Defining the Mechanism of ISGs Induction and Type I Interferon Inhibition in HIV-1 Infected Mononuclear Phagocytes

Dendritic cells (DC) and macrophages are mononuclear phagocytes present in the tissues of the anogenital tract where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and act as the primary opportunity for the virus to inhibit the innate recognition. Our lab has previously shown that both cell types fail to produce type I interferons (IFN) in response to HIV-1. However, it stimulates them to produce specific subsets of interferonstimulated genes (ISGs) some of which have direct antiviral activity. Our lab defined the precise stage in the IFN inducing signalling pathway that HIV-1 targets; blocking the phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1). Finally, we showed that this is mediated by two HIV-1 accessory proteins, Vpr and Vif. In the first results chapter (3) of this thesis, we show that ISG expression is governed by two phases: an initial induction through the detection of the incoming HIV-1 inoculum at 1-6 hpi and then a second phase predominantly by newly transcribed HIV-1 as observed after 72 hpi. We also show that knockdown of either Tat, IRF1 or IRF7 ablated the second phase of ISG subset and that Tat/IRF7 induction of the second phase ISGs is down stream of IRF1. In the second results chapter (4), we demonstrate that Vpr (but not Vif, Nef or Vpu) is the only accessory protein observed to co-immunoprecipitate with TBK1 in the absence of other viral proteins. This was shown using two different Co-IP assay methods (G-Sepharose beads and the Trap system). Therefore, this supports our hypothesis that HIV-1 inhibits IFN signalling in mononuclear phagocytes by physical interactor of its protein with TBK1, thereby blocking its functional role in IFN signalling. Defining the minimal binding regions TBK1 and Vpr interaction could allow the development of small molecule inhibitors of the interaction that would rescue IFNβ production in these cells and reduce HIV replication and spread