Publications by authors named "Nevan J Krogan"

256 Publications

Phospholipidosis is a shared mechanism underlying the antiviral activity of many repurposed drugs against SARS-CoV-2.

bioRxiv 2021 Mar 24. Epub 2021 Mar 24.

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost.

One Sentence Summary: Drug-induced phospholipidosis is a single mechanism that may explain the efficacy of a wide-variety of therapeutics repurposed for COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.03.23.436648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010720PMC
March 2021

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

Virology 2021 Jun 17;558:145-151. Epub 2021 Mar 17.

Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2021.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967279PMC
June 2021

Creating collaboration by breaking down scientific barriers.

Cell 2021 Mar 18. Epub 2021 Mar 18.

Quantitative Biosciences Institute (QBI), San Francisco, CA 94158, USA; QBI COVID-19 Research Group (QCRG), University of California, San Francisco, San Francisco, CA 94158, USA; School of Pharmacy, University of California, San Francisco, San Francisco, CA 94158, USA; David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

The scientific world rewards the individual while often discouraging collaboration. However, times of crisis show us how much more we can accomplish when we work together. Here, we describe our approach to breaking down silos and fostering global collaborations and share the lessons we have learned, especially pertaining to research on SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2021.02.022DOI Listing
March 2021

Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity .

J Virol 2021 Feb 3. Epub 2021 Feb 3.

Gladstone Institute of Virology, Gladstone Institutes, San Francisco, California, USA

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective latency-reversing strategy remains to be determined. If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02393-20DOI Listing
February 2021

SUMO is a pervasive regulator of meiosis.

Elife 2021 Jan 27;10. Epub 2021 Jan 27.

Howard Hughes Medical Institute, University of California Davis, Davis, United States.

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here, we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.57720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924959PMC
January 2021

Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant.

bioRxiv 2021 Jan 24. Epub 2021 Jan 24.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.01.24.427991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836107PMC
January 2021

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Science 2021 02 25;371(6532):926-931. Epub 2021 Jan 25.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abf4058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963220PMC
February 2021

Proteomic Approaches to Study SARS-CoV-2 Biology and COVID-19 Pathology.

J Proteome Res 2021 02 19;20(2):1133-1152. Epub 2021 Jan 19.

QBI COVID-19 Research Group (QCRG), San Francisco, California 94158, United States.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), was declared a pandemic infection in March 2020. As of December 2020, two COVID-19 vaccines have been authorized for emergency use by the U.S. Food and Drug Administration, but there are no effective drugs to treat COVID-19, and pandemic mitigation efforts like physical distancing have had acute social and economic consequences. In this perspective, we discuss how the proteomic research community can leverage technologies and expertise to address the pandemic by investigating four key areas of study in SARS-CoV-2 biology. Specifically, we discuss how (1) mass spectrometry-based structural techniques can overcome limitations and complement traditional structural approaches to inform the dynamic structure of SARS-CoV-2 proteins, complexes, and virions; (2) virus-host protein-protein interaction mapping can identify the cellular machinery required for SARS-CoV-2 replication; (3) global protein abundance and post-translational modification profiling can characterize signaling pathways that are rewired during infection; and (4) proteomic technologies can aid in biomarker identification, diagnostics, and drug development in order to monitor COVID-19 pathology and investigate treatment strategies. Systems-level high-throughput capabilities of proteomic technologies can yield important insights into SARS-CoV-2 biology that are urgently needed during the pandemic, and more broadly, can inform coronavirus virology and host biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.0c00764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839417PMC
February 2021

Mass spectrometry-based protein-protein interaction networks for the study of human diseases.

Mol Syst Biol 2021 01;17(1):e8792

Quantitative Biosciences Institute (QBI), University of California San Francisco, San Francisco, CA, USA.

A better understanding of the molecular mechanisms underlying disease is key for expediting the development of novel therapeutic interventions. Disease mechanisms are often mediated by interactions between proteins. Insights into the physical rewiring of protein-protein interactions in response to mutations, pathological conditions, or pathogen infection can advance our understanding of disease etiology, progression, and pathogenesis and can lead to the identification of potential druggable targets. Advances in quantitative mass spectrometry (MS)-based approaches have allowed unbiased mapping of these disease-mediated changes in protein-protein interactions on a global scale. Here, we review MS techniques that have been instrumental for the identification of protein-protein interactions at a system-level, and we discuss the challenges associated with these methodologies as well as novel MS advancements that aim to address these challenges. An overview of examples from diverse disease contexts illustrates the potential of MS-based protein-protein interaction mapping approaches for revealing disease mechanisms, pinpointing new therapeutic targets, and eventually moving toward personalized applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/msb.20188792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803364PMC
January 2021

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

Cell 2021 01 9;184(1):106-119.e14. Epub 2020 Dec 9.

Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address:

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723770PMC
January 2021

Genetic interaction mapping informs integrative structure determination of protein complexes.

Science 2020 12;370(6522)

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaz4910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946025PMC
December 2020

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

Science 2020 12 5;370(6523):1473-1479. Epub 2020 Nov 5.

Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abe3255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857409PMC
December 2020

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Proc Natl Acad Sci U S A 2020 11 23;117(45):28344-28354. Epub 2020 Oct 23.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2016650117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668094PMC
November 2020

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

Authors:
David E Gordon Joseph Hiatt Mehdi Bouhaddou Veronica V Rezelj Svenja Ulferts Hannes Braberg Alexander S Jureka Kirsten Obernier Jeffrey Z Guo Jyoti Batra Robyn M Kaake Andrew R Weckstein Tristan W Owens Meghna Gupta Sergei Pourmal Erron W Titus Merve Cakir Margaret Soucheray Michael McGregor Zeynep Cakir Gwendolyn Jang Matthew J O'Meara Tia A Tummino Ziyang Zhang Helene Foussard Ajda Rojc Yuan Zhou Dmitry Kuchenov Ruth Hüttenhain Jiewei Xu Manon Eckhardt Danielle L Swaney Jacqueline M Fabius Manisha Ummadi Beril Tutuncuoglu Ujjwal Rathore Maya Modak Paige Haas Kelsey M Haas Zun Zar Chi Naing Ernst H Pulido Ying Shi Inigo Barrio-Hernandez Danish Memon Eirini Petsalaki Alistair Dunham Miguel Correa Marrero David Burke Cassandra Koh Thomas Vallet Jesus A Silvas Caleigh M Azumaya Christian Billesbølle Axel F Brilot Melody G Campbell Amy Diallo Miles Sasha Dickinson Devan Diwanji Nadia Herrera Nick Hoppe Huong T Kratochvil Yanxin Liu Gregory E Merz Michelle Moritz Henry C Nguyen Carlos Nowotny Cristina Puchades Alexandrea N Rizo Ursula Schulze-Gahmen Amber M Smith Ming Sun Iris D Young Jianhua Zhao Daniel Asarnow Justin Biel Alisa Bowen Julian R Braxton Jen Chen Cynthia M Chio Un Seng Chio Ishan Deshpande Loan Doan Bryan Faust Sebastian Flores Mingliang Jin Kate Kim Victor L Lam Fei Li Junrui Li Yen-Li Li Yang Li Xi Liu Megan Lo Kyle E Lopez Arthur A Melo Frank R Moss Phuong Nguyen Joana Paulino Komal Ishwar Pawar Jessica K Peters Thomas H Pospiech Maliheh Safari Smriti Sangwan Kaitlin Schaefer Paul V Thomas Aye C Thwin Raphael Trenker Eric Tse Tsz Kin Martin Tsui Feng Wang Natalie Whitis Zanlin Yu Kaihua Zhang Yang Zhang Fengbo Zhou Daniel Saltzberg Anthony J Hodder Amber S Shun-Shion Daniel M Williams Kris M White Romel Rosales Thomas Kehrer Lisa Miorin Elena Moreno Arvind H Patel Suzannah Rihn Mir M Khalid Albert Vallejo-Gracia Parinaz Fozouni Camille R Simoneau Theodore L Roth David Wu Mohd Anisul Karim Maya Ghoussaini Ian Dunham Francesco Berardi Sebastian Weigang Maxime Chazal Jisoo Park James Logue Marisa McGrath Stuart Weston Robert Haupt C James Hastie Matthew Elliott Fiona Brown Kerry A Burness Elaine Reid Mark Dorward Clare Johnson Stuart G Wilkinson Anna Geyer Daniel M Giesel Carla Baillie Samantha Raggett Hannah Leech Rachel Toth Nicola Goodman Kathleen C Keough Abigail L Lind Reyna J Klesh Kafi R Hemphill Jared Carlson-Stevermer Jennifer Oki Kevin Holden Travis Maures Katherine S Pollard Andrej Sali David A Agard Yifan Cheng James S Fraser Adam Frost Natalia Jura Tanja Kortemme Aashish Manglik Daniel R Southworth Robert M Stroud Dario R Alessi Paul Davies Matthew B Frieman Trey Ideker Carmen Abate Nolwenn Jouvenet Georg Kochs Brian Shoichet Melanie Ott Massimo Palmarini Kevan M Shokat Adolfo García-Sastre Jeremy A Rassen Robert Grosse Oren S Rosenberg Kliment A Verba Christopher F Basler Marco Vignuzzi Andrew A Peden Pedro Beltrao Nevan J Krogan

Science 2020 12 15;370(6521). Epub 2020 Oct 15.

Quantitative Biosciences Institute (QBI) COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abe9403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808408PMC
December 2020

Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses.

bioRxiv 2020 Sep 24. Epub 2020 Sep 24.

Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA.

The are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.09.24.312298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523113PMC
September 2020

The E3 Ubiquitin-Protein Ligase Cullin 3 Regulates HIV-1 Transcription.

Cells 2020 09 1;9(9). Epub 2020 Sep 1.

Immunity and Pathogenesis Program, Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.

The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells, such as CD4+ T cells, monocytes, and macrophages and depletion of Cul3 using siRNA or CRISPR/Cas9 increases HIV-1 infection in immortalized cells and primary CD4+ T cells. Conversely, overexpression of Cul3 reduces HIV-1 infection in single replication cycle assays. Importantly, the antiviral effect of Cul3 was mapped to the transcriptional stage of the viral life cycle, an effect which is independent of its role in regulating the G1/S cell cycle transition. Using isogenic viruses that only differ in their promotor region, we find that the NF-κB/NFAT transcription factor binding sites in the LTR are essential for Cul3-dependent regulation of viral gene expression. Although Cul3 effectively suppresses viral gene expression, HIV-1 does not appear to antagonize the antiviral function of Cul3 by targeting it for degradation. Taken together, these results indicate that Cul3 is a negative regulator of HIV-1 transcription which governs productive viral replication in infected cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9092010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564853PMC
September 2020

Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.

Nat Biotechnol 2020 10 27;38(10):1174-1183. Epub 2020 Aug 27.

J. David Gladstone Institutes, San Francisco, CA, USA.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-020-0659-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7740072PMC
October 2020

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation.

bioRxiv 2020 Aug 10. Epub 2020 Aug 10.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.08.08.238469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430568PMC
August 2020

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

Cell 2020 08 28;182(3):685-712.e19. Epub 2020 Jun 28.

QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321036PMC
August 2020

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Nature 2020 09 8;585(7825):414-419. Epub 2020 Jul 8.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7 mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2457-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501154PMC
September 2020

The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae.

PLoS Genet 2020 06 24;16(6):e1008840. Epub 2020 Jun 24.

Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.

The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1's essential function. Gfa1 is phosphorylated in an Isr1-dependent fashion and mutation of Isr1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cell's energy resources are converted into structural carbohydrates in response to changing cellular needs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1008840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340321PMC
June 2020

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Cancer Cell 2020 07 11;38(1):129-143.e7. Epub 2020 Jun 11.

Department of Pediatrics, Stanford University, 265 Campus Drive, Stanford, CA 94305-5457, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address:

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363571PMC
July 2020

Test performance evaluation of SARS-CoV-2 serological assays.

medRxiv 2020 May 17. Epub 2020 May 17.

J. David Gladstone Institutes, San Francisco, CA 94158, USA.

Background: Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data.

Method: We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system.

Results: Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed.

Conclusion: Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.04.25.20074856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273265PMC
May 2020

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing.

bioRxiv 2020 Mar 22. Epub 2020 Mar 22.

QBI COVID-19 Research Group (QCRG), San Francisco, CA, 94158, USA.

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 disruption. 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.03.22.002386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239059PMC
March 2020

The Landscape of Human Cancer Proteins Targeted by SARS-CoV-2.

Cancer Discov 2020 07 22;10(7):916-921. Epub 2020 May 22.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-0559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357668PMC
July 2020

mRNA display with library of even-distribution reveals cellular interactors of influenza virus NS1.

Nat Commun 2020 05 15;11(1):2449. Epub 2020 May 15.

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, 90095, USA.

A comprehensive examination of protein-protein interactions (PPIs) is fundamental for the understanding of cellular machineries. However, limitations in current methodologies often prevent the detection of PPIs with low abundance proteins. To overcome this challenge, we develop a mRNA display with library of even-distribution (md-LED) method that facilitates the detection of low abundance binders with high specificity and sensitivity. As a proof-of-principle, we apply md-LED to IAV NS1 protein. Complementary to AP-MS, md-LED enables us to validate previously described PPIs as well as to identify novel NS1 interactors. We show that interacting with FASN allows NS1 to directly regulate the synthesis of cellular fatty acids. We also use md-LED to identify a mutant of NS1, D92Y, results in a loss of interaction with CPSF1. The use of high-throughput sequencing as the readout for md-LED enables sensitive quantification of interactions, ultimately enabling massively parallel experimentation for the investigation of PPIs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16140-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229031PMC
May 2020

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

Authors:
David E Gordon Gwendolyn M Jang Mehdi Bouhaddou Jiewei Xu Kirsten Obernier Kris M White Matthew J O'Meara Veronica V Rezelj Jeffrey Z Guo Danielle L Swaney Tia A Tummino Ruth Hüttenhain Robyn M Kaake Alicia L Richards Beril Tutuncuoglu Helene Foussard Jyoti Batra Kelsey Haas Maya Modak Minkyu Kim Paige Haas Benjamin J Polacco Hannes Braberg Jacqueline M Fabius Manon Eckhardt Margaret Soucheray Melanie J Bennett Merve Cakir Michael J McGregor Qiongyu Li Bjoern Meyer Ferdinand Roesch Thomas Vallet Alice Mac Kain Lisa Miorin Elena Moreno Zun Zar Chi Naing Yuan Zhou Shiming Peng Ying Shi Ziyang Zhang Wenqi Shen Ilsa T Kirby James E Melnyk John S Chorba Kevin Lou Shizhong A Dai Inigo Barrio-Hernandez Danish Memon Claudia Hernandez-Armenta Jiankun Lyu Christopher J P Mathy Tina Perica Kala Bharath Pilla Sai J Ganesan Daniel J Saltzberg Ramachandran Rakesh Xi Liu Sara B Rosenthal Lorenzo Calviello Srivats Venkataramanan Jose Liboy-Lugo Yizhu Lin Xi-Ping Huang YongFeng Liu Stephanie A Wankowicz Markus Bohn Maliheh Safari Fatima S Ugur Cassandra Koh Nastaran Sadat Savar Quang Dinh Tran Djoshkun Shengjuler Sabrina J Fletcher Michael C O'Neal Yiming Cai Jason C J Chang David J Broadhurst Saker Klippsten Phillip P Sharp Nicole A Wenzell Duygu Kuzuoglu-Ozturk Hao-Yuan Wang Raphael Trenker Janet M Young Devin A Cavero Joseph Hiatt Theodore L Roth Ujjwal Rathore Advait Subramanian Julia Noack Mathieu Hubert Robert M Stroud Alan D Frankel Oren S Rosenberg Kliment A Verba David A Agard Melanie Ott Michael Emerman Natalia Jura Mark von Zastrow Eric Verdin Alan Ashworth Olivier Schwartz Christophe d'Enfert Shaeri Mukherjee Matt Jacobson Harmit S Malik Danica G Fujimori Trey Ideker Charles S Craik Stephen N Floor James S Fraser John D Gross Andrej Sali Bryan L Roth Davide Ruggero Jack Taunton Tanja Kortemme Pedro Beltrao Marco Vignuzzi Adolfo García-Sastre Kevan M Shokat Brian K Shoichet Nevan J Krogan

Nature 2020 07 30;583(7816):459-468. Epub 2020 Apr 30.

QBI COVID-19 Research Group (QCRG), San Francisco, CA, USA.

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 disruption. 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2286-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431030PMC
July 2020

Cyclophilin A Prevents HIV-1 Restriction in Lymphocytes by Blocking Human TRIM5α Binding to the Viral Core.

Cell Rep 2020 03;30(11):3766-3777.e6

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

Disruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand this mechanism, we utilize human Jurkat cells, peripheral blood mononuclear cells (PBMCs), and CD4 T cells. Our results show that inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human tripartite motif 5α (TRIM5α), showing that TRIM5α restricts HIV-1 in CD4 T cells. Accordingly, depletion of TRIM5α in CD4 T cells rescues HIV-1 that fail to interact with CypA, such as HIV-1-P90A. We found that TRIM5α binds to the HIV-1 core. Disruption of CypA-capsid interactions fail to affect HIV-1-A92E/G94D infection, correlating with the loss of TRIM5α binding to HIV-1-A92E/G94D cores. Disruption of CypA-capsid interactions in primary cells has a greater inhibitory effect on HIV-1 when compared to Jurkat cells. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4 T cells inhibits reverse transcription. Overall, our results reveal that CypA binding to the core protects HIV-1 from TRIM5α restriction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.02.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363000PMC
March 2020

A Quantitative Genetic Interaction Map of HIV Infection.

Mol Cell 2020 04 20;78(2):197-209.e7. Epub 2020 Feb 20.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Gladstone Institutes, San Francisco, CA 94158, USA; Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Electronic address:

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 T cells 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 how different pathogens hijack and rewire the host during infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462049PMC
April 2020