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Structures, Functions, and Druggability of the SARS-CoV-2 Replication–Transcription Complex
Full text linkSince its emergence in late 2019, the COVID-19 pandemic continues to devastate communities worldwide. Despite the successes in both identifying new therapeutics and immunization strategies that have alleviated the toll on livelihoods, the threat of novel coronavirus (CoV) variants triggering future pandemics necessitates research into the lifecycle of the causative agent, SARS-CoV-2, and more broadly related CoVs. An essential aspect of viral propagation is the replication process in which viral enzymes orchestrate the production of nascent viral RNA genomes. CoV RNA synthesis is mediated by the RNA-dependent RNA polymerase (RdRp) which requires a coterie of viral nucleic acid-metabolizing enzymes to maintain the integrity of the genome. The RdRp and other viral replication enzymes are postulated to interact as part of a large assembly known as the replication-transcription complex (RTC). In chapter II, I explore the basis by which RTC components may interact and reveal coupling of the essential viral helicase, non-structural protein (nsp) 13, with the RdRp in which two copies of nsp13 associate with the holoenzyme-RdRp complex bound to its RNA substrate (referred to as the nsp132-RTC) using cryo-electron microscopy (cryo-EM). Intriguingly, we observed that one of the bound helicases is orientated with an opposing polarity to the RdRp on the template RNA (t-RNA) strand. In chapter III, we investigated the effect of this configuration on the RdRp and identified that nsp13 precipitates RdRp backtracking, which is a universal regulatory feature of many nucleic acid polymerases that describes their reverse motion on the nucleic acid template. The backtracking observation prompted questions as to how the polymerase can elongate RNA in the presence of the helicase. To investigate this, we performed further structural analysis of the nsp132-RTC complex using a combination of single particle cryo-electron microscopy and molecular dynamics (MD) simulation analysis. Our results identified several distinct conformational states of the nsp13 helicase and suggested a mechanism for the nsp132-RTC to turn backtracking on and off; allosterically orchestrating either RNA synthesis or backtracking in response to stimuli at the RdRp active site. As the critical element to viral replication, the RTC is a target for clinically used antivirals such as remdesivir and molnupiravir. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA, while antiviral nucleoside analogs must compete with the natural NTPs to be effective. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Therefore, I used cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation in chapter IV. Furthermore, I investigated the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP. My results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. To further our understanding on remdesivir\u27s mechanism of action, I analyzed the effect of remdesivir when incorporated in the newly replicated viral RNA to establish how it may impede viral RNA synthesis following treatment (template-dependent inhibition). My structural studies illustrate how remdesivir perturbs nucleotide incorporation by stalling the polymerase active site in a catalytically unfavorable state. In the final chapter, I explore the implications of our investigations and how they may spur further studies on the underlying mechanisms of CoV RNA synthesis. In particular, I will discuss how the helicase may be involved in the process of template switching required to produce subgenomic-mRNAs (sg-mRNAs) during \u27transcription\u27 as well as how helicase induced backtracking may promote replication coupled repair. Furthermore, I will conclude by describing the remaining gaps in our knowledge to help guide new research on the CoV replication cycle
Enrichment and Transcriptome Analysis of CD4+ T Cell Clones Harboring Intact HIV-1 Proviruses
Full text linkMore than 40 years after its onset, the Human Immunodeficiency Virus Type 1 (HIV-1) epidemic remains a major global health burden. HIV-1 is a retrovirus that infects and destroys predominantly CD4+ T cells. Since CD4+ T cells are crucial for the innate and adaptive immune system, their progressive loss in the course of HIV-1 infection leads to the Acquired Immune Deficiency Syndrome (AIDS). Antiretroviral therapy can suppress the virus in people living with HIV and prevent progression to AIDS, but does not provide a cure. Therefore, lifelong antiretroviral therapy is necessary. The barrier to HIV-1 cure is the HIV-1 reservoir, a scarce pool of HIV-1 infected CD4+ T cells that has a very long lifetime, can multiply, and is not affected by antiretroviral therapy. These persisting infected cells have been the focus of HIV research, with the aim to identify potential biomarkers and characterize their gene expression profile in order to tailor a therapeutic approach to HIV-1. Since these cells are exceedingly rare and cannot be distinguished from uninfected CD4+ T cells, ex vivo analysis on a single cell level has not been feasible. In the first part of this thesis, a method for the enrichment of clonally expanded cells harboring an intact HIV-1 provirus that does not require latency reversal is presented. The enrichment is based on CD45RA, a marker that distinguishes naïve from memory CD4+ T cells, as well as on the variable and constant domain of the T cell receptor (TCR) b chain, that are shared by all members of a given infected clone. The resulting enrichment enables the identification of the unique TCRab sequence of infected clones, which is presented in the second part. The TCRab sequence is a unique molecular identifier of a given infected clone and can be read out by commercially available high throughput single cell gene expression sequencing platforms. In the third part, the gene expression profile of six enriched infected clones harboring an intact provirus from six individuals living with HIV-1 is analyzed by single cell gene expression and TCR sequencing on the 10x Genomics platform. Cells pertaining to expanded infected clones harboring an intact provirus are predominantly found in the CD4+ T effector memory compartment, but also belong to other CD4+ memory cell populations. Thus, expanded infected clones display a diverse gene expression profile and are not a homogenous cell population. Furthermore, the infected clones analyzed in this study did not display a unique gene expression profile that would set them apart from uninfected CD4+ T cells. Thus, proviral integration does not appear to shape the host cell neither in a uniform, nor in a unique way
Multiplexed Mapping of the Interactome of G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins
Full text linkReceptor activity-modifying proteins (RAMPs) have emerged as modulators of many aspects of G protein-coupled receptor (GPCR) biology and pharmacology. The RAMP family was discovered more than two decades ago and GPCR-RAMP interactions and their functional consequences on receptor trafficking and ligand selectivity have been documented for several GPCRs, mostly belonging to the secretin family. However, the pervasiveness of GPCR-RAMP interactions and the mechanisms of their effects are not well understood. Recent bioinformatics and experimental studies suggest that GPCR-RAMP interactions might be much more widespread than previously anticipated. However, potential direct interactions among the three known RAMPs (RAMP1, RAMP2 and RAMP3) and hundreds of GPCRs have never been investigated. To address this gap in knowledge about the GPCR-RAMP interactome, we developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized cell lysates. To enable multiplexing, we engineered a library of GPCRs and validated a library of anti- GPCR antibodies (Abs). We first prepared a library of dual epitope-tagged GPCR clones and complementary dual epitope-tagged RAMPs. We expressed each clone in the library in cultured cells to generate solubilized membranes harboring each GPCR alone, or together with each RAMP. We next tested \u3e400 anti-GPCR Abs from the Human Protein Atlas targeting our customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound offtarget, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs. With the libraries of solubilized cell lysates and validated anti-GPCR Abs in hand, we proceeded to map the GPCR-RAMP interactome. First, we conducted a proof-of-concept study with 23 GPCRs, mainly belonging to the secretin family, and successfully mapped their interactions with each RAMP. Then we scaled up the procedure and mapped the interactions between the 215 GPCRs in our library and the three RAMPs. We detected novel GPCR-RAMP interactions across all subfamilies, as detected by both anti-epitope tag capture methods and anti- GPCR capture methods. We then applied the anti-GPCR Ab library to detect native GPCRRAMP complexes in solubilized membranes from neuroepithelioma and neuroblastoma cells. To follow up on our findings, we selected one newly identified RAMP-interacting GPCR and functionally characterized the consequences of RAMP interaction on its pharmacology. We studied Mas-related GPCR subtype X4 (MRGPRX4), which has been recently identified as a receptor for bile acids and has been implicated in itch in cholestatic liver diseases. Using the SBA assay and a proximity ligation assay, we first showed that MRGPRX4 interacts with RAMPs. We then found that the interaction of MRGPRX4 with RAMP2 causes attenuation of both basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. We then used AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 downregulates MRGPRX4 surface expression may have direct implications for future drug development for cholestatic itch. The application of the multiplexed SBA assay methodology to study membrane proteinprotein interactions suggests that RAMPs interact with many more GPCRs than had been previously known. These findings, especially when combined with recent structural studies of membrane protein complexes, have significant implications for advancing GPCR-targeted drug discovery and the understanding of GPCR pharmacology, biology and regulation. Moreover, the multiplexed platform that we established can be readily adapted for a range of basic and translational applications, including screening of pathological anti-GPCR autoantibodies and interrogation of other protein-protein interactions
Details of the Exhibit
No full textDescription
Details of the exhibit E.G.D. Cohen: A Leader in Statistical Physics
Idea, design - Olga Nilova, Special Collections Librarian
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/cohen-leader-in-statistical-physics/1011/thumbnail.jp
Brentano String Quartet
No full text2023 November 17
Tri-Institutional Recital in Honor of John L. Gerlach (1838-2023)
Brentano String Quartet performed Haydn Quartet in C Major, op. 33, no. 3 ( Bird ); Mendelssohn Quartet in D Major, op. 44, no. 1; Beethoven Cavatina (from Quartet in B-flat).https://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1048/thumbnail.jp
Albert Einstein Award
No full textDr. Vincent P. Dole\u27s Albert Einstein Award For Excellence in Psychiatry and Related Disciplines
A gift from Mary Lee Guptahttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1037/thumbnail.jp
Lasker-DeBakey Clinical Medical Research Award
No full textAlbert Lasker-DeBakey Clinical Medical Research Award was given to Dr. Vincent P. Dole in 1988
A gift from Mary Lee Gupta
Lasker-DeBakey Clinical Medical Research Award is one of four annual awards presented by the Lasker Foundation. The Lasker-DeBakey award is given to honor outstanding work for the understanding, diagnosis, prevention, treatment, and cure of disease. This award was renamed in 2008 in honor of Michael E. DeBakey. It was previously known as the Albert Lasker Award for Clinical Medical Research.
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1039/thumbnail.jp
Beatrice Bishop Berle Award
No full textThe Beatrice Bishop Berle Award was presented to Dr. Vincent P. Dole in 1995 by the Albert Einstein College of Medicine, Division of Substance Abuse
A gift from Mary Lee Gupta
Beatrice Bishop Berle (1902-1993), was an American physician, teacher and author. Dr. Berle, who ran a neighborhood health clinic in East Harlem from 1953 until 1962, took a pioneering approach to family medicine by treating the entire family for the effects of heroin abuse by a member. She also helped to establish methadone maintenance as a significant treatment for heroin abuse.
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/artifacts-ephemera/1041/thumbnail.jp
Rarely Acquired Type II-A CRISPR-Cas Spacers Mediate Anti-Viral Immunity Through the Targeting of a Non-Canonical PAM Sequence
Full text linkViruses that infect prokaryotes, called bacteriophages or phages, are thought to outnumber bacteria by a ratio of 10:1 and provide a constant threat to their hosts. In response, bacteria have evolved numerous defense mechanisms, attacking all major steps of the phage infection and replication cycle. In turn, phages have evolved their own counter defenses. One phage defense system, only recently characterized, has proved revolutionary for our understanding of prokaryote-phage dynamics and our ability to edit genomes of nearly any cell type. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) functions as an adaptive immune system by identifying and degrading specific sequences in invading phage nucleic acids. CRISPR loci consist of repeat sequences interspersed with short fragments of phage DNA (spacers). The cas genes code for proteins that regulate 1) the acquisition of new spacers during phage infection ( immunization ) and 2) the use of spacer transcripts to identify and degrade complementary foreign DNA sequences (protospacers) to prevent infection ( immunity ). In the Streptococcus pyogenes type II-A CRISPR-Cas system, the protospacer targets on the viral genome are followed by a conserved N-G-G DNA motif, known as the protospacer adjacent motif (PAM). The PAM identifies the phage DNA as foreign and triggers the Cas nuclease, Cas9, to cleave the target phage DNA. Most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by N-G-G PAMs. As a consequence, the great majority of acquired spacers target such sequences. However, there is a small fraction of acquired spacers that target non- canonical PAMs. Whether these non-canonical spacers originate through accidental acquisition of phage sequences and/or provide efficient defense has been unknown. In the present body of work, we found that many of the identified non-canonical spacers match phage target regions flanked by a PAM of N-A-G-G. Despite being scarcely present in bacterial populations, these NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro. Moreover, during phage infection competition assays, bacteria harboring NAGG spacers are similarly fit as, and sometimes even outcompete, those organisms carrying spacers targeting canonical AGG sequences. In contrast, when we tested the acquisition efficiency of NAGG spacers, we found that they are infrequently incorporated into the CRISPR array, several orders of magnitude less often than AGG spacers. We therefore conclude that, while NAGG PAMs facilitate robust targeting of phage DNA, these particular non-canonical sequences are selected against during spacer acquisition. Thus, they seem to operate under different mechanisms during each stage of phage defense. Our findings demonstrate that NAGG PAMs can mediate efficient immunity against invading phages, and at times, result in immune levels previously only seen with canonical PAMs. This indicates a lesser degree of stringency in PAM recognition by Cas9 than is commonly thought. Additionally, our results reveal unexpected, and hitherto unappreciated, differences in PAM recognition during the spacer acquisition versus targeting stages of the type II-A CRISPR-Cas immune response, suggesting a different role of Cas9-PAM interactions in these stages. Together, this work furthers our understanding of how bacteria generate, store, and use memories of interactions with foreign DNA, thereby driving the co-evolution of phage, bacteria, and in some cases, human hosts. Moreover, because of CRISPR-Cas9\u27s many biotechnological applications, deciphering the biological relevance of non-canonical PAMs to Cas9 also has wide-ranging implications, from modulating the specificity and efficiency of gene editing, to tracking the recording of cell memory, to developing potentially life-saving phage therapies that combat antibiotic resistance
Antibody-Effector Functions in Antiviral Response
Full text linkAntibodies are a fundamental component of the adaptive arm of the immune system. Interactions of the antibody\u27s fragment crystallizable (Fc) domain with Fc receptors (FcR) on effector leukocytes is essential for proper immunological function and bridges the humoral with the cellular components of immunity. While the Fc domain of immunoglobulin G (IgG) has historically been considered to be relatively constant, recent studies have demonstrated that in conjunction with IgG subclasses, the single conserved N-linked glycan present on the CH2 domain imparts a considerable amount of heterogeneity in the molecule. Given the stepwise construction of this N-glycan structures, families of highly related, but non-equivalent, glycoproteins (known as glycoforms), are produced which can elicit vastly different effector functions. In the first part of this thesis, I examine the expression of FcγRs on T cells in a variety of immunological settings. Molecular studies between mouse and human FcγRs and accessory molecules (Fc common gamma chain and CD3ζ), demonstrate considerable interspecies differences in heterooligomer pairing which may account for the differences observed between species in FcγR expression patterns. Furthermore, using a FcγRhumanized model, I demonstrate that FcγRIIIa is induced on CD8+ T cells in the context of viral infection, and functions as a novel co-stimulatory molecule which can lower the threshold of TCR stimulation required for cellular activation. In the context of viral infections, host outcome is often not dictated by viral cytopathicity, but instead by the quality and the magnitude of the immune response. The abundance of IgG glycoforms produced during an infection can vary greatly, modulating the immune effector repertoire throughout the course of infection. Notably, several studies have demonstrated the increase of afucosylated IgG glycoforms, which exhibit enhanced FcγRIIIa binding, during viral infections and inflammation. Despite the importance of studying these glycoforms and the potential role that they may play in signaling through FcγRIIIa on T cells, there is a clear scarcity of tools to selectively manipulate these molecules. To address this, in the second part of this thesis, I highlight the discovery of a novel class of engineered nanobodies which can discriminate between IgG glycoforms. Using this platform, I define two classes of nanobodies, one which can recognize IgG lacking its core-fucose and one which can recognize IgG bearing terminal sialic acid residues. Structural studies of these nanobodies reveal that these molecules have a novel mode of recognition, utilizing protein-protein and protein-carbohydrate contacts for binding. Lastly, I demonstrate that these nanobodies can be used as powerful prognostic tools in the settings of dengue virus and SARS-CoV2 infection, as novel therapeutics for the selective disruption of Fc-FcγR interactions, and as research tools for interrogating the B cell receptor (BCR) glycan structure