Nucleic acid sensing in CD4 T cells during HIV-1 and other viral infections

Abstract

Viruses are small intracellular parasites and use the host cell’s biosynthesis machinery to replicate and spread. Therefore, viral particles incorporate structures that are similar to the ones that naturally occur within host cells. A major mechanism to identify viral entry and replication within an infected cell is the recognition of viral nucleic acids. Sensors of the innate immune system can detect foreign nucleic acids by their unusual subcellular localisation or modification or both. Once innate immune sensors are activated, they induce distinct signalling cascades which modulate cellular responses to invading pathogens like viruses. In this thesis, we studied the role of RNA sensors RIG-I (retinoic acid-inducible gene 1) and MDA5 (melanoma differentiation-associated protein 5) in CD4 T cells during infections with SeV (Sendai virus) or HIV-1 (Human Immunodeficiency virus 1). <br /> HIV-1 is the causative agent for the acquired immunodeficiency syndrome (AIDS). Globally, more than 30 million people are living with HIV-1 and hundreds of thousands of people are newly infected every year. Today, HIV-1 infection is a chronic and manageable disease. The progression to AIDS is prevented by ART (antiretroviral therapy) which inhibits viral replication but is unable to clear the latent viral reservoir – inactive HIV-1 proviruses within long-lived subsets of immune cells. These latent viruses are not detected by innate and adaptive immunity. Furthermore, HIV-1 manipulates cellular restriction factors and sensors of viral infection to evade immune recognition. This highlights the demand for new approaches to restore innate immune sensing during latency reversal to allow the specific killing of infected cells to the clearance of the latent reservoir. <br /> We first studied the RIG-I signalling pathway in human CD4 T cells, the main reservoir for HIV-1 infection in vivo. Using SeV, a specific activator of RIG-I, and a cell-based type-I interferon reporter assay we showed that the RIG-I signalling pathway was functional in activated CD4 T cells. In resting CD4 T cells, we did not detect the release of type-I IFNs and used next generation sequencing (NGS) to verify the expression of members of the RIG-I signalling pathway. A typical XVII type-I IFN signature was observed in resting CD4 T cells following the stimulation of RIG-I with SeV. These data also showed the downregulation of pathways relevant for T cell activation. We next evaluated how the activation of the RIG-I signalling pathway affects the biology of CD4 T cells. RIG-I activation diminished proliferation, metabolic activity and release of effector cytokine IFNγ in CD4 T cells. <br /> RIG-I and MDA5 are potential sensors for HIV-1 and their role during HIV-1 infection is not fully understood to date. We discovered that HIV-1 protease (PR) directly degrades RIG-I and MDA5 independently of other cellular factors. We showed this by co-expression of HIV-1 PR and RIG-I or MDA5 in HEK293T cells and in an in vitro assay using purified recombinant HIV-1 PR and RIG-I or MDA5 proteins. The degradation of RIG-I and MDA5 by HIV-1 PR sequestrated the sensing of stimulatory RNAs in an in vitro reporter assay. These data indicate that the degradation of RIG-I and MDA5 is a potential immune evasion mechanism for HIV-1 which could be exploited in novel HIV-1 cure approaches. Furthermore, we generated RIG-I and MDA5 knockouts in primary human CD4 T cells and Jurkat cells and performed initial characterisations of those cell lines. Knockout cell lines will be useful in future studies on the role of RIG-I and MDA5 during HIV-1 infection.