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1.
Cell ; 174(6): 1522-1536.e22, 2018 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-30146161

RESUMEN

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.


Asunto(s)
Cromatina/metabolismo , Genoma Humano , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Sitios de Unión , Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Flavonoides/farmacología , Humanos , Interferón beta/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperidinas/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Cohesinas
2.
Trends Biochem Sci ; 46(7): 519-521, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33895084

RESUMEN

The flavivirus genus consists of several major human pathogens including dengue (DENV) and Zika viruses. The flavivirus nonstructural protein 1 (NS1) plays an important role in disease progression, for example, in the development of severe dengue disease. Anti-NS1 antibodies have been shown to confer protection, and two new studies by Biering et al. and Modhiran et al. on the structure of NS1:antibody complexes reveal their mechanism of neutralization.


Asunto(s)
Virus del Dengue , Dengue , Flavivirus , Infección por el Virus Zika , Virus Zika , Anticuerpos Antivirales , Virus del Dengue/inmunología , Humanos , Proteínas no Estructurales Virales
3.
J Virol ; 98(5): e0011624, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38591880

RESUMEN

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.


Asunto(s)
Autofagia , Barrera Hematoencefálica , Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Proteínas no Estructurales Virales , Animales , Humanos , Ratones , Barrera Hematoencefálica/virología , Barrera Hematoencefálica/metabolismo , Encéfalo/virología , Encéfalo/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Encefalitis Japonesa/virología , Encefalitis Japonesa/metabolismo , Células Endoteliales/virología , Células Endoteliales/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , FN-kappa B/metabolismo , Proteínas no Estructurales Virales/metabolismo
4.
J Virol ; 98(3): e0169523, 2024 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-38349085

RESUMEN

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.


Asunto(s)
Lisina Acetiltransferasa 5 , Infecciones por Parvoviridae , Animales , Perros , Acetilación , Acetiltransferasas/metabolismo , Cromatina , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Infecciones por Parvoviridae/metabolismo , Infecciones por Parvoviridae/veterinaria , Infecciones por Parvoviridae/virología , Tirosina/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/virología , Lisina Acetiltransferasa 5/metabolismo
5.
J Virol ; 98(5): e0190123, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38629840

RESUMEN

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.


Asunto(s)
Virus de la Influenza A , Proteínas no Estructurales Virales , Humanos , Células A549 , Interacciones Huésped-Patógeno , Virus de la Influenza A/genética , Gripe Humana/virología , Gripe Humana/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Replicación Viral , Interacciones Huésped-Parásitos
6.
J Virol ; 98(5): e0009324, 2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38591899

RESUMEN

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.


Asunto(s)
Virus de la Panleucopenia Felina , Proteínas no Estructurales Virales , Replicación Viral , Animales , Gatos , Línea Celular , Virus de la Panleucopenia Felina/genética , Virus de la Panleucopenia Felina/inmunología , Inmunidad Innata , Mutación , Infecciones por Parvoviridae/virología , Infecciones por Parvoviridae/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/inmunología
7.
J Infect Dis ; 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38842497

RESUMEN

BACKGROUND: Dengue vascular permeability syndrome is the primary cause of death in severe dengue infections. The protective versus potentially pathogenic role of dengue NS1 antibodies are not well understood. The main goal of this analysis was to characterize the relationship between free NS1 concentration and NS1 antibody titers in primary and secondary dengue infection in order to better understand the presence and duration of NS1 antibody complexes in clinical dengue infections. METHODS: Hospitalized participants with acute dengue infection were recruited from Northern Colombia between 2018 to 2020. Symptom assessment including dengue signs and symptoms, chart review and blood collection was performed. Primary versus secondary Dengue was assessed serologically. NS1 titers and anti-NS1 antibodies were measured daily. RESULTS: Patients with secondary infection have higher antibody titers than those in primary infection, and we find a negative correlation between anti-NS1 antibody titer and NS1 protein. We demonstrate that in a subset of secondary infection, there are indeed NS1 antibody-antigen complexes at the admission day during the febrile phase that are not detectable by the recovery phase. Furthermore, dengue infection status is associated with higher circulating sialidases. DISCUSSION: The negative correlation between antibody and protein suggests that antibodies may play a role in clearing this viral protein.

8.
J Infect Dis ; 2024 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-38478732

RESUMEN

BACKGROUND: Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. METHODS: We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. RESULTS: We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. CONCLUSIONS: These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

9.
J Proteome Res ; 2024 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-39132695

RESUMEN

Dengue fever is a rapidly emerging tropical disease and an important cause of morbidity in its severe form worldwide. A wide spectrum of the pathophysiology is associated with the transition of dengue fever to severe dengue, which is driven by the host immune response and might reflect in patients' proteome profile. This study aims to analyze the plasma from different phases of dengue-infected patients at two time points. A mass-spectrometry-based proteomic approach was utilized to understand the involvement of probable candidate proteins toward developing a more severe, hemorrhagic form of dengue fever. Dengue-infected hospital-admitted patients with <5 days of fever were included in this study. Patient samples from the acute phase were screened for the presence of NS1 antigen using ELISA and subjected to molecular serotyping. Dengue molecular serotype-confirmed patient samples, pairwise from acute and critical phases with healthy control were subjected to qualitative and quantitative proteomic analysis, and then pathway analysis was performed. The protein-protein interaction network between the dengue virus and host proteins was depicted in the search for proteins associated with severe dengue pathophysiology. An array of apolipoprotein, cytokines, and endothelial proteins in association with virus replication and endothelial dysfunction were validated as biomolecules involved in severe dengue pathophysiology.

10.
Mol Biol Evol ; 40(3)2023 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-36795614

RESUMEN

Non-structural protein (NS1) is a 350 amino acid long conserved protein in the dengue virus. Conservation of NS1 is expected due to its importance in dengue pathogenesis. The protein is known to exist in dimeric and hexameric states. The dimeric state is involved in its interaction with host proteins and viral replication, and the hexameric state is involved in viral invasion. In this work, we performed extensive structure and sequence analysis of NS1 protein, and uncovered the role of NS1 quaternary states in its evolution. A three-dimensional modeling of unresolved loop regions in NS1 structure is performed. "Conserved" and "Variable" regions within NS1 protein were identified from sequences obtained from patient samples and the role of compensatory mutations in selecting destabilizing mutations were identified. Molecular dynamics (MD) simulations were performed to extensively study the effect of a few mutations on NS1 structure stability and compensatory mutations. Virtual saturation mutagenesis, predicting the effect of every individual amino acid substitution on NS1 stability sequentially, revealed virtual-conserved and variable sites. The increase in number of observed and virtual-conserved regions across NS1 quaternary states suggest the role of higher order structure formation in its evolutionary conservation. Our sequence and structure analysis could enable in identifying possible protein-protein interfaces and druggable sites. Virtual screening of nearly 10,000 small molecules, including FDA-approved drugs, permitted us to recognize six drug-like molecules targeting the dimeric sites. These molecules could be promising due to their stable interactions with NS1 throughout the simulation.


Asunto(s)
Dengue , Mutación , Biología Computacional , Proteínas no Estructurales Virales/genética
11.
Biochem Biophys Res Commun ; 690: 149312, 2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38016247

RESUMEN

Zika virus (ZIKV), has gained global attention due to its association with severe disorders, including microcephaly and congenital Zika syndrome. We investigated the role of ZIKV nonstructural protein 1 (NS1) in altering the host's antioxidant response. Using a stable cell line expressing NS1, we found that NS1 significantly reduced the expression of antioxidant-related genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and sequestosome-1 (SQSTM1), which are regulated NRF2. Interestingly, this effect was attributed to increased expression of BACH1, a factor that competes with NRF2 for binding to certain antioxidant responsive elements (ARE). Thus, ZIKV NS1-mediated disruption of the antioxidant system is linked to BACH1 overexpression. These findings offer insights into ZIKV pathogenesis and suggest potential therapeutic strategies targeting the NRF2-BACH1 axis.


Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/metabolismo , Antioxidantes , Factor 2 Relacionado con NF-E2/metabolismo , Línea Celular , Proteínas no Estructurales Virales/genética
12.
Biochem Biophys Res Commun ; 706: 149728, 2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38479246

RESUMEN

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.


Asunto(s)
Virus de la Influenza A , eIF-2 Quinasa , Animales , Humanos , eIF-2 Quinasa/metabolismo , Proteínas no Estructurales Virales/química , Virus de la Influenza A/genética , Microscopía por Crioelectrón , Línea Celular , Antivirales/metabolismo , Replicación Viral , Mamíferos/metabolismo
13.
J Virol ; 97(5): e0033723, 2023 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-37166301

RESUMEN

In the influenza virus life cycle, viral RNA (vRNA) transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the viral RNA-dependent RNA polymerase in the host cell nucleus, are delicately controlled, and the levels of the three viral RNA species display very distinct synthesis dynamics. However, the underlying mechanisms remain elusive. Here, we demonstrate that in the context of virus infection with cycloheximide treatment, the expression of viral nonstructural protein 1 (NS1) can stimulate primary transcription, while the expression of viral NS2 inhibits primary transcription. It is known that the NS1 and NS2 proteins are expressed with different timings from unspliced and spliced mRNAs of the viral NS segment. We then simulated the synthesis dynamics of NS1 and NS2 proteins during infection by dose-dependent transfection experiments in ribonucleoprotein (RNP) reconstitution systems. We found that the early-expressed NS1 protein can stimulate viral mRNA synthesis, while the late-expressed NS2 protein can inhibit mRNA synthesis but can promote vRNA synthesis in a manner highly consistent with the dynamic changes in mRNA/vRNA in the virus life cycle. Furthermore, we observed that the coexistence of sufficient NS1 and NS2, close to the status of the NS1 and NS2 levels in the late stage of infection, could boost vRNA synthesis to the highest efficiency. We also identified key functional amino acids of NS1 and NS2 involved in these regulations. Together, we propose that the stoichiometric changes in the viral NS1 and NS2 proteins during infection are responsible for the fine regulation of viral RNA transcription and replication. IMPORTANCE In order to ensure efficient multiplication, influenza virus transcribes and replicates its segmented, negative-sense viral RNA genome in highly ordered dynamics across the virus life cycle. How the virus achieves such regulation remains poorly understood. Here, we demonstrate that the stoichiometric changes in the viral NS1 and NS2 proteins during infection could be responsible for the fine regulation of the distinct dynamics of viral RNA transcription and replication. We thus propose a fundamental mechanism exploited by influenza virus to dynamically regulate the synthesis of its viral RNA through the delicate control of viral NS1 and NS2 protein expression.


Asunto(s)
Virus de la Influenza A , Orthomyxoviridae , Proteínas no Estructurales Virales , Virus de la Influenza A/metabolismo , Orthomyxoviridae/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genética
14.
FASEB J ; 37(9): e23126, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37594040

RESUMEN

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.


Asunto(s)
Trampas Extracelulares , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Enfermedades Neuroinflamatorias , Nervio Ciático
15.
J Biomed Sci ; 31(1): 60, 2024 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-38849802

RESUMEN

BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.


Asunto(s)
Proteínas no Estructurales Virales , Replicación Viral , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Glicosilación , Humanos , Animales , Compartimentos de Replicación Viral/metabolismo , Células HeLa , Chlorocebus aethiops , Flavivirus/fisiología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Células Vero
16.
Virus Genes ; 60(1): 9-17, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37938470

RESUMEN

Most wild strains of Japanese encephalitis virus (JEV) produce NS1' protein, which plays an important role in viral infection and immune escape. The G66A nucleotide mutation in NS2A gene of the wild strain SA14 prevented the ribosomal frameshift that prevented the production of NS1' protein, thus reduced the virulence. In this study, the 66th nucleotide of the NS2A gene of SA14 was mutated into A, U or C, respectively. Both the G66U and G66C mutations cause the E22D mutation of the NS2A protein. Subsequently, the expression of NS1' protein, plaque size, replication ability, and virulence to mice of the three mutant strains were examined. The results showed that the three mutant viruses could not express NS1' protein, and their proliferation ability in nerve cells and virulence to mice were significantly reduced. In addition, the SA14(G66C) was less virulent than the other two mutated viruses. Our results indicate that only when G is the 66th nucleotide of NS2A, the JEV can produce NS1' protein, which affects the virulence.


Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Animales , Ratones , Virus de la Encefalitis Japonesa (Especie)/genética , Nucleótidos/metabolismo , Virulencia/genética , Línea Celular , Proteínas no Estructurales Virales/metabolismo , Proliferación Celular
17.
Mol Divers ; 2024 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-39017952

RESUMEN

Dengue fever is a serious health hazard on a global scale and its primary causative agent is the dengue virus (DENV). The non-structural protein 1 (NS1) of DENV plays a pivotal role in pathogenesis. It is associated with several autoimmune events, endothelial cell apoptosis, and vascular leakage, which increase mainly during the critical phase of infection. In this study, important residues of the oligomerization domain of NS1 protein were identified by literature searches. Virtual screening has been conducted using the entire dataset of the DrugBank database and the potential small-molecule inhibitors against the NS1 protein have been chosen on the basis of binding energy values. This is succeeded by molecular dynamics (MD) simulations of the shortlisted compounds, ultimately giving rise to five compounds. These five compounds were further subjected to RAMD simulations by applying a random direction force of specific magnitude on the ligand center of mass in order to push the ligand out of the protein-binding pocket, for the quantitative estimation of their binding energy values to determine the interaction strength between protein and ligand which prevents ligand unbinding from its binding site, ultimately leading to the selection of three major compounds, DB00826 (Natamycin), DB11274 (Dihydro-alphaergocryptine), and DB11275 (Epicriptine), with the DB11274 having a role against idiopathic Parkinson's disease, and thus may have possible important roles in the prevention of dengue-associated Parkinsonism. These compounds may act as prospective drugs against dengue, by preventing the oligomerization of the NS1 protein, thereby preventing disease progression and pathogenesis.

18.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-34479996

RESUMEN

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.


Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Virus de la Influenza A/genética , Neuraminidasa/metabolismo , Células A549 , Retículo Endoplásmico/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología , Proteínas no Estructurales Virales/genética , Replicación Viral/genética
19.
Mikrochim Acta ; 191(1): 72, 2024 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-38170245

RESUMEN

Non-structural 1 (NS1) is a protein biomarker that can be found in blood in the early stages of dengue and related infections (Zika and Chikungunya). This study aims to develop a biosensor to selectively quantify NS1 using DNA aptamer co-immobilized on gold electrodes with 6-(ferrocenyl)hexanethiol (FCH) using electrochemical capacitive spectroscopy. This technique uses a redox probe (FCH) immobilized on the self-assembled monolayer to convert impedance into capacitance information. The developed platform was blocked with bovine serum albumin before NS1 exposure and the ratio between aptamers and FCH was optimized. The aptasensor was tested using commercial NS1 serotype 4 in phosphate-buffered saline and commercial undiluted human serum. Using the optimum applied potential provides high sensitivity (3 and 4 nF per decade) and low limit of detection (30.9 and 41.8 fg/mL) with a large linear range (10 pg to 1 µg/mL and 10 pg to 100 ng/mL, respectively). Both results exhibit a residual standard deviation value < 1%. The results suggested that this aptasensor was capable of detecting NS1 in the clinical range and can be applied to any other specific aptamer with FCH, opening the path for label-free miniaturized point-of-care devices with high sensitivity and specificity.


Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Dengue , Infección por el Virus Zika , Virus Zika , Humanos , Límite de Detección , Aptámeros de Nucleótidos/química , Espectroscopía Dieléctrica/métodos , Técnicas Biosensibles/métodos , Dengue/diagnóstico
20.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-38473707

RESUMEN

Influenza type A virus (IAV) infection is a major cause of morbidity and mortality during influenza epidemics. Recently, a specific link between IAV infection and neurodegenerative disease progression has been established. The non-structural NS1 protein of IAV regulates viral replication during infection and antagonizes host antiviral responses, contributing to influenza virulence. In the present study, we have prepared a mouse lung-to-lung adapted to the NS1-truncated virus (NS80ad). Transcriptome analysis of the gene expression in the lungs revealed that infection with wild-type A/WSN/33 (WSN), NS80, and NS80ad viruses resulted in different regulation of genes involved in signaling pathways associated with the cell proliferation, inflammatory response, and development of neurodegenerative diseases. NS1 protein did not influence the genes involved in the RIG-I-like receptor signaling pathway in the brains. Lethal infection with IAVs dysregulated expression of proteins associated with the development of neurodegenerative diseases (CX3CL1/Fractalkine, Coagulation factor III, and CD105/Endoglin, CD54/ICAM-1, insulin-like growth factor-binding protein (IGFBP)-2, IGFBP-5, IGFBP-6, chitinase 3-like 1 (CHI3L1), Myeloperoxidase (MPO), Osteopontin (OPN), cystatin C, and LDL R). Transcription of GATA3 mRNA was decreased, and expression of MPO was inhibited in the brain infected with NS80 and NS80ad viruses. In addition, the truncation of NS1 protein led to reduced expression of IGFBP-2, CHI3L1, MPO, and LDL-R proteins in the brains. Our results indicate that the influenza virus influences the expression of proteins involved in brain function, and this might occur mostly through the NS1 protein. These findings suggest that the abovementioned proteins represent a promising target for the development of potentially effective immunotherapy against neurodegeneration.


Asunto(s)
Virus de la Influenza A , Gripe Humana , Enfermedades Neurodegenerativas , Animales , Ratones , Humanos , Virus de la Influenza A/genética , Inmunidad Innata , Interacciones Huésped-Patógeno/genética , Encéfalo
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