One of the largest deficiencies in our understanding of HIV infection is in the area of HIV particle assembly. HIV assembles at and buds from the plasma membrane of infected cells. The development of new therapies that inhibit this process will require elucidation of the cellular factors involved. Previous biochemical studies and molecular genetic experiments illustrate a requirement for cellular factors in HIV assembly. However, the identity of these cellular factors is unknown. Here we propose a direct and sensitive approach to identify cellular factors involved in particle formation, based on affinity purification of human immunodeficiency virus-like particles (VLP), and identification of VLP constituents by mass spectrometry. To accomplish this, we will engineer a tagged version of the HIV envelope that will provide a means of purifying VLP to high purity. A combination of highly sensitive mass spectrometric methods (MALDI-MS and ESI-MS) will be used to identify virus-associated proteins. We will then corroborate the results with a preliminary list of candidate assembly cofactors from a yeast two-hybrid screen and verify the biological relevance of the identified proteins with biochemical and molecular genetic analyses. By analyzing which polypeptides are identified in the virus particle, we hope to uncover key cellular factors involved in the budding process.
| Law, K M; Satija, N; Esposito, A M et al. (2016) Cell-to-Cell Spread of HIV and Viral Pathogenesis. Adv Virus Res 95:43-85 |
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