| The human immunodeficiency virus (HIV)-1 enters cells through a multi-step fusion process where the viral Envelope (Env) binds: A) the primary receptor CD4 leading to significant conformational changes and B) engages in a complex interaction with either the chemokine receptors CCR5, or CXCR4. This second step is the result of direct interaction of the V3 with amino acids in the pocket formed by the transmembrane spanning domains of the coreceptor (CoR), particularly extracellular loop (ECL)-2. A second Env-CoR interaction occurs between the CoR amino terminus (NT) and a pocket formed at the base of V3 and the bridging sheet. This thesis presents two approaches to dissecting the NT-Env interaction; the first primarily through gp120/coreceptor mutagenesis and the second approach through a completely novel adaptation of HIV-1.;First we utilized a clone of HIV-1 R3A DeltaV3 (9,9), TA1, which was adapted to replicate with the distal half of its V3 removed. The parental R3A is a R5/X4 dual tropic Env where as the DeltaV3 Env was restricted to R5 tropism and became resistant to all ECL targeted fusion inhibitors. Alanine scanning mutagenesis on the ECLs of CCR5 showed that fusion by TA1 was altered compared to parental R3A. Alanine substitutions of the tyrosines at positions Y10, Y14, and Y15 in the NT of CCR5 were able to block TA1 replication in both cell-cell fusion and viral replication assays. This is in contrast to the effect seen by the parental R3A. This data shows that the altered phenotype of TA1 is the result of greater CCR5 NT dependence.;In the second project we describe the first derivation of an HIV-1 Env that is able to fuse and promote replication with only the NT region of CCR5, or CXCR4. Chimeric proteins and novel human T-cell lines were created for the adaptation process. The NT only viruses, CMAX and CMAR, were derived from the previously described HIV-1 R3A DeltaV3 (9,9) 7.10 viral swarm. With these viruses a novel assay was developed to examine a new class of small molecule fusion inhibitors that bind to Env competing with the NT of CCR5 and CXCR4. |
