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Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.

Summary: The mapping of SARS-CoV-2 human protein–protein interactions by Gordon and colleagues revealed druggable targets that are hijacked by the virus. Here, we highlight several oncogenic pathways identified at the host–virus interface of SARS-CoV-2 to enable cancer biologists to apply their knowledge for rapid drug repurposing to treat COVID-19, and help inform the response to potential long-term complications of the disease.

Summary The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies., Graphical Abstract, Highlights • Phosphoproteomics analysis of SARS-CoV-2-infected cells uncovers signaling rewiring • Infection promotes host p38 MAPK cascade activity and shutdown of mitotic kinases • Infection stimulates CK2-containing filopodial protrusions with budding virus • Kinase activity analysis identifies potent antiviral drugs and compounds, Phosphoproteomics analysis of SARS-CoV-2-infected Vero E6 cells reveals host cellular pathways hijacked by viral infection, leading to the identification of small molecules that target dysregulated pathways and elicit potent antiviral efficacy.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary Tcells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of howdifferent pathogens hijack and rewire the host during infection.

Author(s): Gordon, David E; Jang, Gwendolyn M; Bouhaddou, Mehdi; Xu, Jiewei; Obernier, Kirsten; O'Meara, Matthew J; Guo, Jeffrey Z; Swaney, Danielle L; Tummino, Tia A; Huttenhain, Ruth; Kaake, Robyn M; Richards, Alicia L; Tutuncuoglu, Beril; Foussard, Helene; Batra, Jyoti; Haas, Kelsey; Modak, Maya; Kim, Minkyu; Haas, Paige; Polacco, Benjamin J; Braberg, Hannes; Fabius, Jacqueline M; Eckhardt, Manon; Soucheray, Margaret; Bennett, Melanie J; Cakir, Merve; McGregor, Michael J; Li, Qiongyu; Naing, Zun Zar Chi; Zhou, Yuan; Peng, Shiming; Kirby, Ilsa T; Melnyk, James E; Chorba, John S; Lou, Kevin; Dai, Shizhong A; Shen, Wenqi; Shi, Ying; Zhang, Ziyang; Barrio-Hernandez, Inigo; Memon, Danish; Hernandez-Armenta, Claudia; Mathy, Christopher JP; Perica, Tina; Pilla, Kala B; Ganesan, Sai J; Saltzberg, Daniel J; Ramachandran, Rakesh; Liu, Xi; Rosenthal, Sara B; Calviello, Lorenzo; Venkataramanan, Srivats; Lin, Yizhu; Wankowicz, Stephanie A; Bohn, Markus; Trenker, Raphael; Young, Janet M; Cavero, Devin; Hiatt, Joe; Roth, Theo; Rathore, Ujjwal; Subramanian, Advait; Noack, Julia; Hubert, Mathieu; Roesch, Ferdinand; Vallet, Thomas; Meyer, Bjorn; White, Kris M; Miorin, Lisa; Agard, David; Emerman, Michael; Ruggero, Davide; Garcia-Sastre, Adolfo; Jura, Natalia; von Zastrow, Mark; Taunton, Jack; Schwartz, Olivier; Vignuzzi, Marco; d'Enfert, Christophe; Mukherjee, Shaeri; Jacobson, Matt; Malik, Harmit S; Fujimori, Danica G; Ideker, Trey; Craik, Charles S | Abstract: An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity- purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

SummaryGenome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

SUMMARY Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies., In brief Hiatt et al. report a method for genome editing in primary human monocytes using CRISPR-Cas9 ribonucleoproteins (RNPs). These cells can be differentiated into macrophages or dendritic cells for downstream phenotypic assays. They demonstrate the value for functional host-pathogen studies through knockout of the HIV-1 restriction factor SAMHD1., Graphical Abstract

ER-to-Golgi transport is the first step in the constitutive secretory pathway which, unlike regulated secretion, is believed to proceed non-stop regardless of Ca2+ flux. Rowever, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adjusting the ER export rate of COPII-sorted cargos up or down by ~50%. At steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. During Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication, following secretory increases, or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Within the NRK cell model, distinct Ca2+ signaling patterns can produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased COPTT outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPTT outer shell and decreased peflin at ERES. PEF protein complexes represent a true regulator of transport as they are dispensable for secretion yet adjust the secretion rate to physiological conditions. Their dynamics affects secretion of important physiological cargoes such as collagen T and significantly impacts ER stress.

CRISPR interference screens with pairs of sgRNAs allow for quantitative characterization of genetic interactions. We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein–protein-interaction data revealed a functional map of chromatin regulation.

Summary The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFs complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4 AMBRA 1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4 AMBRA 1 is required to disrupt the assembly and attenuate the ligase activity of human CRL5 SOCS 3 and HIV‐1 CRL5 VIF complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4 AMBRA 1 modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5 SOCS 3 and CRL5 VIF , respectively. Thus, by discovering a substrate of CRL4 AMBRA 1 , ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway.

The evolutionarily conserved SIRT1–FoxO1 axis plays a new role in human CD8+ T cell metabolism and function. Progression from the naive to the terminally differentiated memory state is accompanied by the loss of SIRT1 and FoxO1 expression, which derepresses glycolytic and cytotoxic capacities of CD8+CD28– T cells under resting conditions., The expansion of CD8+CD28– T cells, a population of terminally differentiated memory T cells, is one of the most consistent immunological changes in humans during aging. CD8+CD28– T cells are highly cytotoxic, and their frequency is linked to many age-related diseases. As they do not accumulate in mice, many of the molecular mechanisms regulating their fate and function remain unclear. In this paper, we find that human CD8+CD28– T cells, under resting conditions, have an enhanced capacity to use glycolysis, a function linked to decreased expression of the NAD+-dependent protein deacetylase SIRT1. Global gene expression profiling identified the transcription factor FoxO1 as a SIRT1 target involved in transcriptional reprogramming of CD8+CD28– T cells. FoxO1 is proteasomally degraded in SIRT1-deficient CD8+CD28– T cells, and inhibiting its activity in resting CD8+CD28+ T cells enhanced glycolytic capacity and granzyme B production as in CD8+CD28– T cells. These data identify the evolutionarily conserved SIRT1–FoxO1 axis as a regulator of resting CD8+ memory T cell metabolism and activity in humans.

α-Synuclein impairs autophagosome formation and mislocalizes Atg9 by inhibiting Rab1a., Parkinson’s disease (PD) is characterized pathologically by intraneuronal inclusions called Lewy bodies, largely comprised of α-synuclein. Multiplication of the α-synuclein gene locus increases α-synuclein expression and causes PD. Thus, overexpression of wild-type α-synuclein is toxic. In this study, we demonstrate that α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein. Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation. Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.