RESUMO
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , Multimerização Proteica , RNA Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , RNA Viral/metabolismo , RNA Viral/química , RNA Viral/genética , Ligação Proteica , Sítios de Ligação , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Montagem de Vírus/genética , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , COVID-19/virologiaRESUMO
The Mpox virus can cause severe disease in the susceptible population with dermatologic and systemic manifestations. Furthermore, ophthalmic manifestations of mpox infection are well documented. Topical trifluridine (TFT) eye drops have been used for therapy of ophthalmic mpox infection in patients, however, its efficacy against mpox virus infection in this scenario has not been previously shown. In the present study, we have established ophthalmic cell models suitable for the infection with mpox virus. We show, that TFT is effective against a broad range of mpox isolates in conjunctival epithelial cells and keratocytes. Further, TFT remained effective against a tecovirimat-resistant virus strain. In the context of drug combinations, a nearly additive effect was observed for TFT combinations with brincidofovir and tecovirimat in conjunctival epithelial cells, while a slight antagonism was observed for both combinations in keratocytes. Altogether, our findings demonstrate TFT as a promising drug for treatment of ophthalmic mpox infection able to overcome tecovirimat resistance. However, conflicting results regarding the effect of drug combinations with approved compounds warrant close monitoring of such use in patients.
Assuntos
Mpox , Trifluridina , Humanos , Trifluridina/farmacologia , Trifluridina/uso terapêutico , Olho , Combinação de Medicamentos , Benzamidas , Isoindóis , Monkeypox virusRESUMO
Colloidal aggregation is one of the largest contributors to false positives in early drug discovery. Here, we consider aggregation's role in cell-based infectivity assays in Covid-19 drug repurposing. We investigated the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate the impact of aggregation on antiviral efficacy in cells, we presaturated the colloidal drug suspensions with BSA or spun them down by centrifugation and measured the effects on spike pseudovirus infectivity. Antiviral potencies diminished by at least 10-fold following both BSA and centrifugation treatments, supporting a colloid-based mechanism. Aggregates induced puncta of the labeled spike protein in fluorescence microscopy, consistent with sequestration of the protein on the colloidal particles. These observations suggest that colloidal aggregation is common among cell-based antiviral drug repurposing and offers rapid counter-screens to detect and eliminate these artifacts.
Assuntos
Antivirais , Coloides , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , Humanos , SARS-CoV-2/efeitos dos fármacos , Coloides/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Tratamento Farmacológico da COVID-19 , Reposicionamento de Medicamentos , COVID-19/virologiaRESUMO
Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled "smart" therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo-designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.
RESUMO
During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Infecções por HIV , HIV , Fator 2 Associado a Receptor de TNF , Ubiquitina-Proteína Ligases , Latência Viral , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linfócitos T CD4-Positivos , Sistemas CRISPR-Cas , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Replicação Viral , HIV/fisiologiaRESUMO
As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Inibidor de NF-kappaB alfa , Organoides , SARS-CoV-2 , Replicação Viral , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , COVID-19/virologia , COVID-19/metabolismo , COVID-19/genética , NF-kappa B/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/genética , Organoides/virologia , Organoides/metabolismo , SARS-CoV-2/fisiologiaRESUMO
In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2-dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Pulmão , Macrófagos Alveolares , Células Mieloides , SARS-CoV-2 , Humanos , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/virologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Células Mieloides/virologia , Células Mieloides/metabolismo , Células Mieloides/imunologia , Internalização do Vírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Tropismo ViralRESUMO
SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results pharmacologically validate Mac1 as a therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.