Retroviruses encode a number of genes that are crucial for their replication in mammalian cells. Gag, Pol and Env are the main components of virions; during viral assembly, Gag and Gag-Pol polyproteins are targeted to the membrane of infected cells where they interact with Env and encapsidate genomic RNA to form a nascent viral particle. Gag molecules multimerize at the cell surface to form the shell of the virion. This step requires an intact central domain in Gag, the capsid domain (CA), which is processed from Gag in late stages of assembly into a separate protein that assembles to form the retroviral core. Infectious retroviral particles from different families are morphologically distinct and are characterized by the shape of their central CA cores. Mutations that disrupt the core shape result in the loss of infectivity. We, in collaboration with Nico Tjandra?s group at NHLBI, previously solved the structure of N-terminal domain of Human T Cell Leukemia Virus (HTLV-I) CA by NMR and determined that the protein shares a highly conserved, predominantly helical structure with an N-terminal beta-hairpin, with Human Immunodeficiency Virus (HIV-1) CA. Subsequently we studied the functional role of the beta-hairpin by mutating residues in HTLV-I CA that caused the hairpin to unfold. These conformational and backbone changes in the CA structure were also detected in mature CA expressed in mammalian cells and in released viral particles. We showed that the N-terminal beta-hairpin contributes to the overall structure and function of HTLV-I CA during assembly. We plan to further analyze other regions in CA that determine core shape, assembly and virion release. Mutations are being generated in the HTLV-I proviral genome and the assembly and release of infectious virions from human cells will be examined. We also plan to use crystallography techniques to examine the structure of the full length HTLV-I CA. These studies should help delineate the role of core shape in viral infectivity. A second project focuses on understanding the late stages of retroviral budding and release of particles. We and others have previously reported that the HIV-1 Gag polyprotein binds the cellular protein Tsg101 via direct interaction with the p6 domain of Gag; this interaction is required for releasing the nascent viral particle from the surface of infected cells. Tsg101 is part of a multi-protein complex called Endosomal Complex Required for Transport I (ESCRT-I) that is essential for sorting of cargo proteins into the endosome. ESCRT proteins are recruited to the surface of the endosome through interaction between the cellular protein Tsg101 and its natural partner in the cell Hrs. Mutants of Hrs that interfered with Tsg101 binding to HIV-1 Gag were identified and, as a consequence, potently inhibited HIV-1 particle release. Scanning electron microscopy of cells expressing a mutant Hrs protein showed an accumulation of HIV-1 particles in abnormal structures at the cell surface. These findings are described in a manuscript that is under review. Additional Hrs mutant proteins are being generated to identify those that interfere with Tsg101 binding to HIV-1 Gag and inhibit viral release.
