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1.
Int J Mol Sci ; 23(14)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35887322

RESUMO

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.


Assuntos
Vírus Sincicial Respiratório Humano , Transporte Ativo do Núcleo Celular , Idoso , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Fosforilação
2.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30541831

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of hospitalization of infants and young children, causing considerable respiratory disease and repeat infections that may lead to chronic respiratory conditions such as asthma, wheezing, and bronchitis. RSV causes ∼34 million new episodes of lower respiratory tract illness (LRTI) in children younger than 5 years of age, with >3 million hospitalizations due to severe RSV-associated LRTI. The standard of care is limited to symptomatic relief as there are no approved vaccines and few effective antiviral drugs; thus, a safe and efficacious RSV therapeutic is needed. Therapeutic targeting of host proteins hijacked by RSV to facilitate replication is a promising antiviral strategy as targeting the host reduces the likelihood of developing drug resistance. The nuclear export of the RSV M protein, mediated by the nuclear export protein exportin 1 (XPO1), is crucial for RSV assembly and budding. Inhibition of RSV M protein export by leptomycin B correlated with reduced RSV replication in vitro In this study, we evaluated the anti-RSV efficacy of Verdinexor (KPT-335), a small molecule designed to reversibly inhibit XPO1-mediated nuclear export. KPT-335 inhibited XPO1-mediated transport and reduced RSV replication in vitro KPT-335 was effective against RSV A and B strains and reduced viral replication following prophylactic or therapeutic administration. Inhibition of RSV replication by KPT-335 was due to a combined effect of reduced XPO1 expression, disruption of the nuclear export of RSV M protein, and inactivation of the NF-κB signaling pathway.IMPORTANCE RSV is an important cause of LRTI in infants and young children for which there are no suitable antiviral drugs offered. We evaluated the efficacy of KPT-335 as an anti-RSV drug and show that KPT-335 inhibits XPO1-mediated nuclear export, leading to nuclear accumulation of RSV M protein and reduction in RSV levels. KPT-335 treatment also resulted in inhibition of proinflammatory pathways, which has important implications for its effectiveness in vivo.


Assuntos
Acrilamidas/farmacologia , Hidrazinas/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Acrilamidas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/imunologia , Humanos , Hidrazinas/metabolismo , Carioferinas/efeitos dos fármacos , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Células Vero , Proteína Exportina 1
3.
Parasitology ; 145(11): 1378-1387, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29565001

RESUMO

Entamoeba histolytica is the protozoan parasite that causes human amoebiasis. It is one of the leading parasitic disease burdens in tropical regions and developing countries, with spread to developed countries through migrants from and travellers to endemic regions.Understanding E. histolytica's invasion mechanisms requires an understanding of how it interacts with external cell components and how it engulfs and kills cells (phagocytosis). Recent research suggests that optimal phagocytosis requires signalling events from the cell surface to the nucleus via the cytoplasm, and the induction of several factors that are transported to the plasma membrane. Current research in other protozoans suggests the presence of proteins with nuclear localization signals, nuclear export signals and Ran proteins; however, there is limited literature on their functionality and their functional similarity to higher eukaryotes.Based on learnings from the development of antivirals, nuclear transport elements in E. histolytica may present viable, specific, therapeutic targets.In this review, we aim to summarize our limited knowledge of the eukaryotic nuclear transport mechanisms that are conserved and may function in E. histolytica.


Assuntos
Núcleo Celular/metabolismo , Entamoeba histolytica/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas de Ligação ao Cálcio/metabolismo , Citoplasma/metabolismo , Humanos , Proteína ran de Ligação ao GTP/metabolismo
4.
J Virol ; 90(24): 11032-11042, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27681132

RESUMO

The human rhinovirus (HRV) 3C and 2A proteases (3Cpro and 2Apro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cpro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Apro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cpro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3Cpro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Apro fusion proteins, we demonstrate formally that 2Apro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cpro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Apro activity and not 3Cpro activity, which is observed only later in infection. The results thus define the temporal activities of 2Apro and 3CD/3Cpro activities in HRV serotype16 infection. IMPORTANCE: The human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.


Assuntos
Cisteína Endopeptidases/genética , Citoplasma/virologia , Regulação Viral da Expressão Gênica , Rhinovirus/genética , Proteínas Virais/genética , Proteases Virais 3C , Núcleo Celular/virologia , Cisteína Endopeptidases/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Transporte Proteico , Proteólise , Rhinovirus/classificação , Rhinovirus/metabolismo , Sorogrupo , Proteínas Virais/metabolismo , Replicação Viral , Proteína Vermelha Fluorescente
5.
Virol J ; 13(1): 161, 2016 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-27670781

RESUMO

Respiratory syncytial virus (RSV) is one of the major pathogens responsible for lower respiratory tract infections (LRTI) in young children, the elderly, and the immunosuppressed. Currently, there are no antiviral drugs or vaccines available that effectively target RSV infections, proving a significant challenge in regards to prevention and treatment. An in-depth understanding of the host-virus interactions that underlie assembly and budding would inform new targets for antiviral development.Current research suggests that the polymerised form of actin, the filamentous or F-actin, plays a role in RSV assembly and budding. Treatment with cytochalasin D, which disrupts F-actin, has been shown to inhibit virus release. In addition, the actin cytoskeleton has been shown to interact with the RSV matrix (M) protein, which plays a central role in RSV assembly. For this reason, the interaction between these two components is hypothesised to facilitate the movement of viral components in the cytoplasm and to the budding site. Despite increases in our knowledge of RSV assembly and budding, M-actin interactions are not well understood. In this review, we discuss the current literature on the role of actin cytoskeleton during assembly and budding of RSV with the aim to integrate disparate studies to build a hypothetical model of the various molecular interactions between actin and RSV M protein that facilitate RSV assembly and budding.

6.
Front Microbiol ; 14: 1321531, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38249483

RESUMO

Human Rhinoviruses (RV) are a major cause of common colds and infections in early childhood and can lead to subsequent development of asthma via an as yet unknown mechanism. Asthma is a chronic inflammatory pulmonary disease characterized by significant airway remodeling. A key component of airway remodeling is the transdifferentiation of airway epithelial and fibroblast cells into cells with a more contractile phenotype. Interestingly, transforming growth factor-beta (TGF-ß), a well characterized inducer of transdifferentiation, is significantly higher in airways of asthmatics compared to non-asthmatics. RV infection induces TGF-ß signaling, at the same time nucleoporins (Nups), including Nup153, are cleaved by RV proteases disrupting nucleocytoplasmic transport. As Nup153 regulates nuclear export of SMAD2, a key intermediate in the TGF-ß transdifferentiation pathway, its loss of function would result in nuclear retention of SMAD2 and dysregulated TGF-ß signaling. We hypothesize that RV infection leads to increased nuclear SMAD2, resulting in sustained TGF-ß induced gene expression, priming the airway for subsequent development of asthma. Our hypothesis brings together disparate studies on RV, asthma and Nup153 with the aim to prompt new research into the role of RV infection in development of asthma.

7.
Phytomed Plus ; 2(3): 100286, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35474908

RESUMO

BACKGROUND: While Molnupiravir and Paxlovid have recently been approved for use in some countries, there are no widely available treatments for COVID-19, the disease caused by SARS-CoV-2 infection. Herbal extracts have been used to treat respiratory clinical indications by Ayurvedic medicine practitioners with minimal adverse reactions and intense research efforts are currently under way to develop some of these formulations for COVID-19 treatment. METHODS: Literature search for in silico, in vitro, in vivo, and clinical studies on the topic of Ayurvedic formulations for potential COVID-19 treatment, in order to present the current state of current knowledge by integrating information across all systems. RESULTS: The search yielded 20 peer reviewed articles on in silico studies examining the interaction of phytoconstituents of popular Ayurvedic formulations with SARS-CoV-2 components and its receptors; five articles on preclinical investigations of the ability of selected Ayurvedic formulations to inhibit functions of SARS-CoV-2 proteins; and 51 completed clinical trials on the efficacy of using Ayurvedic formulations for treatment of mild to moderate COVID-19. Clinical data was available from 17 of the 51 trials. There was a considerable overlap between formulations used in the in silico studies and the clinical trials. This finding was unexpected as there is no clearly stated alignment between studies and the traditional pathway to drug discovery- basic discovery leading to in vitro and in vivo proof of concept, followed by validation in clinical trials. This was further demonstrated in the majority of the in silico studies where focus was on potential antiviral mechanisms, while the clinical trials were focused on patient recovery using oral treatments. In all 17 clinical trials where data was available, Ayurvedic treatments lead to a shorter period to recovery in participants with COVID-19. CONCLUSION: The most commonly used Ayurvedic treatments for management of respiratory symptoms associated with SARS-CoV-2 infection appear to have prophylactic and/or therapeutic properties. It would be of particular interest to assess synergistic and concomitant systemic effects and antiviral activities of individual phytoconstituents and their combinations in the Ayurvedic treatments.

8.
Viruses ; 13(12)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34960671

RESUMO

Rhinoviruses (RV), like many other viruses, modulate programmed cell death to their own advantage. The viral protease, 3C has an integral role in the modulation, and we have shown that RVA-16 3C protease cleaves Receptor-interacting protein kinase-1 (RIPK1), a key host factor that modulates various cell death and cell survival pathways. In the current study, we have investigated whether this cleavage is conserved across selected RV strains. RIPK1 was cleaved in cells infected with strains representing diversity across phylogenetic groups (A and B) and receptor usage (major and minor groups). The cleavage was abrogated in the presence of the specific 3C protease inhibitor, Rupintrivir. Interestingly, there appears to be involvement of another protease (maybe 2A protease) in RIPK1 cleavage in strains belonging to genotype B. Our data show that 3C protease from diverse RV strains cleaves RIPK1, highlighting the importance of the cleavage to the RV lifecycle.


Assuntos
Proteases Virais 3C/metabolismo , Infecções por Picornaviridae/enzimologia , Rhinovirus/enzimologia , Proteases Virais 3C/genética , Antivirais/química , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Isoxazóis/química , Isoxazóis/farmacologia , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/virologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Rhinovirus/química , Rhinovirus/efeitos dos fármacos , Rhinovirus/genética , Valina/análogos & derivados , Valina/química , Valina/farmacologia
9.
Viruses ; 13(4)2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916958

RESUMO

Rhinoviruses (RVs) are the etiological agents of upper respiratory tract infections, particularly the common cold. Infections in the lower respiratory tract is shown to cause severe disease and exacerbations in asthma and COPD patients. Viruses being obligate parasites, hijack host cell pathways such as programmed cell death to suppress host antiviral responses and prolong viral replication and propagation. RVs are non-enveloped positive sense RNA viruses with a lifecycle fully contained within the cytoplasm. Despite decades of study, the details of how RVs exit the infected cell are still unclear. There are some diverse studies that suggest a possible role for programmed cell death. In this review, we aimed to consolidate current literature on the impact of RVs on cell death to inform future research on the topic. We searched peer reviewed English language literature in the past 21 years for studies on the interaction with and modulation of cell death pathways by RVs, placing it in the context of the broader knowledge of these interconnected pathways from other systems. Our review strongly suggests a role for necroptosis and/or autophagy in RV release, with the caveat that all the literature is based on RV-A and RV-B strains, with no studies to date examining the interaction of RV-C strains with cell death pathways.


Assuntos
Morte Celular , Infecções por Picornaviridae/virologia , Rhinovirus/patogenicidade , Replicação Viral , Autofagia , Humanos , Necroptose , Infecções por Picornaviridae/complicações , Rhinovirus/fisiologia
10.
Sci Rep ; 11(1): 19223, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584169

RESUMO

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract disease in infants, young children, the elderly and immunocompromised individuals. Therapy for RSV infections is limited to high risk infants and there are no safe and efficacious vaccines. Matrix (M) protein is a major RSV structural protein with a key role in virus assembly. Interestingly, M is localised to the nucleus early in infection and its export into the cytoplasm by the nuclear exporter, exportin-1 (XPO1) is essential for RSV assembly. We have shown previously that chemical inhibition of XPO1 function results in reduced RSV replication. In this study, we have investigated the anti-RSV efficacy of Selective Inhibitor of Nuclear Export (SINE) compounds, KPT-335 and KPT-185. Our data shows that therapeutic administration of the SINE compounds results in reduced RSV titre in human respiratory epithelial cell culture. Within 24 h of treatment, RSV replication and XPO1 expression was reduced, M protein was partially retained in the nucleus, and cell cycle progression was delayed. Notably, the effect of SINE compounds was reversible within 24 h after their removal. Our data show that reversible inhibition of XPO1 can disrupt RSV replication by affecting downstream pathways regulated by the nuclear exporter.


Assuntos
Acrilatos/farmacologia , Carioferinas/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Triazóis/farmacologia , Proteínas da Matriz Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Células A549 , Acrilatos/uso terapêutico , Núcleo Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/metabolismo , Triazóis/uso terapêutico , Proteína Exportina 1
11.
Front Microbiol ; 12: 715450, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335553

RESUMO

Hepatitis B virus (HBV) infection is a global public health problem that plagues approximately 240 million people. Chronic hepatitis B (CHB) often leads to liver inflammation and aberrant repair which results in diseases ranging from liver fibrosis, cirrhosis, to hepatocellular carcinoma. Despite its narrow species tropism, researchers have established various in vivo models for HBV or its related viruses which have provided a wealth of knowledge on viral lifecycle, pathogenesis, and immunity. Here we briefly revisit over five decades of endeavor in animal model development for HBV and summarize their advantages and limitations. We also suggest directions for further improvements that are crucial for elucidation of the viral immune-evasion strategies and for development of novel therapeutics for a functional cure.

12.
Curr Res Food Sci ; 4: 532-542, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34430875

RESUMO

Honey is a supersaturated sugar solution produced from plant nectar, with its composition influenced by geographic and floral origins, and with several properties contributing to its health-related abilities. This study aimed to determine the bioactive composition, antioxidant characteristics, antibacterial activity, and physicochemical properties of commercial Australian honeys. In total, 42 commercial Australian honeys were selected, and categorised according to front-label descriptions. Honeys were analysed: quality (Hydroxymethylfurfural); colour (colour intensity, L*,a*,b*); bioactive composition (phenolic, flavonoid, and carotenoid content); antioxidant characteristics (DPPH, CUPRAC, FRAP); antibacterial activity (MIC50); physicochemical properties (pH, TSS, viscosity, a w). Colour intensity correlated with each assessed bioactive compound and antioxidant characteristic (p ≤ 0.001). MIC50 (S. aureus) was associated with FRAP and a w, suggesting mechanisms of action for honey's antibacterial activity. Manuka-type honeys had higher colour intensity (1440 (98.5) mAU) than other categories (p ≤ 0.05), and consistently higher bioactive and antioxidant properties. This provides the potential to inform antioxidant-related health outcomes.

13.
Cells ; 10(10)2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34685766

RESUMO

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110-183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M's RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.


Assuntos
Cromatina/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Gênica , Proteínas da Matriz Viral/metabolismo , Animais , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , DNA Viral/metabolismo , Modelos Animais de Doenças , Humanos , Lisina/metabolismo , Camundongos Endogâmicos BALB C , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Domínios Proteicos , RNA Viral/metabolismo , Células Vero , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Viremia/virologia
14.
Life Sci ; 265: 118750, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33188836

RESUMO

BACKGROUND: Lipopolysaccharide (LPS) is an endotoxin that leads to inflammation in many organs, including liver. It binds to pattern recognition receptors, that generally recognise pathogen expressed molecules to transduce signals that result in a multifaceted network of intracellular responses ending up in inflammation. Aim In this study, we used lauric acid (LA), a constituent abundantly found in coconut oil to determine its anti-inflammatory role in LPS-induced liver inflammation in Sprague Dawley (SD) rats. METHOD: Male SD rats were divided into five groups (n = 8), injected with LPS and thereafter treated with LA (50 and 100 mg/kg) or vehicle orally for 14 days. After fourteen days of LA treatment, all the groups were humanely killed to investigate biochemical parameters followed by pro-inflammatory cytokine markers; tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß. Moreover, liver tissues were harvested for histopathological studies and evaluation of targeted protein expression with western blot and localisation through immunohistochemistry (IHC). RESULTS: The study results showed that treatment of LA 50 and 100 mg/kg for 14 days were able to reduce the elevated level of pro-inflammatory cytokines, liver inflammation, and downregulated the expression of TLR4/NF-κB mediating proteins in liver tissues. CONCLUSION: These findings suggest that treatment of LA has a protective role against LPS-induced liver inflammation in rats, thus, warrants further in-depth investigation through mechanistic approaches in different study models.


Assuntos
Inflamação/tratamento farmacológico , Ácidos Láuricos/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocinas/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ácidos Láuricos/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
J Virol ; 83(11): 5353-62, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19297465

RESUMO

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin beta1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.


Assuntos
Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Proteínas da Matriz Viral/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Chlorocebus aethiops , Citoplasma/metabolismo , Mutação/genética , Vírus Sinciciais Respiratórios/genética , Células Vero , Proteínas da Matriz Viral/genética , Replicação Viral , Proteína Exportina 1
16.
J Virol ; 83(14): 7349-52, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19403669

RESUMO

The degradation of nuclear pore components and disruption of nucleocytoplasmic trafficking during rhinovirus infection have been attributed to viral 2A protease. Here we show for the first time that rhinovirus 3C protease may also have a role. Specifically, we show that 3C and its precursor, 3CD, can target green fluorescent protein to the nucleus of living cells, leading to degradation of nuclear pore components, and that incubation with recombinant 3C disrupts active and passive nucleocytoplasmic transport in a semi-intact cell nuclear transport system dependent on 3C protease activity. 3C may thus contribute to host cell shutoff in infected cells by localizing in the nucleus and facilitating nuclear pore breakdown.


Assuntos
Núcleo Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Infecções por Picornaviridae/metabolismo , Rhinovirus/enzimologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Transporte Ativo do Núcleo Celular , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Cisteína Endopeptidases/genética , Humanos , Infecções por Picornaviridae/virologia , Transporte Proteico , Rhinovirus/genética , Proteínas Virais/genética
17.
J Immunol ; 181(10): 7356-66, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18981159

RESUMO

Despite steady progress in elimination of measles virus globally, measles infection still causes 500,000 annual deaths, mostly in developing countries where endemic measles strains still circulate. Many adults are infected every year in China, with symptoms more severe than those observed in children. In this study, we have used blood samples from adult measles patients in Shanghai and age-matched healthy controls to gain an understanding of the immune status of adult measles patients. IFN-alpha mRNA was reduced in patient PBMC compared with healthy controls. In contrast, gene expression and plasma production of IL-2, IL-10, and IFN-gamma were elevated in patient blood. A similar cytokine profile was observed at early times when cultured PBMC were infected with a clinical isolate of measles virus. In contrast to previous studies in pediatric patients, we did not find a reduction in total CD4(+) and CD8(+) T cells in patient PBMC. Interestingly, we found that CD4(+)CD25(+)CD127(low) regulatory T cells were significantly increased in patient PBMC compared with controls. Using intracellular cytokine staining we also show that the measles virus induces IL-10-producing CD14(+) and CD4(+)CD25(+) cells in PBMC. Our results show that adult measles patients in the acute phase of the disease have a mixed Th1/Th2 type response, accompanied with severe immunosuppression of both innate and adaptive responses including suppression of type I IFN. Both regulatory T cells and plasma IL-10 may contribute to the immunosuppression.


Assuntos
Interleucina-10/biossíntese , Sarampo/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Leucócitos Mononucleares/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Am J Respir Cell Mol Biol ; 41(3): 339-47, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19168696

RESUMO

Individuals with asthma are prone to viral and bacterial infections, and most asthma exacerbations have been linked to viruses, particularly rhinovirus. Excess transforming growth factor (TGF)-beta present in asthmatic airways may cause immune suppression, as well as transdifferentiate fibroblasts to myofibroblasts, thereby augmenting proinflammatory responses after rhinovirus infection. After rhinovirus infection we examined virus replication and host cell immune responses in airway fibroblasts in the presence of TGF-beta1 and in myofibroblasts. Primary culture fibroblasts were pretreated with TGF-beta1 or transdifferentiated into myofibroblasts, and then infected with rhinovirus. Viral replication, virus release, chemokine production, and interferon (IFN) responses were measured over 72 hours. Rhinovirus replication and virus release into supernatants were enhanced in fibroblasts incubated with TGF-beta1 and in fibroblasts obtained from patients with asthma. Myofibroblasts also showed more rhinovirus replication, and infected myofibroblasts produced excess neutrophil chemokines. Examination of innate responses revealed blunting of type I IFN reactions with dissociated viral RNA and IFN mRNA responses. Addition of type I IFN restituted antiviral responses, and the effect of TGF-beta1 appeared to be mediated via actions on IFN regulatory factor-3 pathways. These data demonstrate that TGF-beta1 mediates enhanced virus replication and proinflammatory responses in airway cells. TGF-beta may act as an endogenous immunosuppressant promoting virus replication and inflammation during the evolution of acute severe asthma associated with rhinovirus infection.


Assuntos
Imunidade Inata/fisiologia , Infecções por Picornaviridae/imunologia , Rhinovirus/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Asma/imunologia , Asma/virologia , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Quimiocinas/imunologia , Criança , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Interleucina-8/genética , Interleucina-8/metabolismo , Neutrófilos/imunologia , Mucosa Respiratória/citologia , Transdução de Sinais/fisiologia , Replicação Viral/fisiologia
19.
Biochim Biophys Acta ; 1784(1): 213-27, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17933596

RESUMO

The study of viral proteins and host cell factors that interact with them has represented an invaluable contribution to understanding of the physiology as well as associated pathology of key eukaryotic cell processes such as cell cycle regulation, signal transduction and transformation. Similarly, knowledge of nucleocytoplasmic transport is based largely on pioneering studies performed on viral proteins that enabled the first sequences responsible for the facilitated transport through the nuclear pore to be identified. The study of viral proteins has also enabled the discovery of several nucleocytoplasmic regulatory mechanisms, the best characterized being through phosphorylation. Recent delineation of the mechanisms whereby phosphorylation regulates nuclear import and export of key viral gene products encoded by important human pathogens such as human cytomegalovirus dengue virus and respiratory syncytial virus has implications for the development of antiviral therapeutics. In particular, the development of specific and effective kinase inhibitors makes the idea of blocking viral infection by inhibiting the phosphorylation-dependent regulation of viral gene product nuclear transport a real possibility. Additionally, examination of a chicken anemia virus (CAV) protein able to target selectively into the nucleus of tumor but not normal cells, as specifically regulated by phosphorylation, opens the exciting possibility of cancer cell-specific nuclear targeting. The study of nucleoplasmic transport may thus enable the development not only of new antiviral approaches, but also contribute to anti-cancer strategies.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteínas Virais/metabolismo , Vírus/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Regulação Viral da Expressão Gênica , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , Transporte Proteico , Transdução de Sinais , Replicação Viral , Vírus/patogenicidade
20.
J Virol ; 82(17): 8863-70, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18579594

RESUMO

Cytoplasmic inclusions in respiratory syncytial virus-infected cells comprising viral nucleocapsid proteins (L, N, P, and M2-1) and the viral genome are sites of viral transcription. Although not believed to be necessary for transcription, the matrix (M) protein is also present in these inclusions, and we have previously shown that M inhibits viral transcription. In this study, we have investigated the mechanisms for the association of the M protein with cytoplasmic inclusions. Our data demonstrate for the first time that the M protein associates with cytoplasmic inclusions via an interaction with the M2-1 protein. The M protein colocalizes with M2-1 in the cytoplasm of cells expressing only the M and M2-1 proteins and directly interacts with M2-1 in a cell-free binding assay. Using a cotransfection system, we confirmed that the N and P proteins are sufficient to form cytoplasmic inclusions and that M2-1 localizes to these inclusions; additionally, we show that M associates with cytoplasmic inclusions only in the presence of the M2-1 protein. Using truncated mutants, we show that the N-terminal 110 amino acids of M mediate the interaction with M2-1 and the subsequent association with nucleocapsids. The interaction of M2-1 with M and, in particular, the N-terminal region of M may represent a target for novel antivirals that block the association of M with nucleocapsids, thereby inhibiting virus assembly.


Assuntos
Nucleocapsídeo/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Citoplasma/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Vetores Genéticos , Humanos , Corpos de Inclusão Viral/metabolismo , Dados de Sequência Molecular , Mutação , Nucleocapsídeo/química , Nucleocapsídeo/genética , Plasmídeos , Ligação Proteica , Transfecção
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