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1.
Cell ; 187(8): 1936-1954.e24, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38490196

RESUMO

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.


Assuntos
Encéfalo , Interferon Tipo I , Microglia , Animais , Camundongos , Interferon Tipo I/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Peixe-Zebra , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento
2.
Cell ; 186(1): 1-4, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608647

RESUMO

1988, the World Health Assembly committed to eradicate poliomyelitis, a viral disease that can cause permanent paralysis. Today, only type 1 of the three wild poliovirus types remains circulating in limited geographic areas following widespread use of different poliovirus vaccines. While we are close to zero new cases of wild polio, it is a fragile situation, and there are many remaining and new hurdles to overcome. Here, experts discuss how to address them.


Assuntos
Poliomielite , Vacinas contra Poliovirus , Poliovirus , Humanos , Poliomielite/epidemiologia , Poliomielite/prevenção & controle , Saúde Global , Erradicação de Doenças
3.
Cell ; 186(1): 112-130.e20, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36580912

RESUMO

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.


Assuntos
COVID-19 , Sistema Respiratório , SARS-CoV-2 , Humanos , Cílios/fisiologia , Cílios/virologia , COVID-19/virologia , Sistema Respiratório/citologia , Sistema Respiratório/virologia , SARS-CoV-2/fisiologia , Microvilosidades/fisiologia , Microvilosidades/virologia , Internalização do Vírus , Células Epiteliais/fisiologia , Células Epiteliais/virologia
4.
Cell ; 185(9): 1539-1548.e5, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35429436

RESUMO

Virus-like particle (VLP) and live virus assays were used to investigate neutralizing immunity against Delta and Omicron SARS-CoV-2 variants in 259 samples from 128 vaccinated individuals. Following Delta breakthrough infection, titers against WT rose 57-fold and 3.1-fold compared with uninfected boosted and unboosted individuals, respectively, versus only a 5.8-fold increase and 3.1-fold decrease for Omicron breakthrough infection. Among immunocompetent, unboosted patients, Delta breakthrough infections induced 10.8-fold higher titers against WT compared with Omicron (p = 0.037). Decreased antibody responses in Omicron breakthrough infections relative to Delta were potentially related to a higher proportion of asymptomatic or mild breakthrough infections (55.0% versus 28.6%, respectively), which exhibited 12.3-fold lower titers against WT compared with moderate to severe infections (p = 0.020). Following either Delta or Omicron breakthrough infection, limited variant-specific cross-neutralizing immunity was observed. These results suggest that Omicron breakthrough infections are less immunogenic than Delta, thus providing reduced protection against reinfection or infection from future variants.


Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos
5.
Cell ; 184(25): 6037-6051.e14, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34852237

RESUMO

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.


Assuntos
Proteínas do Capsídeo/genética , Vírus Defeituosos Interferentes/metabolismo , Replicação Viral/efeitos dos fármacos , Administração Intranasal , Animais , Antivirais/farmacologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/farmacologia , COVID-19 , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Vírus Defeituosos Interferentes/patogenicidade , Modelos Animais de Doenças , Genoma Viral/genética , Humanos , Influenza Humana , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliovirus/genética , Poliovirus/metabolismo , Infecções Respiratórias/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade
6.
Cell ; 184(13): 3426-3437.e8, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33991487

RESUMO

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.


Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/transmissão , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Humanos , Mutação/genética , Sequenciamento Completo do Genoma/métodos
7.
Cell ; 178(2): 275-289.e16, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31204099

RESUMO

Positive-stranded RNA viruses extensively remodel host cell architecture to enable viral replication. Here, we examined the poorly understood formation of specialized membrane compartments that are critical sites for the synthesis of the viral genome. We show that the replication compartments (RCs) of enteroviruses are created through novel membrane contact sites that recruit host lipid droplets (LDs) to the RCs. Viral proteins tether the RCs to the LDs and interact with the host lipolysis machinery to enable transfer of fatty acids from LDs, thereby providing lipids essential for RC biogenesis. Inhibiting the formation of the membrane contact sites between LDs and RCs or inhibition of the lipolysis pathway disrupts RC biogenesis and enterovirus replication. Our data illuminate mechanistic and functional aspects of organelle remodeling in viral infection and establish that pharmacological targeting of contact sites linking viral and host compartments is a potential strategy for antiviral development.


Assuntos
Enterovirus/fisiologia , Gotículas Lipídicas/metabolismo , Replicação Viral , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Lipólise , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus
8.
Cell ; 175(7): 1931-1945.e18, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30550790

RESUMO

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.


Assuntos
Vírus da Dengue , Dengue , Proteínas de Membrana , Proteínas Nucleares , Proteínas não Estruturais Virais , Infecção por Zika virus , Zika virus , Animais , Linhagem Celular Tumoral , Culicidae , Dengue/genética , Dengue/metabolismo , Dengue/patologia , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Zika virus/genética , Zika virus/metabolismo , Zika virus/patogenicidade , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia
9.
Cell ; 169(2): 314-325.e13, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388413

RESUMO

Effective antiviral protection in multicellular organisms relies on both cell-autonomous and systemic immunity. Systemic immunity mediates the spread of antiviral signals from infection sites to distant uninfected tissues. In arthropods, RNA interference (RNAi) is responsible for antiviral defense. Here, we show that flies have a sophisticated systemic RNAi-based immunity mediated by macrophage-like haemocytes. Haemocytes take up dsRNA from infected cells and, through endogenous transposon reverse transcriptases, produce virus-derived complementary DNAs (vDNA). These vDNAs template de novo synthesis of secondary viral siRNAs (vsRNA), which are secreted in exosome-like vesicles. Strikingly, exosomes containing vsRNAs, purified from haemolymph of infected flies, confer passive protection against virus challenge in naive animals. Thus, similar to vertebrates, insects use immune cells to generate immunological memory in the form of stable vDNAs that generate systemic immunity, which is mediated by the vsRNA-containing exosomes.


Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/virologia , Hemócitos/imunologia , Sindbis virus/fisiologia , Imunidade Adaptativa , Animais , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Exossomos/metabolismo , Hemócitos/efeitos dos fármacos , Hemócitos/virologia , Memória Imunológica , Interferência de RNA , RNA Viral/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Transcrição Reversa/efeitos dos fármacos , Sindbis virus/genética , Zidovudina/farmacologia
10.
Cell ; 169(1): 35-46.e19, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28340348

RESUMO

Paralytic polio once afflicted almost half a million children each year. The attenuated oral polio vaccine (OPV) has enabled world-wide vaccination efforts, which resulted in nearly complete control of the disease. However, poliovirus eradication is hampered globally by epidemics of vaccine-derived polio. Here, we describe a combined theoretical and experimental strategy that describes the molecular events leading from OPV to virulent strains. We discover that similar evolutionary events occur in most epidemics. The mutations and the evolutionary trajectories driving these epidemics are replicated using a simple cell-based experimental setup where the rate of evolution is intentionally accelerated. Furthermore, mutations accumulating during epidemics increase the replication fitness of the virus in cell culture and increase virulence in an animal model. Our study uncovers the evolutionary strategies by which vaccine strains become pathogenic and provides a powerful framework for rational design of safer vaccine strains and for forecasting virulence of viruses. VIDEO ABSTRACT.


Assuntos
Poliomielite/virologia , Vacina Antipólio Oral/efeitos adversos , Vacina Antipólio Oral/genética , Poliovirus/patogenicidade , Animais , Evolução Biológica , Camundongos , Filogenia , Poliomielite/prevenção & controle , Poliovirus/genética , Vacina Antipólio Oral/classificação , Vacina Antipólio Oral/imunologia
11.
Cell ; 163(5): 1108-1123, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26582131

RESUMO

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals.


Assuntos
Dengue/virologia , Proteínas de Choque Térmico HSP70/metabolismo , Replicação Viral , Animais , Proteínas do Capsídeo/metabolismo , Culicidae/virologia , Dengue/metabolismo , Vírus da Dengue , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Humanos , Replicação Viral/efeitos dos fármacos
12.
Nature ; 619(7968): 135-142, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37316671

RESUMO

Vaccination with Sabin, a live attenuated oral polio vaccine (OPV), results in robust intestinal and humoral immunity and has been key to controlling poliomyelitis. As with any RNA virus, OPV evolves rapidly to lose attenuating determinants critical to the reacquisition of virulence1-3 resulting in vaccine-derived, virulent poliovirus variants. Circulation of these variants within underimmunized populations leads to further evolution of circulating, vaccine-derived poliovirus with higher transmission capacity, representing a significant risk of polio re-emergence. A new type 2 OPV (nOPV2), with promising clinical data on genetic stability and immunogenicity, recently received authorization from the World Health Organization for use in response to circulating, vaccine-derived poliovirus outbreaks. Here we report the development of two additional live attenuated vaccine candidates against type 1 and 3 polioviruses. The candidates were generated by replacing the capsid coding region of nOPV2 with that from Sabin 1 or 3. These chimeric viruses show growth phenotypes similar to nOPV2 and immunogenicity comparable to their parental Sabin strains, but are more attenuated. Our experiments in mice and deep sequencing analysis confirmed that the candidates remain attenuated and preserve all the documented nOPV2 characteristics concerning genetic stability following accelerated virus evolution. Importantly, these vaccine candidates are highly immunogenic in mice as monovalent and multivalent formulations and may contribute to poliovirus eradication.


Assuntos
Poliomielite , Vacina Antipólio Oral , Poliovirus , Vacinas Atenuadas , Animais , Camundongos , Modelos Animais de Doenças , Poliomielite/imunologia , Poliomielite/prevenção & controle , Poliomielite/virologia , Poliovirus/classificação , Poliovirus/genética , Poliovirus/imunologia , Vacina Antipólio Oral/química , Vacina Antipólio Oral/genética , Vacina Antipólio Oral/imunologia , Vacinas Atenuadas/química , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Erradicação de Doenças
13.
Nature ; 607(7918): 351-355, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35584773

RESUMO

SARS-CoV-2 Delta and Omicron are globally relevant variants of concern. Although individuals infected with Delta are at risk of developing severe lung disease, infection with Omicron often causes milder symptoms, especially in vaccinated individuals1,2. The question arises of whether widespread Omicron infections could lead to future cross-variant protection, accelerating the end of the pandemic. Here we show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. Sera from mice overexpressing the human ACE2 receptor and infected with Omicron neutralize only Omicron, but not other variants of concern, whereas broader cross-variant neutralization was observed after WA1 and Delta infections. Unlike WA1 and Delta, Omicron replicates to low levels in the lungs and brains of infected animals, leading to mild disease with reduced expression of pro-inflammatory cytokines and diminished activation of lung-resident T cells. Sera from individuals who were unvaccinated and infected with Omicron show the same limited neutralization of only Omicron itself. By contrast, Omicron breakthrough infections induce overall higher neutralization titres against all variants of concern. Our results demonstrate that Omicron infection enhances pre-existing immunity elicited by vaccines but, on its own, may not confer broad protection against non-Omicron variants in unvaccinated individuals.


Assuntos
COVID-19 , Proteção Cruzada , SARS-CoV-2 , Vacinação , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Proteção Cruzada/imunologia , Citocinas , Humanos , Camundongos , SARS-CoV-2/classificação , SARS-CoV-2/imunologia , Vacinação/estatística & dados numéricos
14.
Nature ; 596(7873): 558-564, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34408324

RESUMO

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.


Assuntos
Flavivirus/crescimento & desenvolvimento , Flavivirus/metabolismo , Interações Hospedeiro-Patógeno , Hidroxilação , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas , Linhagem Celular , Colágeno/química , Colágeno/metabolismo , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Flavivirus/química , Regulação Viral da Expressão Gênica , Genômica , Fatores Celulares Derivados do Hospedeiro/antagonistas & inibidores , Fatores Celulares Derivados do Hospedeiro/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Sítios Internos de Entrada Ribossomal , Chaperonas Moleculares/metabolismo , Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , Poliovirus/crescimento & desenvolvimento , Polirribossomos/química , Polirribossomos/metabolismo , Agregados Proteicos , Dobramento de Proteína , Mapas de Interação de Proteínas , Proteólise , Proteômica , Zika virus/genética , Zika virus/crescimento & desenvolvimento
15.
Nature ; 596(7870): 103-108, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34153975

RESUMO

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-21-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/farmacologia , Anticorpos Antivirais/uso terapêutico , COVID-19/virologia , Testes de Neutralização , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , COVID-19/genética , COVID-19/imunologia , COVID-19/prevenção & controle , Chlorocebus aethiops , Feminino , Humanos , Masculino , Mesocricetus/imunologia , Mesocricetus/virologia , Camundongos , Camundongos Transgênicos , Profilaxia Pós-Exposição , Profilaxia Pré-Exposição , SARS-CoV-2/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
16.
PLoS Pathog ; 20(2): e1011535, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38335237

RESUMO

A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection.


Assuntos
COVID-19 , RNA Viral , Humanos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/genética , Fatores de Iniciação de Peptídeos , Proteínas Virais
17.
Proc Natl Acad Sci U S A ; 120(31): e2304667120, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37487061

RESUMO

RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.


Assuntos
Evolução Molecular , Vírus de RNA , Genoma , Taxa de Mutação , Mutação INDEL , RNA Viral/genética , Vírus de RNA/genética
18.
Cell ; 140(5): 652-65, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20211135

RESUMO

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/metabolismo , Animais , Crise Blástica , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo
19.
Mol Ther ; 32(6): 1790-1804, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38605519

RESUMO

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.


Assuntos
Anticorpos Antivirais , Linfócitos T CD8-Positivos , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Linfócitos T CD8-Positivos/imunologia , Camundongos , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Feminino , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas contra COVID-19/imunologia , Masculino , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos C57BL , Humanos , Modelos Animais de Doenças
20.
PLoS Pathog ; 18(12): e1010598, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36455064

RESUMO

Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is the formation of stress granules (SG), which are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket paralysis virus (CrPV), is a multifunctional protein that can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and CrPV infection in both Drosophila S2 cells and adult flies. Here, we uncoupled CrPV-1A's functions and provide insight into its underlying mechanism for SG inhibition. CrPV-1A mediated inhibition of SGs requires the E3 ubiquitin-ligase binding domain and the R146 residue, but not the Ago-2 binding domain. Wild-type but not mutant CrPV-1A R146A localizes to the nuclear membrane which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome changes in CrPV-infected cells are dependent on the R146 residue. Finally, Nup358/RanBP2 is targeted and degraded in CrPV-infected cells in an R146-dependent manner and the depletion of Nup358 blocks SG formation. We propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with a nuclear event that contributes to SG inhibition in order to promote infection.


Assuntos
Proteínas Virais , Replicação Viral , Animais , Proteínas Virais/metabolismo , Grânulos de Estresse , Linhagem Celular , Drosophila , Grânulos Citoplasmáticos/metabolismo
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