Retroviral Egress via Multivesicular Bodies

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DESCRIPTION (provided by applicant): Retroviral assembly and budding are driven by the Gag protein that induces assembly of the viral capsid and recruits cellular proteins that are essential for pinching off from the cellular membrane. These host factors include ubiquitin ligases that mediate ubiquitination of the capsid and the vacuolar protein sorting machinery responsible for the sorting of proteins into the late endosome. While Gag can recruit these factors to the plasma membrane, assembly and budding also occur at intracellular late endosomal vesicles called multivesicular bodies (MVB). Using a visual approach based on fluorescently labeled virions, we have reported that retroviral Gag proteins of the murine leukemia virus (MLV) and the human immunodeficiency virus (HIV) accumulate on and bud into late endosomes. In mammalian cells, MVBs are capable of fusing with the plasma membrane to release their contents. As such, budding into MVBs provides an alternative pathway to budding from the plasma membrane. A number of important questions now need to be addressed. How are MVBs carrying virus activated to fuse with the plasma membrane in order to release infectious virus? Can these fusion events be captured visually to reveal the dynamics in living cells? What cellular factors are involved in the activation? Are factors that play a role in exocytosis via secretory lysosomes involved? Finally, are the MVBs of antigen-presenting cells specifically mobilized in response to an interaction with T-cells? To answer these questions, we will study primary cells such as macrophages in which the MVB pathway is primarily responsible for the release of infectious viruses. To this end, we have established technologies that allow us to visualize HIV and MLV egress in primary cells using total internal reflection fluorescent microscopy. To gain insights into the cell biology of viral egress we will take advantage of the availability of mouse models for defects in secretory lyososomes and determine if MLV release is affected in these models in vivo and in vitro.