POSTBINDING EVENTS MEDIATED BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ARE SENSITIVE TO MODIFICATIONS IN THE D4-TRANSMEMBRANE LINKER REGION OF CD4

Evidence from both structural and functional studies of the CD4 molecu le suggests that several domains, including the transmembrane (TM) dom ain and the adjoining extracellular region (D4-TM linker), contribute to the post-gp120-binding events leading to human immunodeficiency vir us-mediated membrane fusion. To investigate such a role in syncytium f ormation and cell-free infectivity, we generated several deletion and substitution mutations in the TM and D4-TM linker regions of the CD4 m olecule. We found that while the TM domain of CD4 was dispensable for cell-cell and virus-cell interactions, modifications in the D4-TM link er led to perturbations in both processes. Deletion of the five amino acid residues linking D4 to the TM domain resulted in a delayed and re duced capacity to form syncytia, whereas replacement of the residues w ith the heterologous sequence from the CD8 molecule restored the kinet ic profile to wild-type CD4 levels. On the other hand, both mutants of the CD4 D4-TM linker demonstrated delayed cell-free human immunodefic iency virus type 1 infectivity profiles. The defective fusion capacity may be linked to structural perturbations identified with anti-CD4 mo noclonal antibodies in the D1-D2 interface and D3 domain of the deleti on mutant yet absent in D1 and D4. While all cells were found to bind comparable levels of gp120, both D4-TM linker mutants appeared to indu ce a decrease in the V3 loop exposure of bound gp120. This underexposu re may explain the delays in cell-free infectivities observed for both of these mutants. Together, these findings confirm a role for regions of the CD4 molecule located outside D1 in post-gp120-binding events a nd suggest that the D4-TM interface contributes to the conformational changes that direct the fusion process.