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Host Cell Peptidylprolyl cis-trans Isomerases as Immune Modulators of HIV-1 Infection

Bunten, Sarah Simone

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Abstract

Since the 1980s, the human immunodeficiency virus 1 (HIV-1) has been acknowledged as the trigger for AIDS, the acquired immunodeficiency syndrome. Every year, worldwide approximately 700,000 people die from late effects of HIV-1 infection and AIDS (UNAIDS, 2018). Thus, continuous research is important to better understand the interaction of the virus with the human host and to develop a cure. Host cell proteins that promote or fight infection are referred to as co- and restriction factors, respectively. Innate immunity restriction factors are, for example, TRIM5 or tetherin and some of them are induced by interferons (IFNs). Cyclophilin A (CypA), a small protein that influences the folding and thus the function of several cellular proteins, is a co-factor for HIV-1 infection. According to current knowledge, CypA shields cellular HIV-1 capsid cores form restriction factors after viral cell entry and thus ensures the safe transport of the virus genome into the cell nucleus. There, the HIV-1 genome can integrate into the host genome. In addition to CypA, the protein family of cyclophilins (Cyps) contains at least 16 other proteins in humans, all of which have similar cyclophilin domain structures, but have diverse cellular functions. While the role of CypA during HIV-1 infection is reasonably well characterized, almost no information is available for the other Cyps. This work investigated the influence of Cyps on early HIV-1 infection events in connection with the antiviral effects of type I IFNs. In general, some type I IFN-stimulated cells exert a significantly reduced HIV-1 infection. Interestingly, this early block to infection is amplified in CypA deficient cells. This indicates a role of CypA in the immune defense against HIV-1. Furthermore, an increase in infection after treatment with a cyclophilin inhibitor, Cyclosporin A (CsA) was observed. This can also be observed in the absence of CypA, the supposedly main target of CsA inhibition. This suggested the presence of CsA-sensitive factors that affect HIV-1 infection in type I IFN treated cells. Since both, CsA and type I IFNs have been proposed and tested as possible therapy strategies, however with little success, this observation warrants further investigation to reveal the underlying mechanisms, which could lead towards an adapted therapeutic strategy. The most obvious candidate targets are other members of the cyclophilin family. Therefore, CypB, CypC, CypD, CypE and CypH deficient THP-1 cells were generated using CRISPR/Cas9, and the effect of type I IFN treatment and CsA stimulation on HIV-1 infection was examined. While knockout of CypB, CypC and CypD modulated infection but showed no effect in response to IFN or CsA, CypE and CypH knockout cells showed a significantly increased sensitivity of HIV-1 infection to type I IFN-induced blocks. Due to these different phenotypes, the interplay of several Cyps was examined by generating double knockout cell lines. Depletion of CypB alone had no effect on HIV-1 infection, but a significantly increased sensitivity to type I IFN-induced post entry blocks was observed in the absence of both, CypA and CypB. The same could be observed for the double knockout of CypA and CypE. This suggests that Cyp functions on early HIV-1 infection events are complex and that some functions may depend on other members of this protein family. Furthermore, the results of this study show that CypA is not the only member of this family that has a function during early HIV-1 infection. The results from this study suggest that knockout of single cyclophilin genes was insufficient to render THP-1 cells insensitive to the CsA-induced increase in HIV-1 infection in type I IFN-induced cells, i.e. the phenotype could not be explained by one the candidates tested. To conduct a more unbiased approach, a mass spectrometry screen based on thermal protein stability was carried out covering the entire cellular proteome. In addition to known CsA targets, several new factors could be identified for which protein stability was sometimes dramatically altered upon CsA treatment of cells, indicating possible functional sensitivity to CsA. Some of these were IFN-induced proteins, such as members of the Retinoic Acid inducible gene I (RIG-I) signaling pathway. RIG-I recognizes viral RNA and induces an antiviral signaling cascade within the cell, which among other things leads to type I IFN production. Since this signaling pathway has been already associated with HIV-1 infection in the literature, some members of this signaling pathway were examined in more detail regarding HIV-1 infection and sensitivity to type I IFN and CsA. Knockout of RIG-I, MDA5, MAVS, TRADD or IRF3 in CypA deficient THP-1 cells showed increased sensitivity to type I IFN-induced early infection blocks. In addition, an increased infection in IRF3 knockout cells was observed, which indicates a function of IRF3 in the restriction of HIV-1. It was also seen that the CsA-induced effects in CypA knockout cells were no longer observable when either RIG-I, MDA5, MAVS or IRF3 were knocked out on top. While a mechanism of action of CsA on the RIG-I signaling pathway could unfortunately not be identified due to time limitations, the generated cell lines in this study are excellent tools for future studies that will aim to reveal mechanistic insights. The complex interplay between HIV-1 co-factors and type I IFN-induced cellular restriction factors in early infection events may yet again underline how perfectly well HIV-1 has adapted to exploit cellular pathways.

Document type: Dissertation
Supervisor: Kräusslich, Prof. Dr. Hans-Georg
Place of Publication: Heidelberg
Date of thesis defense: 14 September 2020
Date Deposited: 02 Oct 2020 09:07
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
DDC-classification: 500 Natural sciences and mathematics
570 Life sciences
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