electroporation-based-crispr-cas9-mediated-gene-knockout-thp-1-cells

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

Université Paris-Saclay

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection. Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.

A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic TE mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells.

TASOR, a component of the HUSH repressor epigenetic complex, and SAMHD1, a cellular triphosphohydrolase (dNTPase), are both anti-HIV proteins antagonized by HIV-2/SIVsmm Viral protein X. As a result, the same viral protein is able to relieve two different blocks along the viral life cell cycle, one at the level of reverse transcription, by degrading SAMHD1, the other one at the level of proviral expression, by degrading TASOR. Phosphorylation of SAMHD1 at T592 has been shown to downregulate its antiviral activity. The discovery that T819 in TASOR was lying within a SAMHD1 T592-like motif led us to ask whether TASOR is phosphorylated on this residue and whether this post-translational modification could regulate its repressive activity.

Alessia Zamborlini

Inserm,
CEA,
Center for Immunology of Viral,
Auto-immune,
Hematological and Bacterial diseases (IMVA-HB/IDMIT),
INSERM, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT)

Université Paris-Saclay

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

Alessia Zamborlini

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Inserm,CEA,Center for Immunology of Viral,Auto-immune,Hematological and Bacterial diseases (IMVA-HB/IDMIT),INSERM, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT)

Université Paris-Saclay

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

Inserm,
CEA,
Center for Immunology of Viral,
Auto-immune,
Hematological and Bacterial diseases (IMVA-HB/IDMIT),
INSERM, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT)