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SAMHD1: HIV-1 restriction, post-translational regulation and function
Despite enormous efforts to develop curative treatments or effective vaccination strategies against human immunodeficiency virus 1 (HIV-1), the virus still is a substantial global health thread and a social and economic burden. Understanding host-HIV-1 interactions will be essential to advance treat...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2022
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| Zusammenfassung: | Despite enormous efforts to develop curative treatments or effective vaccination strategies against human immunodeficiency virus 1 (HIV-1), the virus still is a substantial global health thread and a social and economic burden. Understanding host-HIV-1 interactions will be essential to advance treatment options. Numerous pro- and anti-viral host factors are known to date, the latter are referred to as restriction factors.
Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for HIV-1, active in myeloid and resting CD4+ T cells, which both are important HIV-1 target cells. To complete its obligate reverse transcription step, HIV-1 requires deoxyribonucleoside triphosphate (dNTPs) as a substrate for DNA synthesis. SAMHD1 is a dNTP triphosphate triphosphohydrolase (dNTPase). Therefore, SAMHD1 was proposed to limit cellular dNTP levels, which is supported by correlative data.
The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on its dNTPase activity is still under debate. This might be in part due to limitations in the myeloid models and genetic tools, which are available to genetically study the relationship between SAMHD1 T592 phosphorylation, anti-viral restriction and enzymatic functions of SAMHD1. Also, additional cellular functions of SAMHD1, as well as additional post-translational modifications could impact anti-viral restriction.
To overcome technical limitations and the lack of knowledge, we used BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1, strongly inhibiting HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1 replication.
We developed a pipeline to introduce specific mutations into the genomic SAMHD1 locus via CRISPR/Cas9-mediated homologous recombination and were able to generate homozygous phosphomimetic SAMHD1 T592E and phosphoablative SAMHD1 T592A mutants. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. In stark contrast however, T592E KI cells retain wild type dNTP levels and dNTP pool composition. Thus, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled.
To further understand if SAMHD1 dNTPase activity contributes to HIV-1 restriction, we developed an advanced overexpression system to reliably screen mutants of SAMHD1 for their restrictive potential in macrophage-like BlaER1 cells. Indeed, we could identify mutants, which lack dNTPase activity, but seemed to maintain their restrictive potential. In line with this, we were unable to show an additional T592 phospho-regulation independent SAMHD1 function.
SAMHD1 was recently proposed to play a role in the resolution of R-loops, tertiary DNA:RNA structures, which occur at sites of transcription-replication conflicts. As a surrogate model for the role of SAMHD1 in R-loop biology, we tested the effect of SAMHD1 depletion on CRISPR/Cas9 induced R-loops. We did not detect alterations of CRISPR/Cas9 KO and KI efficiency in absence of SAMHD1. Nevertheless, modulation of R-loop stability might still contribute to cellular and anti-viral functions of SAMHD1.
The lack of experimental correlation between SAMHD1 dNTPase activity, HIV-1 restriction and T592 phospho-status might also be due to an incomplete understanding of the complexity of SAMHD1 regulation. To obtain a more sophisticated image of SAMHD1 phosphorylation sites, we performed a targeted mass spectrometric analysis of endogenous SAMHD1 phosphopeptides in restrictive versus non-restrictive myeloid cells. We were able to identify several differentially phosphorylated residues in addition to T592. The N-terminal phospho-hub consistent of T21, T25 and S33 seemed not to influence SAMHD1 localization, expression, T592 phosphorylation or HIV-1 replication. In contrast, T579 emerged as a potential phosphorylation site regulating HIV-1 restriction. However, mutants of T579 might affect SAMHD1 stability and further experiments will be necessary to confirm the role of pT579.
We think that BlaER1 as a novel cell model for SAMHD1-mediated HIV-1 restriction and CRISPR/Cas9-mediated KI, will be helpful to unravel the interplay between SAMHD1-mediated HIV-1 restriction, SAMHD1 post-translational regulation and cellular functions of SAMHD1. This knowledge might be essential for the future development of curative or preventive strategies against HIV-1. |
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| Umfang: | 211 Seiten |
| DOI: | 10.17192/z2023.0021 |