RESUMO
Avian influenza viruses continue to cross the species barrier and infect mammals. In this issue of Cell, Sun and colleagues demonstrate that viruses obtained from humans infected with an emergent avian H3N8 viruses exhibit increasing accumulation of mutations that promote respiratory droplet transmission and disease in mammals.
Assuntos
Vírus da Influenza A Subtipo H3N8 , Vírus da Influenza A , Animais , Humanos , Vírus da Influenza A/genética , Mutação , MamíferosRESUMO
My coworkers and I have used animal viruses and their interaction with host cells to investigate cellular processes difficult to study by other means. This approach has allowed us to branch out in many directions, including membrane protein characterization, endocytosis, secretion, protein folding, quality control, and glycobiology. At the same time, our aim has been to employ cell biological approaches to expand the fundamental understanding of animal viruses and their pathogenic lifestyles. We have studied mechanisms of host cell entry and the uncoating of incoming viruses as well as the synthesis, folding, maturation, and intracellular movement of viral proteins and molecular assemblies. I have had the privilege to work in institutions in four different countries. The early years in Finland (the University of Helsinki) were followed by 6 years in Germany (European Molecular Biology Laboratory), 16 years in the United States (Yale School of Medicine), and 16 years in Switzerland (ETH Zurich).
Assuntos
Calnexina/genética , Calreticulina/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/genética , Picornaviridae/genética , Proteínas Virais/genética , Virologia/história , Animais , Calnexina/química , Calnexina/metabolismo , Calreticulina/química , Calreticulina/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Regulação da Expressão Gênica , História do Século XX , História do Século XXI , Humanos , Vírus da Influenza A/metabolismo , Picornaviridae/metabolismo , Dobramento de Proteína , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Internalização do VírusRESUMO
Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
Assuntos
Vírus da Influenza A/genética , Transcrição Gênica/genética , Replicação Viral/genética , Domínio Catalítico , Simulação por Computador , Microscopia Crioeletrônica/métodos , Genoma Viral/genética , Humanos , Vírus da Influenza A/metabolismo , Influenza Humana/genética , Influenza Humana/virologia , Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.
Assuntos
Vírus da Influenza A/genética , Necroptose/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/genética , Morte Celular/genética , Linhagem Celular Tumoral , Feminino , Vírus da Influenza A/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , RNA/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologiaRESUMO
Animals such as raccoon dogs, mink and muskrats are farmed for fur and are sometimes used as food or medicinal products1,2, yet they are also potential reservoirs of emerging pathogens3. Here we performed single-sample metatranscriptomic sequencing of internal tissues from 461 individual fur animals that were found dead due to disease. We characterized 125 virus species, including 36 that were novel and 39 at potentially high risk of cross-species transmission, including zoonotic spillover. Notably, we identified seven species of coronaviruses, expanding their known host range, and documented the cross-species transmission of a novel canine respiratory coronavirus to raccoon dogs and of bat HKU5-like coronaviruses to mink, present at a high abundance in lung tissues. Three subtypes of influenza A virus-H1N2, H5N6 and H6N2-were detected in the lungs of guinea pig, mink and muskrat, respectively. Multiple known zoonotic viruses, such as Japanese encephalitis virus and mammalian orthoreovirus4,5, were detected in guinea pigs. Raccoon dogs and mink carried the highest number of potentially high-risk viruses, while viruses from the Coronaviridae, Paramyxoviridae and Sedoreoviridae families commonly infected multiple hosts. These data also reveal potential virus transmission between farmed animals and wild animals, and from humans to farmed animals, indicating that fur farming represents an important transmission hub for viral zoonoses.
Assuntos
Pelo Animal , Animais Domésticos , Animais Selvagens , Reservatórios de Doenças , Especificidade de Hospedeiro , Zoonoses Virais , Animais , Cães , Cobaias , Humanos , Animais Domésticos/virologia , Animais Selvagens/virologia , Arvicolinae/virologia , Quirópteros/virologia , Coronavirus/isolamento & purificação , Coronavirus/genética , Coronavirus/classificação , Reservatórios de Doenças/virologia , Reservatórios de Doenças/veterinária , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Pulmão/virologia , Vison/virologia , Orthoreovirus/genética , Orthoreovirus/isolamento & purificação , Filogenia , Cães Guaxinins/virologia , Zoonoses Virais/transmissão , Zoonoses Virais/virologiaRESUMO
Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.
Assuntos
Aves , Interações entre Hospedeiro e Microrganismos , Vírus da Influenza A , Influenza Aviária , Influenza Humana , Zoonoses Virais , Animais , Humanos , Aves/virologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/prevenção & controle , Influenza Humana/transmissão , Influenza Humana/virologia , Primatas , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Medição de Risco , Zoonoses Virais/prevenção & controle , Zoonoses Virais/transmissão , Zoonoses Virais/virologia , Replicação ViralRESUMO
The advent of H7N9 in early 2013 is of concern for a number of reasons, including its capability to infect humans, the lack of clarity in the etiology of infection, and because the human population does not have pre-existing immunity to the H7 subtype. Earlier sequence analyses of H7N9 hemagglutinin (HA) point to amino acid changes that predicted human receptor binding and impinge on the antigenic characteristics of the HA. Here, we report that the H7N9 HA shows limited binding to human receptors; however, should a single amino acid mutation occur, this would result in structural changes within the receptor binding site that allow for extensive binding to human receptors present in the upper respiratory tract. Furthermore, a subset of the H7N9 HA sequences demarcating coevolving amino acids appears to be in the antigenic regions of H7, which, in turn, could impact effectiveness of the current WHO-recommended prepandemic H7 vaccines.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/classificação , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Receptores Virais/metabolismo , Sequência de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Especificidade de Hospedeiro , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vacinas contra Influenza/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Filogenia , Polissacarídeos/metabolismo , Receptores Virais/química , Traqueia/virologiaRESUMO
Vaccination stands as the most effective and economical strategy for prevention and control of influenza. The primary target of neutralizing antibodies is the surface antigen hemagglutinin (HA). However, ongoing mutations in the HA sequence result in antigenic drift. The success of a vaccine is contingent on its antigenic congruence with circulating strains. Thus, predicting antigenic variants and deducing antigenic clusters of influenza viruses are pivotal for recommendation of vaccine strains. The antigenicity of influenza A viruses is determined by the interplay of amino acids in the HA1 sequence. In this study, we exploit the ability of convolutional neural networks (CNNs) to extract spatial feature representations in the convolutional layers, which can discern interactions between amino acid sites. We introduce PREDAC-CNN, a model designed to track antigenic evolution of seasonal influenza A viruses. Accessible at http://predac-cnn.cloudna.cn, PREDAC-CNN formulates a spatially oriented representation of the HA1 sequence, optimized for the convolutional framework. It effectively probes interactions among amino acid sites in the HA1 sequence. Also, PREDAC-CNN focuses exclusively on physicochemical attributes crucial for the antigenicity of influenza viruses, thereby eliminating unnecessary amino acid embeddings. Together, PREDAC-CNN is adept at capturing interactions of amino acid sites within the HA1 sequence and examining the collective impact of point mutations on antigenic variation. Through 5-fold cross-validation and retrospective testing, PREDAC-CNN has shown superior performance in predicting antigenic variants compared to its counterparts. Additionally, PREDAC-CNN has been instrumental in identifying predominant antigenic clusters for A/H3N2 (1968-2023) and A/H1N1 (1977-2023) viruses, significantly aiding in vaccine strain recommendation.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Vacinas , Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H3N2/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Estações do Ano , Estudos Retrospectivos , Antígenos Virais/genética , Redes Neurais de Computação , AminoácidosRESUMO
Highly pathogenic avian influenza viruses (HPAIVs) cause severe hemorrhagic disease in terrestrial poultry and are a threat to the poultry industry, wild life, and human health. HPAIVs arise from low pathogenic avian influenza viruses (LPAIVs), which circulate in wild aquatic birds. HPAIV emergence is thought to occur in poultry and not wild aquatic birds, but the reason for this species-restriction is not known. We hypothesized that, due to species-specific tropism and replication, intrahost HPAIV selection is favored in poultry and disfavored in wild aquatic birds. We tested this hypothesis by co-inoculating chickens, representative of poultry, and ducks, representative of wild aquatic birds, with a mixture of H7N7 HPAIV and LPAIV, mimicking HPAIV emergence in an experimental setting. Virus selection was monitored in swabs and tissues by RT-qPCR and immunostaining of differential N-terminal epitope tags that were added to the hemagglutinin protein. HPAIV was selected in four of six co-inoculated chickens, whereas LPAIV remained the major population in co-inoculated ducks on the long-term, despite detection of infectious HPAIV in tissues at early time points. Collectively, our data support the hypothesis that HPAIVs are more likely to be selected at the intrahost level in poultry than in wild aquatic birds and point towards species-specific differences in HPAIV and LPAIV tropism and replication levels as possible explanations.
Assuntos
Vírus da Influenza A Subtipo H7N7 , Vírus da Influenza A , Influenza Aviária , Doenças das Aves Domésticas , Animais , Humanos , Galinhas , Patos , Vírus da Influenza A/genética , Animais Selvagens , Aves DomésticasRESUMO
Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.
Assuntos
Deriva Genética , Infecções por Orthomyxoviridae , Doenças dos Suínos , Animais , Suínos , Infecções por Orthomyxoviridae/virologia , Doenças dos Suínos/virologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H1N1/genética , Variação Genética , Evolução Molecular , Seleção Genética , FilogeniaRESUMO
An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Influenza Humana , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Célula Única/métodos , Animais , Células Madin Darby de Rim Canino , Vírus da Influenza A/genética , Cães , Replicação ViralRESUMO
The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.
Assuntos
Vírus da Influenza A , Influenza Humana , Animais , Humanos , Camundongos , Antivirais/metabolismo , Vírus da Influenza A/genética , Chaperonas Moleculares/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Nucleotídeos de Uracila , Animais , Camundongos , Humanos , Vírus da Influenza A/genética , Antivirais/uso terapêutico , Vírus da Influenza A Subtipo H1N1/genética , Furões , Infecções por Orthomyxoviridae/tratamento farmacológicoRESUMO
Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.
Assuntos
Vírus da Influenza A , Influenza Humana , RNA Mensageiro , RNA Viral , Proteínas de Ligação a RNA , Animais , Humanos , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Vírus da Influenza A/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Influenza Humana/genética , Transporte de RNA , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Viral/metabolismo , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Replicação ViralRESUMO
Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. However, functions of lncRNAs-encoded small proteins or micropeptides in viral pathogenesis remain largely unexplored. Here, we identified a 110-amino acid small protein as a key regulator of influenza A virus (IAV) replication. This small protein that we call PESP was encoded by the putative lncRNA PCBP1-AS1. It was observed that both PCBP1-AS1 and PESP were significantly upregulated by IAV infection. Furthermore, they were markedly induced by treatment with either type I or type III interferon. Overexpression of either PCBP1-AS1 or PESP alone significantly enhanced IAV replication. In contrast, shRNA-mediated knockdown of PCBP1-AS1 or CRISPR/Cas9-mediated knockout of PESP markedly inhibited the viral production. Moreover, the targeted deletion or mutation of the sORF within the PCBP1-AS1 transcript, which resulted in the disruption of PESP expression, significantly diminished the capacity of PCBP1-AS1 to enhance IAV replication, underscoring the indispensable role of PESP in the facilitation of IAV replication by PCBP1-AS1. Interestingly, overexpression of PESP enhanced the IAV-induced autophagy by increasing the expression of ATG7, an essential autophagy effector enzyme. We also found that the 7-22 amino acids at the N-terminus of PESP were crucial for its functionality in modulating ATG7 expression and action as an enhancer of IAV replication. Additionally, HSP90AA1, a protein identified previously as a facilitator of autophagy, was found to interact with PESP, resulting in the stabilization of PESP and consequently an increase in the production of IAV. These data reveal a critical lncRNA-encoded small protein that is induced and exploited by IAV during its infection, and provide a significant insight into IAV-host interaction network.
Assuntos
Autofagia , Vírus da Influenza A , RNA Longo não Codificante , Proteínas de Ligação a RNA , Replicação Viral , Replicação Viral/fisiologia , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Influenza Humana/metabolismo , Influenza Humana/genética , Células A549 , Animais , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a DNARESUMO
Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.
Assuntos
Imunidade Inata/imunologia , Microtúbulos/imunologia , RNA Viral/imunologia , Fatores de Troca de Nucleotídeo Guanina Rho/imunologia , Transdução de Sinais/imunologia , Animais , Células COS , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Expressão Gênica/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunidade Inata/genética , Immunoblotting , Vírus da Influenza A/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microtúbulos/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genéticaRESUMO
Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.
Assuntos
Aptidão Genética , Interações Hospedeiro-Patógeno , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/metabolismo , Interferon gama/metabolismo , Animais , Análise por Conglomerados , Citocinas/biossíntese , Modelos Animais de Doenças , Resistência à Doença/genética , Resistência à Doença/imunologia , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Mediadores da Inflamação/metabolismo , Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Interferon gama/genética , Interferon gama/farmacologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Carga Viral , Replicação ViralRESUMO
During influenza A virus infection, the viral RNA polymerase transcribes the viral negative-sense segmented RNA genome and replicates it in a two-step process via complementary RNA within viral ribonucleoprotein (vRNP) complexes. While numerous viral and host factors involved in vRNP functions have been identified, dissecting the roles of individual factors remains challenging due to the complex cellular environment in which vRNP activity has been studied. To overcome this challenge, we reconstituted viral transcription and a full cycle of replication in a test tube using vRNPs isolated from virions and recombinant factors essential for these processes. This novel system uncovers the minimal components required for influenza virus replication and also reveals new roles of regulatory factors in viral replication. Moreover, it sheds light on the molecular interplay underlying the temporal regulation of viral transcription and replication. Our highly robust in vitro system enables systematic functional analysis of factors modulating influenza virus vRNP activity and paves the way for imaging key steps of viral transcription and replication.
Assuntos
Vírus da Influenza A , Influenza Humana , Orthomyxoviridae , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ribonucleoproteínas/genética , Replicação Viral/fisiologia , RNA Viral/genéticaRESUMO
It is now established that many viruses that threaten public health establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid condensates known as viral inclusions, concentrate the 8 distinct viral ribonucleoproteins (vRNPs) that form IAV genome and are viewed as sites dedicated to the assembly of the 8-partite genomic complex. Despite not being delimited by host membranes, IAV liquid inclusions accumulate host membranes inside as a result of vRNP binding to the recycling endocytic marker Rab11a, a driver of the biogenesis of these structures. We lack molecular understanding on how Rab11a-recycling endosomes condensate specifically near the endoplasmic reticulum (ER) exit sites upon IAV infection. We show here that liquid viral inclusions interact with the ER to fuse, divide, and slide. We uncover that, contrary to previous indications, the reported reduction in recycling endocytic activity is a regulated process rather than a competition for cellular resources involving a novel role for the host factor ATG9A. In infection, ATG9A mediates the removal of Rab11a-recycling endosomes carrying vRNPs from microtubules. We observe that the recycling endocytic usage of microtubules is rescued when ATG9A is depleted, which prevents condensation of Rab11a endosomes near the ER. The failure to produce viral inclusions accumulates vRNPs in the cytosol and reduces genome assembly and the release of infectious virions. We propose that the ER supports the dynamics of liquid IAV inclusions, with ATG9A facilitating their formation. This work advances our understanding on how epidemic and pandemic influenza genomes are formed. It also reveals the plasticity of recycling endosomes to undergo condensation in response to infection, disclosing new roles for ATG9A beyond its classical involvement in autophagy.
Assuntos
Vírus da Influenza A , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Vírus da Influenza A/genética , Microtúbulos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.