent carriers that form in the absence of dynamin-2 and test whether dynamin-2 recruitment to CLIC membranes is required for delivery of membrane components and internalized HIV-1 particles to the VCC. The central hypothesis is that disruption of CLIC/GEEC-mediated endocytosis prevents VCC formation and reduces trans-infection efficiency.

Work is performed in Cincinnati, Ohio. The award period runs from September 1, 2026, through August 31, 2027. This is a Project Grant assistance type.

Abstract

Myeloid cells capture free HIV-1 particles via interactions between cell surface siglec-1 and gangliosides on the viral lipid envelope. Following particle capture, the siglec-1-HIV-1 complexes coalesce on the surface and then are taken into a complex intracellular compartment termed the virus-containing compartment (VCC). The VCC is also a prominent feature in HIV-1-infected myeloid cells, sharing identical late endosome and plasma membrane markers with the VCC that is formed following exogenous particle capture. Thin tubules extend from the PM to the VCC, and HIV-1 particles can be found within these surface-connected tubules. Work in our laboratory recently demonstrated that the tubules represent clathrin-independent carriers (clics), part of the clic/geec pathway. Disruption of clic/geec endocytosis prevented VCC formation and greatly diminished trans-infection of autologous t cells. The overall objective of this proposal is to define the mechanisms leading to HIV-1 particle internalization and VCC formation in macrophages, and subsequently for transmission of virus to target cells. Our central hypothesis is that the clic/geec pathway is essential to VCC formation, and dynamin-2 recruitment to clic membranes is required for delivery of membrane components and internalized HIV-1 particles to the VCC. Virions within the VCC contribute to cell-cell transmission, so defining the details of VCC formation in macrophages is an important goal. Experiments in aim 1 will define the role of CD98 heavy chain (CD98HC) and its binding partners in VCC formation. Novel chimeric approaches in ipsc-derived macrophages (imacs) will be used to identify critical domains and key interactors of CD98HC that contribute to VCC formation. Experiments in aim 2 will evaluate the clics that form in the absence of dynamin-2 and test the hypothesis that the site of HIV-1 assembly on VCC membranes requires clic/geec endocytosis and dynamin-2. Gene editing approaches will facilitate experiments testing if the gtpase activity of dynamin-2 is required for delivery of HIV-1 particles to the VCC. In aim 3, the contribution of GRAF1, a clic/geec regulator that is known to interact directly with dynamin- 2, will be evaluated. The recruitment of GRAF1 to the VCC will be evaluated by super resolution microscopy techniques. GRAF1 depletion in infected mdms and knockout in imacs will establish if IT is essential for VCC formation and function. Reconstitution with WT or GAP domain mutant GRAF1 will define the role of the RHO gtpase function of GRAF1 in leading to VCC formation. Together these studies will provide important new insights into HIV interactions with myeloid cells.