RESUMO
The controlled release of mitochondrial content into the cytosol has emerged as one of the key steps in mitochondrial signaling. In particular, the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-ß) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) infection induces the release of mtDNA into the cytosol. The release of mtDNA is mediated by the viral minor capsid protein VI (pVI), which localizes to mitochondria. The presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release, whereas the viral E1B-19K protein blocked pVI-mediated mtDNA release. Surprisingly, the pVI-mediated mtDNA release did not increase but inhibited the IFN-ß gene expression. Notably, the pVI expression caused mitochondrial leakage of the HSP60 protein. The latter prevented specific phosphorylation of the interferon regulatory factor 3 (IRF3) needed for IFN-ß gene expression. Overall, we assign a new mitochondria and IFN-ß signaling-modulating function to the HAdV-C5 minor capsid protein VI. IMPORTANCE: Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.
Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , DNA Mitocondrial , Interferon beta , Mitocôndrias , Transdução de Sinais , Humanos , Adenovírus Humanos/fisiologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Mitocôndrias/metabolismo , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Interferon beta/metabolismo , Interferon beta/genética , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Infecções por Adenovirus Humanos/virologia , Infecções por Adenovirus Humanos/metabolismo , Membranas Mitocondriais/metabolismo , Células HEK293 , Fosforilação , Citosol/metabolismo , Citosol/virologiaRESUMO
Upon entry of Human cytomegalovirus (HCMV) into the host cell, the viral genome is transported to the nucleus where it serves as a template for transcription and genome replication. Production of new viral genomes is a coordinated effort between viral and cellular proteins. While the core replication proteins are encoded by the virus, additional cellular proteins support the process of genome synthesis. We used accelerated native isolation of proteins on nascent DNA (aniPOND) to study protein dynamics on nascent viral DNA during HCMV infection. Using this method, we identified specific viral and cellular proteins that are associated with nascent viral DNA. These included transcription factors, transcriptional regulators, DNA damage and repair factors, and chromatin remodeling complexes. The association of these identified proteins with viral DNA was confirmed by immunofluorescent imaging, chromatin-immunoprecipitation analyses, and shRNA knockdown experiments. These data provide evidence for the requirement of cellular factors involved in HCMV replication.
Assuntos
Citomegalovirus/genética , Fibroblastos/metabolismo , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Fatores de Transcrição/genética , Proteínas Virais/genética , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/metabolismo , Proteínas do Citoesqueleto/classificação , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citosol/metabolismo , Citosol/virologia , DNA Viral/genética , DNA Viral/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Ontologia Genética , Histonas/classificação , Histonas/genética , Histonas/metabolismo , Humanos , Anotação de Sequência Molecular , Proteínas Ribossômicas/classificação , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Proteínas Virais/classificação , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.
Assuntos
Citosol/virologia , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Vírus 40 dos Símios/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Citosol/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Interações Hospedeiro-Patógeno , Membranas Intracelulares/ultraestrutura , Membranas Intracelulares/virologia , Masculino , Proteínas de Membrana/genética , Vírus 40 dos Símios/patogenicidade , Vírus 40 dos Símios/ultraestruturaRESUMO
Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Poli dA-dT/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Reconhecimento de Padrão/agonistas , Receptores Virais/agonistas , Animais , Antivirais/uso terapêutico , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citosol/virologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Hepatite B/imunologia , Hepatite B/virologia , Vírus da Hepatite B da Marmota/fisiologia , Hepatócitos/virologia , Imunidade Inata , Interferons/farmacologia , Fígado/efeitos dos fármacos , Fígado/virologia , Marmota , Infecção Persistente , Poli dA-dT/uso terapêutico , Pteridinas/farmacologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Reconhecimento de Padrão/biossíntese , Receptores de Reconhecimento de Padrão/genética , Receptores Virais/biossíntese , Receptores Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
KEY MESSAGE: PSV infection changed the abundance of host plant's transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)-Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The 'omic' results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)-seq data were obtained to provide new insights into PSV-P-satRNA-plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components.
Assuntos
Cucumovirus/patogenicidade , Nicotiana/citologia , Nicotiana/virologia , Biologia de Sistemas/métodos , Núcleo Celular/genética , Núcleo Celular/virologia , Cloroplastos/genética , Cloroplastos/virologia , Citoesqueleto/genética , Citoesqueleto/virologia , Citosol/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/fisiologia , MicroRNAs , Nitrogênio/metabolismo , Fosfoproteínas/metabolismo , Células Vegetais/virologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas/genética , RNA Satélite , Nicotiana/genéticaRESUMO
To understand the role of the HIV-1 capsid in viral replication, we developed a protocol to biochemically track capsid in the nucleus during infection. To this end, we separated HIV-1-infected cells into nuclear and cytosolic fractions. Fractions were analyzed by western blotting for HIV-1 capsid content as well as for nuclear and cytosolic markers to assess the bona fide origin of the fractions. This protocol can be applied in both cycling and non-cycling human cells. For complete details on the use and execution of this protocol, please refer to Selyutina et al. (2020a).
Assuntos
Capsídeo/metabolismo , Núcleo Celular , Citosol , Infecções por HIV , HIV-1/fisiologia , Replicação Viral , Células A549 , Animais , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Núcleo Celular/virologia , Citosol/metabolismo , Citosol/patologia , Citosol/virologia , Cães , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Células HeLa , HumanosRESUMO
Hepatitis B virus (HBV) core protein (Cp) can be found in the nucleus and cytoplasm of infected hepatocytes; however, it preferentially segregates to a specific compartment correlating with disease status. Regulation of this intracellular partitioning of Cp remains obscure. In this paper, we report that cellular compartments are filled and vacated by Cp in a time- and concentration-dependent manner in both transfections and infections. At early times after transfection, Cp, in a dimeric state, preferentially localizes to the nucleolus. Later, the nucleolar compartment is emptied and Cp progresses to being predominantly nuclear, with a large fraction of the protein in an assembled state. Nuclear localization is followed by cell-wide distribution, and then Cp becomes exclusively cytoplasmic. The same trend in Cp movement is seen during an infection. Putative nucleolar retention signals have been identified and appear to be structure dependent. Export of Cp from the nucleus involves the CRM1 exportin. Time-dependent flux can be recapitulated by modifying Cp concentration, suggesting transitions are regulated by reaching a threshold concentration.IMPORTANCE HBV is an endemic virus. More than 250 million people suffer from chronic HBV infection and about 800,000 die from HBV-associated disease each year. HBV is a pararetrovirus; in an infected cell, viral DNA in the nucleus is the template for viral RNA that is packaged in nascent viral capsids in the cytoplasm. Inside those capsids, while resident in cytoplasm, the linear viral RNA is reverse transcribed to form the circular double-stranded DNA (dsDNA) of the mature virus. The HBV core (or capsid) protein plays a role in almost every step of the viral life cycle. Here, we show the core protein appears to follow a programmed, sequential localization from cytoplasmic translation then into the nucleolus, to the nucleus, and back to the cytoplasm. Localization is primarily a function of time, core protein concentration, and assembly. This has important implications for our understanding of the mechanisms of antivirals that target HBV capsid assembly.
Assuntos
Nucléolo Celular/virologia , Núcleo Celular/virologia , Citoplasma/virologia , Vírus da Hepatite B/genética , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas Virais/fisiologia , Antivirais/farmacologia , Citoplasma/fisiologia , Citosol/virologia , DNA Viral/metabolismo , Vírus da Hepatite B/química , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Proteínas Virais/genética , Montagem de Vírus , Replicação ViralRESUMO
The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.
Assuntos
HIV-1/genética , Proteínas Nucleares/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Núcleo Celular/genética , Núcleo Celular/virologia , Citosol/metabolismo , Citosol/virologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Éxons , Redes Reguladoras de Genes , HIV-1/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Íntrons , Células Jurkat , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Transcriptoma , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Bacteriophages (phages) are the most abundant biological entity in the human body, but until recently the role that phages play in human health was not well characterized. Although phages do not cause infections in human cells, phages can alter the severity of bacterial infections by the dissemination of virulence factors amongst bacterial hosts. Recent studies, made possible with advances in genome engineering and microscopy, have uncovered a novel role for phages in the human body - the ability to modulate the physiology of the mammalian cells that can harbor intracellular bacteria. In this review, we synthesize key results on how phages traverse through mammalian cells - including uptake, distribution, and interaction with intracellular receptors - highlighting how these steps in turn influence host cell killing of bacteria. We discuss the implications of the growing field of phage-mammalian cell interactions for phage therapy.
Assuntos
Bacteriófagos/metabolismo , Células/metabolismo , Células/virologia , Interações Hospedeiro-Patógeno , Mamíferos , Animais , Bacteriófagos/genética , Células/citologia , Citosol/microbiologia , Citosol/virologia , DNA Viral , Humanos , Camundongos , Fagossomos/microbiologia , Fagossomos/virologia , Prófagos/genética , Prófagos/metabolismo , Internalização do VírusRESUMO
Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.
Assuntos
Bactérias/isolamento & purificação , Técnicas Biossensoriais , Ácidos Nucleicos/isolamento & purificação , Vírus/isolamento & purificação , Animais , Bactérias/patogenicidade , Citosol/microbiologia , Citosol/virologia , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Proteínas de Ligação a DNA/química , Peixes/genética , Peixes/microbiologia , Peixes/virologia , Ácidos Nucleicos/genética , Proteínas com Motivo de Reconhecimento de RNA/química , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Viral/genética , RNA Viral/isolamento & purificação , Vírus/patogenicidadeRESUMO
The canine distemper virus (CDV) matrix (M) protein is multifunctional; it orchestrates viral assembly and budding, drives the formation of virus-like particles (VLPs), regulates viral RNA synthesis, and may support additional functions. CDV M may assemble into dimers, where each protomer is constituted by N-terminal and C-terminal domains (NTD and CTD, respectively). Here, to investigate whether electrostatic interactions between CDV M and the plasma membrane (PM) may contribute to budding activity, selected surface-exposed positively charged lysine residues, which are located within a large basic patch of CTD, were replaced by amino acids with selected properties. We found that some M mutants harboring amino acids with neutral and positive charge (methionine and arginine, respectively) maintained full functionality, including proper interaction and localization with the PM as well as intact VLP and progeny virus production as demonstrated by employing a cell exit-complementation system. Conversely, while the overall structural integrity remained mostly unaltered, most of the nonconservative M variants (carrying a glutamic acid; negatively charged) exhibited a cytosolic phenotype secondary to the lack of interaction with the PM. Consequently, such M variants were entirely defective in VLP production and viral particle formation. Furthermore, the proteasome inhibitor bortezomib significantly reduced wild-type M-mediated VLP production. Nevertheless, in the absence of the compound, all engineered M lysine variants exhibited unaffected ubiquitination profiles, consistent with other residues likely involved in this functionally essential posttranslational modification. Altogether, our data identified multiple surface-exposed lysine residues located within a basic patch of CDV M-CTD, critically contributing to PM association and ensuing membrane budding activity.IMPORTANCE Although vaccines against some morbilliviruses exist, infections still occur, which can result in dramatic brain disease or fatal outcome. Postexposure prophylaxis with antivirals would support global vaccination campaigns. Unfortunately, there is no efficient antiviral drug currently approved. The matrix (M) protein of morbilliviruses coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, molecular mechanisms supporting these functions remain poorly understood, which preclude the rationale design of inhibitors. Here, to investigate potential interactions between canine distemper virus (CDV) M and the plasma membrane (PM), we combined structure-guided mutagenesis of selected surface-exposed lysine residues with biochemical, cellular, and virological assays. We identified several lysines clustering in a basic patch microdomain of the CDV M C-terminal domain, which contributed to PM association and budding activity. Our findings provide novel mechanistic information of how morbilliviruses assemble and egress from infected cells, thereby delivering bases for future antiviral drug development.
Assuntos
Membrana Celular/virologia , Vírus da Cinomose Canina/fisiologia , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus , Animais , Membrana Celular/metabolismo , Citosol/metabolismo , Citosol/virologia , Cães , Células HEK293 , Humanos , Lisina/genética , Lisina/metabolismo , Células Madin Darby de Rim Canino , Mutação , Inibidores de Proteassoma/farmacologia , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Ubiquitinação , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacosRESUMO
Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -ß1 (Imp-ß1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-ß1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/ß1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/ß1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adenovírus Humanos/efeitos dos fármacos , Antiparasitários/farmacologia , Ivermectina/farmacologia , Replicação Viral/efeitos dos fármacos , alfa Carioferinas/genética , beta Carioferinas/genética , Células A549 , Transporte Ativo do Núcleo Celular/genética , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adenovírus Humanos/patogenicidade , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Citosol/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Transdução de Sinais , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismo , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismoRESUMO
Innate immunity to nucleic acids forms the backbone for anti-viral immunity and several inflammatory diseases. Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type I interferon (IFN-I). TRAF3-interacting protein 3 (TRAF3IP3, T3JAM) is essential for T and B cell development. It is also well-expressed by myeloid cells, where its role is unknown. Here we report that TRAF3IP3 suppresses cytosolic poly(I:C), 5'ppp-dsRNA, and vesicular stomatitis virus (VSV) triggers IFN-I expression in overexpression systems and Traf3ip3-/- primary myeloid cells. The mechanism of action is through the interaction of TRAF3IP3 with endogenous TRAF3 and TBK1. This leads to the degradative K48 ubiquitination of TBK1 via its K372 residue in a DTX4-dependent fashion. Mice with myeloid-specific gene deletion of Traf3ip3 have increased RNA virus-triggered IFN-I production and reduced susceptibility to virus. These results identify a function of TRAF3IP3 in the regulation of the host response to cytosolic viral RNA in myeloid cells.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Interferon Tipo I/genética , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Viral/genética , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Citosol/virologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Células Jurkat , Lisina/genética , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/virologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Viral/metabolismo , Células THP-1 , Ubiquitinação , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA and synthesizes the second messenger cyclic GMP-AMP (cGAMP), which binds to mediator of IRF3 activation (MITA) and initiates MITA-mediated signaling, leading to induction of type I interferons (IFNs) and other antiviral effectors. Human cytomegalovirus (HCMV), a widespread and opportunistic pathogen, antagonizes the host antiviral immune response to establish latent infection. Here, we identified HCMV tegument protein UL94 as an inhibitor of the cGAS-MITA-mediated antiviral response. Ectopic expression of UL94 impaired cytosolic double-stranded DNA (dsDNA)- and DNA virus-triggered induction of type I IFNs and enhanced viral replication. Conversely, UL94 deficiency potentiated HCMV-induced transcription of type I IFNs and downstream antiviral effectors and impaired viral replication. UL94 interacted with MITA, disrupted the dimerization and translocation of MITA, and impaired the recruitment of TBK1 to the MITA signalsome. These results suggest that UL94 plays an important role in the immune evasion of HCMV.IMPORTANCE Human cytomegalovirus (HCMV), a large double-stranded DNA (dsDNA) virus, encodes more than 200 viral proteins. HCMV infection causes irreversible abnormalities of the central nervous system in newborns and severe syndromes in organ transplantation patients or AIDS patients. It has been demonstrated that HCMV has evolved multiple immune evasion strategies to establish latent infection. Previous studies pay more attention to the mechanism by which HCMV evades immune response in the early phase of infection. In this study, we identified UL94 as a negative regulator of the innate immune response, which functions in the late phase of HCMV infection.
Assuntos
Proteínas do Capsídeo/imunologia , Citomegalovirus/imunologia , Genoma Viral , Evasão da Resposta Imune , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/genética , Proteínas do Capsídeo/genética , Núcleo Celular/imunologia , Núcleo Celular/virologia , GMP Cíclico/imunologia , GMP Cíclico/metabolismo , Citomegalovirus/genética , Citomegalovirus/crescimento & desenvolvimento , Citosol/imunologia , Citosol/virologia , DNA/imunologia , DNA/metabolismo , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/genética , Cultura Primária de Células , Ligação Proteica , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Sequenciamento do ExomaRESUMO
During entry, viruses must navigate through the host endomembrane system, penetrate cellular membranes, and undergo capsid disassembly to reach an intracellular destination that supports infection. How these events are coordinated is unclear. Here, we reveal an unexpected function of a cellular motor adaptor that coordinates virus membrane penetration and disassembly. Polyomavirus SV40 traffics to the endoplasmic reticulum (ER) and penetrates a virus-induced structure in the ER membrane called "focus" to reach the cytosol, where it disassembles before nuclear entry to promote infection. We now demonstrate that the ER focus is constructed proximal to the Golgi-associated BICD2 and BICDR1 dynein motor adaptors; this juxtaposition enables the adaptors to directly bind to and disassemble SV40 upon arrival to the cytosol. Our findings demonstrate that positioning of the virus membrane penetration site couples two decisive infection events, cytosol arrival and disassembly, and suggest cargo remodeling as a novel function of dynein adaptors.
Assuntos
Retículo Endoplasmático/genética , Complexo de Golgi/genética , Interações Hospedeiro-Patógeno/genética , Polyomavirus/genética , Animais , Transporte Biológico/genética , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/virologia , Citosol/metabolismo , Citosol/virologia , Endocitose/genética , Retículo Endoplasmático/virologia , Complexo de Golgi/virologia , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Polyomavirus/patogenicidade , Internalização do VírusRESUMO
The nonenveloped polyomavirus simian virus 40 (SV40) must penetrate the host endoplasmic reticulum (ER) membrane to enter the cytosol in order to promote infection. How this is accomplished is not entirely clear. Here, we demonstrate that the cytosolic chaperone Ubiquilin4 (Ubqln4) binds directly to the ER membrane J proteins B12 and B14. Strategically localized at the ER-cytosol interface, Ubqln4 captures SV40 emerging from the ER, thereby facilitating escape of the virus from the ER into the cytosol, which leads to infection. Strikingly, Ubqln4 engages the J proteins in a J-domain-independent manner, in contrast to the previously reported Hsc70-Hsp105-SGTA-Bag2 cytosolic complex that also mediates SV40 ER-to-cytosol transport. Our results also reveal that the H domain and STI1 motif (1-2) of Ubqln4 support J protein binding, essential for SV40 infection. Together, these data further clarify the molecular basis by which a nonenveloped virus escapes a host membrane during infectious entry.IMPORTANCE How a nonenveloped virus escapes from a host membrane to promote infection remains enigmatic. In the case of the nonenveloped polyomavirus SV40, penetration of the ER membrane to reach the cytosol is a decisive virus infection step. In this study, we found a new host factor called Ubqln4 that facilitates escape of SV40 from the ER into the cytosol, thereby providing a path for the virus to enter the nucleus to cause infection.
Assuntos
Proteínas de Transporte/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Nucleares/metabolismo , Infecções por Polyomavirus/metabolismo , Vírus 40 dos Símios/metabolismo , Motivos de Aminoácidos , Transporte Biológico Ativo/genética , Proteínas de Transporte/genética , Linhagem Celular , Citosol/patologia , Citosol/virologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Retículo Endoplasmático/virologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/patologia , Domínios Proteicos , Vírus 40 dos Símios/genéticaRESUMO
One step of the life cycle common to all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of nonstructural protein 5 (NSP5) with the RV core shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, and I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as a dominant-negative element by disrupting the formation of either VLS or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV species A to H. Detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication.IMPORTANCE Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients, adults traveling to high-risk regions, and even for the livestock industry. Aside from structural and physiological divergences among RV species studied before now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV protein NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral.
Assuntos
Proteínas do Capsídeo/química , Citosol/virologia , Rotavirus/fisiologia , Proteínas não Estruturais Virais/química , Proteínas Virais/química , Animais , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Simulação por Computador , Genes Dominantes , Cobaias , Células HEK293 , Humanos , Macaca mulatta , Camundongos , Mutação , Fosforilação , Ligação Proteica , Domínios Proteicos , RNA Viral/biossíntese , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Replicação ViralRESUMO
Microtubules, part of the cytoskeleton, are indispensable for intracellular movement, cell division, and maintaining cell shape and polarity. In addition, microtubules play an important role in viral infection. In this review, we summarize the role of the microtubules' network during polyomavirus infection. Polyomaviruses usurp microtubules and their motors to travel via early and late acidic endosomes to the endoplasmic reticulum. As shown for SV40, kinesin-1 and microtubules are engaged in the release of partially disassembled virus from the endoplasmic reticulum to the cytosol, and dynein apparently assists in the further disassembly of virions prior to their translocation to the cell nucleus-the place of their replication. Polyomavirus gene products affect the regulation of microtubule dynamics. Early T antigens destabilize microtubules and cause aberrant mitosis. The role of these activities in tumorigenesis has been documented. However, its importance for productive infection remains elusive. On the other hand, in the late phase of infection, the major capsid protein, VP1, of the mouse polyomavirus, counteracts T-antigen-induced destabilization. It physically binds microtubules and stabilizes them. The interaction results in the G2/M block of the cell cycle and prolonged S phase, which is apparently required for successful completion of the viral replication cycle.
Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/virologia , Interações Hospedeiro-Patógeno , Microtúbulos/fisiologia , Microtúbulos/virologia , Polyomavirus/patogenicidade , Animais , Proteínas do Capsídeo/genética , Citosol/virologia , Retículo Endoplasmático/virologia , Endossomos/virologia , Humanos , Camundongos , Polyomavirus/genética , Ligação Proteica , Replicação ViralRESUMO
Muscovy duck reovirus (MDRV) causes immunosuppression and results in high mortality among Muscovy ducklings. Cell entry is the first step of virus infection and represents a potential therapeutic target. However, very little is known about the mechanism by which MDRV penetrates the cells. The aim of this study was to explore the mechanism of MDRV cell entry and subsequent infection. DF-1 and Vero cells were pretreated with the inhibitors chlorpromazine (CPZ), cytochalasin D, methyl-beta-cyclodextrin (M-ß-CD), genistein, dynasore, nocodazole, or NH4Cl, and then infected with MDRV. The copy number of the MDRV p10.8 gene and the expression of viral sigma A protein were determined by RT-PCR and western blot, respectively. Both sigma A expression and p10.8 gene copy number were decreased by treatment with M-ß-CD, genistein, dynasore, nocodazole, and NH4Cl. In contrast, no effects on virus infection were detected when inhibitors of clathrin-mediated endocytosis or macropinocytosis were used. In addition, the colocalization between MDRV sigma A protein and caveolin-1 was evaluated by double-label immunofluorescence. Collectively, our data revealed that MDRV can enter susceptible cells through caveolin-dependent endocytosis involving dynamin and microtubules. Moreover, the acidic environment of the endosomes was found to be critical for efficient infection. Our findings provide new insights into the infection process of MDRV.
Assuntos
Cavéolas/metabolismo , Citosol/virologia , Endocitose , Fibroblastos/virologia , Orthoreovirus Aviário/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Galinhas , Chlorocebus aethiops , Dinaminas/fisiologia , Microtúbulos/fisiologia , Células VeroRESUMO
After assembly in the cytosol, some Vaccinia virus particles go through a complex process that leads to virus egress and eventually cell-to-cell transmission. Intracellular particles are fully infectious, and therefore virus mutants lacking essential functions in the exit pathway are unable to form plaques but can multiply intracellularly. We isolated virus mutants in which two of the genes required for virus spread (F13L and A27L) were deleted independently or concurrently. The phenotypes of the mutant viruses were consistent with the need of A27L and F13L for intercellular virus transmission, the effect of the ΔA27L mutation being more severe than that of ΔF13L. Despite their defect in spread, ΔA27L mutant viruses could be expanded by infecting cell cultures at high multiplicity of infection, followed by the release of virions from infected cells by physical means. We developed a novel system for the isolation of recombinant Vaccinia virus in which selection is efficiently achieved by recovering plaque formation capacity after re-introduction of A27L into a ΔA27L virus. This system allowed the insertion of foreign DNA into the viral genome without the use of additional genetic markers. Furthermore, starting with a double mutant (ΔA27L-ΔF13L) virus, A27L selection was used in conjunction with F13L selection to mediate simultaneous dual insertions in the viral genome. This selection system facilitates combined expression of multiple foreign proteins from a single recombinant virus.