Characterization of Innate Cellular Modulators of Retroviral Infection

HIV-1 is a retrovirus that causes AIDS, a condition that is characterized by progressive failure of the immune system and emerging opportunistic infections. Research aiming at understanding mechanisms for retroviral spread and pathogenesis has recently started to focus on the innnate immune system and factors that have been demonstrated to either enhance or block infection in tissue-culture experiments. The work presented in this thesis aimed to characterize the expression and function of three cellular proteins; CD317, SAMHD1, and Siglec-1, that function as modulators of retroviral infection. In the first project, we investigated the in vivo expression pattern of the intrinsic immunity factor CD317 (BST-2, tetherin) in humans. CD317 restricts the release of enveloped viruses including HIV from infected cells. An additional proposed function for CD317 is as a selective target for immunotherapy of multiple myeloma, due to its apparently highly restricted expression on the surface of terminally differentiated B cells. To facilitate further studies of the biological functions, regulation, and therapeutic potential of CD317, we performed microarray-based expression profiling in 468 tissue samples from 25 healthy organs from more than 210 patients. CD317 protein could be detected in all organs investigated and in a number of specialized cell types, several of which are targeted in vivo by pathogenic viruses restricted by CD317 in tissue culture. We found limited co-expression of CD317 with the IFN biomarker MxA in vivo and low or no stimulation in organ explants exposed to recombinant IFNα, indicating that interferons may only partially regulate CD317. The study identified multiple thus far unknown interaction sites of viruses with CD317 and refutes the concept of its limited constitutive expression and strict IFN inducibility. Furthermore, CD317’s widespread expression questions its suitability as a target for immunotherapy. The second project involved characterizations of the restriction factor SAMHD1 in resting CD4+ T cells. SAMHD1 was reported to potently limit productive HIV-1 infection in a myeloid cell-specific manner by restricting HIV at the level of reverse transcription, and this block could be overcome by the Vpx protein of SIV and HIV-2. Our study demonstrated that SAMHD1 is also expressed abundantly in resting CD4+ T cells, where it locates to both the nucleus and cytoplasm. A staining protocol for SAMHD1 detection by flow cytometry was established, which enabled kinetic monitoring of its expression at a single cell level. Endogenous SAMHD1 levels were depleted in cells which either expressed Vpx following plasmid transfection or infection with Vpx-carrying HIV virions. In the latter case the restriction was alleviated, suggesting that reverse transcription is actively suppressed in resting CD4+ T cells, and that SAMHD1 is at least partly responsible for this restriction. The third project focused on the surface receptor Siglec-1 (sialoadhesin, CD169) that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Our study demonstrated that mouse Siglec-1, expressed on macrophages, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating B- and T-lymphocytes. To investigate the interaction between Siglec-1 and virus and its importance for mediating trans-infection, we analyzed the potential of sialic acid precursor analogs to modulate the MLV/Siglec-1 interaction. MLV released from N-substituted D-mannosamine analog-pretreated cells displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection. The N-acyl side chain of sialic acid was thus revealed as a critical functional determinant for the mouse Siglec-1-MLV interaction. In conclusion, the findings presented in this thesis provide new insights into the physiological expression pattern, regulation and function of the innate immunity factors CD317, SAMHD1 and Siglec-1. Characterizing the infection-modifying properties of these proteins might lead to a better understanding of their impact on retroviral spread and pathogenesis.