Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction and cellular dNTP levels

• Type of Material: Article
• Authors: Moritz Schüssler, Paul-Ehrlich-Institut; Kerstin Schott, Paul-Ehrlich-Institut; Nina Verena Fuchs, Paul-Ehrlich-Institut; Adrian Oo, Emory University; Morssal Zahadi, Paul-Ehrlich-Institut; Paula Rauch, Paul-Ehrlich-Institut; Baek Kim, Emory University; Renate König, Paul-Ehrlich-Institut
• Language: English
• Date: 2023-08-25
• Publisher: NIH
• Publication Version: Preprint (Prior to Peer Review)
• Copyright Statement: The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): https://doi.org/10.1101%2F2023.08.24.554731
• Title of Journal or Parent Work: bioRxiv
• Grant/Funding Information: This research was supported by the German Research Foundation SPP1923 Project KO4573/1-2 to R.K. and Project Number 318346496, SFB1292/2 TP04 to R.K, by NIH AI162633 to B.K. and NIH AI136581 to B.K..
• Keywords: gene editing; HIV-1 restriction; SAMHD1
• Research Categories: Biology, Virology

Abstract

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is not completely understood. We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into the SAMHD1 locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of an additional SAMHD1-mediated antiviral activity independent of T592 de-phosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet poor correlation of HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context.