RESUMEN
Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.
Asunto(s)
Adenovirus Humanos , Tropismo Viral , alfa-Defensinas , alfa-Defensinas/metabolismo , Humanos , Adenovirus Humanos/fisiología , Adenovirus Humanos/metabolismo , Animales , Ratones , Infecciones por Adenovirus Humanos/metabolismo , Infecciones por Adenovirus Humanos/virología , Receptores Virales/metabolismo , Internalización del VirusRESUMEN
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.
Asunto(s)
Adenovirus Humanos , Proteínas de la Cápside , ADN Mitocondrial , Interferón beta , Mitocondrias , Transducción de Señal , Humanos , Adenovirus Humanos/fisiología , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Mitocondrias/metabolismo , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Interferón beta/metabolismo , Interferón beta/genética , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Infecciones por Adenovirus Humanos/virología , Infecciones por Adenovirus Humanos/metabolismo , Membranas Mitocondriales/metabolismo , Células HEK293 , Fosforilación , Citosol/metabolismo , Citosol/virologíaRESUMEN
Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.
Asunto(s)
Proteínas E4 de Adenovirus , Adenovirus Humanos , Fosfatidilinositol 3-Quinasa , Proteínas Proto-Oncogénicas c-akt , Proteína p53 Supresora de Tumor , Humanos , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/metabolismo , Línea Celular Tumoral , Células HEK293 , Sistemas de Lectura Abierta/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/agonistas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Replicación ViralRESUMEN
Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.
Asunto(s)
Adenovirus Humanos , Proteínas de la Cápside , Lactoferrina , Receptores Virales , Internalización del Virus , Humanos , Infecciones por Adenovirus Humanos/metabolismo , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/química , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Adenovirus Humanos/ultraestructura , Sitios de Unión/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Lactoferrina/química , Lactoferrina/genética , Lactoferrina/metabolismo , Lactoferrina/ultraestructura , Modelos Biológicos , Mutación , Unión Proteica , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , Receptores Virales/ultraestructura , Solubilidad , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virologíaRESUMEN
The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.
Asunto(s)
Infecciones por Adenoviridae , Proteínas E4 de Adenovirus , Adenovirus Humanos , Humanos , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Infecciones por Adenoviridae/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismoRESUMEN
IMPORTANCE: The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there's still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.
Asunto(s)
Adenovirus Humanos , Desmogleína 2 , Proteína Cofactora de Membrana , Receptores Virales , Humanos , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Línea Celular , Desmogleína 2/genética , Desmogleína 2/metabolismo , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismoRESUMEN
IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas E1A de Adenovirus , Proteínas Portadoras , ADN Helicasas , Inmunidad Innata , Complejo de la Endopetidasa Proteasomal , Estrés Fisiológico , Humanos , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/enzimología , Adenovirus Humanos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , ADN Helicasas/metabolismo , Interferones/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Cuaternaria de Proteína , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Replicación ViralRESUMEN
Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvß3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.
Asunto(s)
Adenovirus Humanos , Humanos , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Línea Celular , Células Endoteliales/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismoRESUMEN
IMPORTANCE: The architecture of sub-nuclear structures of eucaryotic cells is often changed during the infectious cycle of many animal and plant viruses. Cajal bodies (CBs) form a major sub-nuclear structure whose functions may include the regulation of cellular RNA metabolism. During the lifecycle of human adenovirus 5 (Ad5), CBs are reorganized from their spherical-like structure into smaller clusters termed microfoci. The mechanism of this reorganization and its significance for virus replication has yet to be established. Here we show that the major CB protein, p80-coilin, facilitates the nuclear export of Ad5 transcripts. Depletion of p80-coilin by RNA interference led to lowered levels of viral proteins and infectious virus. p80-coilin was found to form a complex with the viral L4-22K protein in Ad5-infected cells and in some reorganized microfoci. These findings assign a new role for p80-coilin as a potential regulator of infection by a human DNA virus.
Asunto(s)
Infecciones por Adenoviridae , Adenovirus Humanos , Animales , Humanos , Adenoviridae/genética , Adenoviridae/metabolismo , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular , Cuerpos Enrollados/genética , Cuerpos Enrollados/metabolismo , Infecciones por Adenoviridae/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismoRESUMEN
The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.
Asunto(s)
Adenovirus Humanos , Proteínas Oncogénicas Virales , Animales , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Transformación Celular Neoplásica/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas Oncogénicas Virales/metabolismo , ADN , Mamíferos/genéticaRESUMEN
Out of the three core proteins in human adenovirus, protein V is believed to connect the inner capsid surface to the outer genome layer. Here, we explored mechanical properties and in vitro disassembly of particles lacking protein V (Ad5-ΔV). Ad5-ΔV particles were softer and less brittle than the wild-type ones (Ad5-wt), but they were more prone to release pentons under mechanical fatigue. In Ad5-ΔV, core components did not readily diffuse out of partially disrupted capsids, and the core appeared more condensed than in Ad5-wt. These observations suggest that instead of condensing the genome, protein V antagonizes the condensing action of the other core proteins. Protein V provides mechanical reinforcement and facilitates genome release by keeping DNA connected to capsid fragments that detach during disruption. This scenario is in line with the location of protein V in the virion and its role in Ad5 cell entry.
Asunto(s)
Adenovirus Humanos , Cápside , Humanos , Cápside/metabolismo , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Adenoviridae/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Adenovirus Humanos/metabolismoRESUMEN
Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.
Asunto(s)
Adenovirus Humanos , Condensados Biomoleculares , Proteínas Virales , Humanos , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Adenovirus Humanos/química , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismoRESUMEN
The emergence and re-emergence of viruses has highlighted the need to develop new broad-spectrum antivirals to mitigate human infections. Pursuing our search for new bioactive plant-derived molecules, we study several diterpene derivatives synthesized from jatropholones A and B and carnosic acid isolated from Jatropha isabellei and Rosmarinus officinalis, respectively. Here, we investigate the antiviral effect of the diterpenes against human adenovirus (HAdV-5) that causes several infections for which there is no approved antiviral therapy yet. Ten compounds are evaluated and none of them present cytotoxicity in A549 cells. Only compounds 2, 5 and 9 inhibit HAdV-5 replication in a concentration-dependent manner, without virucidal activity, whereas the antiviral action takes place after virus internalization. The expression of viral proteins E1A and Hexon is strongly inhibited by compounds 2 and 5 and, in a lesser degree, by compound 9. Since compounds 2, 5 and 9 prevent ERK activation, they might exert their antiviral action by interfering in the host cell functions required for virus replication. Besides, the compounds have an anti-inflammatory profile since they significantly inhibit the levels of IL-6 and IL-8 produced by THP-1 cells infected with HAdV-5 or with an adenoviral vector. In conclusion, diterpenes 2, 5 and 9 not only exert antiviral activity against adenovirus but also are able to restrain pro-inflammatory cytokines induced by the virus.
Asunto(s)
Infecciones por Adenoviridae , Adenovirus Humanos , Diterpenos , Humanos , Antivirales/farmacología , Adenoviridae , Adenovirus Humanos/metabolismo , Diterpenos/farmacología , Replicación ViralRESUMEN
BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow, the incidence of which has further increased in recent years. In multiple myeloma, wild-type functional p53 is often inactivated or dysregulated. Therefore, this study aimed to investigate the role of p53 knockdown or overexpression in multiple myeloma and the therapeutic effect of recombinant adenovirus-p53 (rAd-p53) in combination with Bortezomib. METHODS: SiRNA p53 and rAd-p53 were used to knock down and overexpress p53. RT-qPCR was used to detect gene expression, and western blotting (WB) was used to detect protein expression levels. We also constructed wild-type multiple myeloma cell line-MM1S cell xenograft tumor models and explored the effects of siRNA-p53, rAd-p53, and Bortezomib on multiple myeloma in vivo and in vitro. H&E staining and KI67 immunohistochemical staining were used to assess the anti-myeloma effects of recombinant adenovirus and Bortezomib in vivo. RESULTS: The designed siRNA p53 effectively led to the knockdown of the p53 gene, while rAd-p53 could significantly achieve p53 overexpression. p53 gene inhibited MM1S cell proliferation and promoted apoptosis of wild-type multiple myeloma cell line MM1S. P53 gene inhibited tumor proliferation in vitro by promoting p21 expression and reducing cell cycle protein B1 expression of MM1S. P53 gene overexpression could inhibit tumor growth in vivo. Injection of rAd-p53 in tumor models inhibited tumor development through p21- and cyclin B1-mediated cell proliferation and apoptosis regulation. CONCLUSIONS: We found that overexpression of p53 inhibits MM tumor cell survival and proliferation in vivo and in vitro. Furthermore, the combination of rAd-p53 and Bortezomib significantly improved the efficacy, which provides a new possibility for more effective treatment of MM.
Asunto(s)
Adenovirus Humanos , Mieloma Múltiple , Humanos , Bortezomib/farmacología , Bortezomib/uso terapéutico , Mieloma Múltiple/terapia , Mieloma Múltiple/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Proliferación Celular , Adenoviridae/genética , Adenoviridae/metabolismo , Apoptosis , ARN Interferente PequeñoRESUMEN
Assembly of the adenovirus capsid protein hexon depends on the assistance of the molecular chaperone L4-100K. However, the chaperone mechanisms remain unclear. In this study, we found that L4-100K was involved in the hexon translation process and could prevent hexon degradation by the proteasome in cotransfected human cells. Two nonadjacent domains, 84-133 and 656-697, at the N-terminal and C-terminal regions of human adenovirus type 5 L4-100K, respectively, were found to be crucial and cooperatively responsible for hexon trimer expression and assembly. These two chaperone-related domains were conserved in the sequence of L4-100K and in the function of hexon assembly across different adenovirus serotypes. Different degrees of cross-activity of hexon trimerization with different serotypes were detected in subgroups B, C, and D, which were proven to be controlled by the interaction between the C-terminal chaperone-related domain of L4-100K and hypervariable regions (HVR) of hexon. Additionally, HVR-chimeric hexon mutants were successfully assembled with the assistance of the 1-697 mutant. Structural analysis of 656-697 by nuclear magnetic resonance and structural prediction of L4-100K using Robetta showed that the two conserved domains are mainly composed of α-helices and are located on the surface of the highly folded core region. Our research provides a more complete understanding of hexon assembly and guidance for the development of hexon-chimeric adenovirus vectors that will be safer, smarter, and more efficient. IMPORTANCE Adenovirus vectors have been widely used in clinical trials of vaccines and gene therapy, although some deficiencies remain. Chimeric modification of the hexon was expected to improve the potency of preexisting immune evasion and targeting, but in many cases, viral packaging is prevented by the inability of the chimeric hexon to assemble correctly. So far, few studies have examined the mechanisms of hexon trimer assembly. Here, we show how the chaperone protein L4-100K contributes to the assembly of the adenovirus capsid protein hexon, and these data will provide a guide for novel adenovirus vector design and development, as we desired.
Asunto(s)
Adenovirus Humanos , Chaperonas Moleculares , Proteínas no Estructurales Virales , Humanos , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
INTRODUCTION: Treatment of head and neck squamous cell carcinomas (HNSCC) by oncolytic adenoviral vectors holds promise as an efficient anti-cancer therapy. The epidermal growth factor receptor (EGFR) represents an attractive target receptor since it is frequently overexpressed in many types of HNSCC. METHODS: To achieve EGFR-specific targeting by human adenovirus type 5 (HAdV-5) based vectors, the EGFR affinity ligand Affilin was covalently attached in a position specific manner either to the fiber or the hexon protein of the vector capsid. In vitro and in vivo studies investigated EGFR-specific cancer cell transduction, susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of Affilin-decorated vectors. RESULTS: Affilin-decorated vectors showed strongly enhanced and EGFR-specific cancer cell transduction in vitro and less susceptibility to known sequestration mechanisms of HAdV-5 particles. However, in vivo neither systemic nor intratumoral vector administration resulted in an improved transduction of EGFR-positive tumors. Comprehensive analyses indicated hampered EGFR-targeting by Affilin-decorated vectors was caused by rapid vector particle consumption due to binding to the murine EGFR, insufficient tumor vascularization and poor target accessibility for Affilin in the solid tumor caused by a pronounced tumor stroma. CONCLUSION: In vitro studies yielded proof-of-concept results demonstrating that covalent attachment of a receptor-specific Affilin to the adenoviral capsid provides an effective and versatile tool to address cancer-specific target receptors by adenoviral vectors. Regarding EGFR as the vector target, off-target tissue transduction and low receptor accessibility within the tumor tissue prevented efficient tumor transduction by Affilin-decorated vectors, rendering EGFR a difficult-to-target receptor for adenoviral vectors.
Asunto(s)
Adenovirus Humanos , Neoplasias de Cabeza y Cuello , Viroterapia Oncolítica , Carcinoma de Células Escamosas de Cabeza y Cuello , Animales , Humanos , Ratones , Adenovirus Humanos/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Terapia Genética/métodos , Neoplasias de Cabeza y Cuello/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Distribución Tisular , Transducción GenéticaRESUMEN
It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.
Asunto(s)
Adenovirus Humanos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Replicación Viral , Proteínas Virales/genética , Perfilación de la Expresión Génica , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Severe pneumonia related to human adenoviruses (HAdVs) has a high lethality rate in children and its early diagnosis and treatment remain a major challenge. Circular RNAs (circRNAs) are novel long noncoding RNAs that play important roles in gene regulation and disease pathogenesis. To investigate the roles of circRNAs in HAdV pneumonia, we analyzed the circRNA profiles of healthy children and children with HAdV pneumonia, including both mild and severe cases, and identified 139 significantly upregulated circRNAs in children with HAdV pneumonia vs healthy controls and 18 significantly upregulated circRNAs in children with severe HAdV pneumonia vs mild HAdV pneumonia. In particular, hsa_circ_0002171 was differentially expressed in both groups and might thus be useful as a diagnostic biomarker of HAdV pneumonia and severe HAdV pneumonia. To identify the underlying mechanisms of circRNAs in HAdV pneumonia, we analyzed the transcriptome of children with HAdV pneumonia and established a circRNA-mRNA regulatory network. Enrichment analysis of differentially expressed target mRNAs demonstrated that the differentially expressed genes between healthy controls and HAdV pneumonia patients were mainly involved in RNA splicing while the differentially expressed genes between children with mild and severe HAdV pneumonia were mainly involved in regulating lymphocyte activation. Receiver operating characteristic (ROC) curve analysis suggested that hsa_circ_0002171 had a significant value in the diagnosis of HAdV pneumonia and of severe HAdV pneumonia. Taken together, the circRNA expression profile was altered in children with HAdV pneumonia. These results demonstrated that hsa_circ_0002171 is a potential diagnostic biomarker of HAdV pneumonia.
Asunto(s)
Adenovirus Humanos , Neumonía , Niño , Humanos , ARN Circular/genética , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Biomarcadores , Curva ROC , ARN Mensajero/genética , ARN/genéticaRESUMEN
OBJECTIVE: To investigate the effect of silencing CD46 and desmoglein 2 (DSG2) in host A549 cells on the entry of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and host cell secretion of inflammatory cytokines. METHODS: RNA interference technique was use to silence the expression of CD46 or DSG2 in human epithelial alveolar A549 cells as the host cells of HAdV-3 or HAdV-7. The binding of the viruses with CD46 and DSG2 were observed with immunofluorescence staining at 0.5 and 1 h after viral infection. The viral load in the host cells was determined with qRT-PCR, and IL-8 secretion level was measured using ELISA. RESULTS: In infected A549 cells, immunofluorescent staining revealed colocalization of HAdV-3 and HAdV-37 with their receptors CD46 and DSG2 at 0.5 h and 2 h after infection, and the copy number of the viruses increased progressively after the infection in a time-dependent manner. In A549 cells with CD46 silencing, the virus titers were significantly lower at 2, 6, 12 and 24 h postinfection in comparison with the cells without gene silencing; the virus titers were also significantly decreased in the cells with DSG2 silencing. The secretion level of IL-8 increased significantly in A549 cells without siRNA transfection following infection with HAdV-3 and HAdV-7 (P < 0.0001), but decreased significantly in cells with CD46 and DSG2 silencing (P < 0.0001). CONCLUSION: HAdV-3 and HAdV-7 enter host cells by binding to their receptors CD46 and DSG2, and virus titer and cytokines release increase with infection time. Silencing CD46 and DSG2 can inhibit virus entry and cytokine IL-8 production in host cells.
Asunto(s)
Adenovirus Humanos , Células A549 , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Desmogleína 2/genética , Desmogleína 2/metabolismo , Humanos , Interleucina-8 , Proteína Cofactora de Membrana/genética , ARN Interferente PequeñoRESUMEN
Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvß3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvß3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvß3 integrin-mediated HAdV26 infection. Regardless of αvß3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector. IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvß3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.