Human immunodeficiency virus type 1 (HIV-1), a member of the Lentivirus genus of the Retroviridae family, is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Like many other animal viruses, HIV-1 is able to spread by either a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-cell contacts. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we developed quantitative assays to analyze the relative contribution of HIV-1 cell-free and cell-to-cell transmission in any given co-culture assays. We demonstrated that HIV-1 can spread by a cell-free pathway if all steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when we systematically introduced barriers to the cell-free path at various steps, HIV-1 transmission became contact-dependent. Importantly, we demonstrated that pre-existing barriers in both donor and target cells contribute to contact-dependent transmission. Thus, our model offered an explanation for conflicting reports on the contribution of HIV-1 cell-to-cell transmission, which is the result of specific features in donor and target cell types.;HIV-1 cell-to-cell transmission allows for 2-3 orders of magnitude more efficient virus spreading than the cell-free transmission and can overcome multiple barriers imposed on cell-free virus. We demonstrate that this also includes barriers at the step of assembly and release of cell-free HIV-1. The host factor Tsg101, while essential for cell-free release, is not required for efficient HIV-1 cell-to-cell transmission in various T cells. Our results suggest that the underlying mechanism of Tsg101 independence in HIV-1 cell-to-cell transmission is compensation by another cellular factor ALIX. Our data support a model whereby the localization and concentration of viral and cellular factors at sites of cell-cell contact allows for efficient HIV-1 cell-to-cell transmission.;We then applied our quantitative assays to carefully characterize the efficacy of ART drugs to interfere with HIV-1 cell-to-cell transmission. Our data confirmed the previously reported failure of tenofovir and ziduvudine to efficiently interfere with HIV-1 cell-to-cell transmission and extended this observation to raltegravir. Thus, HIV-1 cell-to-cell transmission is more resistant to various antiretroviral inhibitors, just like it can overcome various barriers in donor and target cells that are effectively against cell-free virus. Interestingly, we also found that efavirenz and saquinavir remain effective against HIV-1 cell-to-cell transmission. Our data suggest that competitive inhibitors are less efficient in interfering with HIV-1 cell-to-cell transmission and the success of allosteric reverse transcriptase and protease inhibitors in ART is in part due to their efficacy against HIV-1 cell-to-cell transmission.;Host factors are potential targets to develop antiretroviral therapies. However, the example of Tsg101 suggest that some host factors that are required for the spread of cell-free HIV-1 are less important during cell-to-cell transmission. Therefore, it is critical to identify novel host factors required for HIV-1 replication that cannot be overcome during cell-to-cell transmission. Towards this end, we further developed a quantitative assay for HIV-1 cell-to-cell transmission suitable for high-throughput screening and performed a kinase/phosphatase genome-wide siRNA screen. We identified WW domain binding protein 11 (WBP11) as a novel factor required for HIV-1 assembly and budding that cannot be overcome in cell-to-cell transmission. Future work should concentrate on identifying a potential role for WBP11 in regulating the viral genome during nuclear export, translation, cytoplasmic trafficking and virus assembly.;In summary, the work accomplished in this thesis project - the establishment o f quantitative assays and a mechanistic understanding o f HIV-1 cell-to-cell transmission provide a powerful framework to outline strategies for the successful therapeutic control o f HIV-1, through the use of current ART regimens and targeting host factors that cannot be overcome during HIV-1 cell-to-cell transmission.
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