Molecular characterization of the HIV-1 Vpu protein and its role in antagonizing the cellular restriction factor Bst-2/tetherin both in vitro and in vivo

The cellular restriction factor BST-2/tetherin exerts a late stage anti-viral activity against enveloped viruses, retaining newly formed virions at the cell surface and effectively lowering virus output from infected cells. It is also a key player in the regulation of interferon production through binding to ILT7 on plasmacytoid dendritic cells, which also impacts the virus-host interaction. A number of pathogenic viruses have been found to express counter strategies to tetherin, with the Human Immunodeficiency Virus 1 using Vpu. This suggests that a strong selective pressure exists for HIV to block tetherin expression.;This potent antagonism of tetherin by Vpu could pose as a novel target for development of additional therapeutic compounds to combat viral infection. The accessory proteins of HIV are not the main targets for current HAART therapy of infected individuals. New compounds targeting these proteins could dramatically aid in the fight against HIV infection, especially considering these proteins are responsible for mediating cellular conditions, permitting efficient viral replication and dissemination.;Current systems to investigate tetherin countermeasures are limited since in vitro cell cultures do not adequately recapitulate certain aspects of viral replication or innate immune activation. Additionally, in vivo testing using SIV or SHIV derivatives in monkey models of infection can be misrepresentative of HIV infections in humans. We are using humanized mice as a small animal model to study HIV-1 infections in vivo. The mice are created by engraftment of NOD/SCID/IL2Rgamma-/- mice with human hematopoietic stem cells, resulting in the development of mature human CD4+ T cells that support infection by HIV-1.;We have created a series of Vpu deficient viruses in the NL4.3 backbone, using either a null mutant or a specific point mutation (A18H) that blocks tetherin antagonism without affecting other functions of Vpu, such as CD4 degradation. Interestingly, neither of these Vpu mutations had any effect on virus replication that was apparent in the Jurkat-based JLTRG reporter cell line, but caused a decrease in replicative fitness in PBMC cultures. By infecting humanized mice with these viruses which vary in their ability to counteract tetherin, we were able to gain a better understanding on the role of tetherin restriction during the course of an in vivo infection. Together, these studies were meant to better characterize the interaction between tetherin and its viral antagonist Vpu by both determining the significance of restriction during infection, and discovering novel ways to disrupt this interaction through therapeutic intervention.