Dendritic cells (DCs) and macrophages are both present in the tissues of the anogenital tracts where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and represent the first opportunity for the virus to interfere with innate recognition. Previously we have shown that both cell types fail to produce type I interferons (IFN) in response to HIV-1.
we have used micropscopy, PCR and western blots to define the stage of IFN inhibition upon HIV infection. In order to fully examine the role of HIV-1 accessory proteins (Vpr, Vif, Vpu and Nef) in interfering with TBK1 function we transfected them separately into 293T cells and found that Vpr only was able to directly interact with TBK1. In order to define the precise interaction between Vpr and TBK1 we then cloned three TBK1 fragment domains; the kinase domain (KD), ubiqutin-like domain (ULD) and dimerization domain (DD) and used a coimmunoprecipitation assay to determine which of these domains bound Vpr.
We 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). We showed that this may be mediated by two HIV-1 accessory proteins, Vpr and Vif in the context of a whole virus infection. Furthermore we found that Vpr bound only to the KD, not the ULD or DD domains of TBK1. These fragments of TBK1 have now been cloned into a third generation lentiviral vector and we aim to restore interferon β production upon transduction of DCs with these lentiviral vectors.
The definition of the minimal binding regions TBK1 and Vpr interaction could allow the development of inhibitors of the interaction that would rescue IFNβ production in these cells and reduce HIV replication and spread with potential clinical applications.