RESUMO
The M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research. IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A , Complexo de Endopeptidases do Proteassoma , Ubiquitinação , Proteínas da Matriz Viral , Replicação Viral , Animais , Antivirais , Linhagem Celular , Embrião de Galinha , Fibroblastos , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Virulência/genéticaRESUMO
Introduction: Antenatal steroid improves respiratory distress syndrome in preterm infants. The molecular mechanism of the process is not well established. The aim of this study is to investigate the possible association between antenatal steroid and fetal Forkhead box M1(Foxm1) expression. Materials and methods: An animal study using mated pregnant New Zealand white rabbits and their fetuses was designed. Fourteen mother rabbits were assigned to four groups to undergo a cesarean section. In groups 1, 2, and 3, preterm pups were harvested on day 27 of gestation. In group 4, term pups were harvested on day 31. Antenatal maternal intramuscular injection was performed in groups 2 (normal saline) and 3 (betamethasone). Using qRT-PCR and Western blot, mRNA transcription and protein expression of surfactant protein (SP) A, B, C, and Foxm1 were compared between the pups of those four groups. Results: Sixty two fetal rabbits were harvested. One-way ANOVA test showed higher mRNA transcription of SPs in groups 3 and 4 than groups 1 and 2. Significantly lower Foxm1 mRNA transcription and protein expression were observed in group 3 or 4 compared with group 1 or 2. Conclusion: Decreased Foxm1 expression was associated in an antenatal betamethasone animal model.
Assuntos
Antiasmáticos/administração & dosagem , Betametasona/administração & dosagem , Proteína Forkhead Box M1/metabolismo , Surfactantes Pulmonares/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Feminino , Exposição Materna , Gravidez , Cuidado Pré-Natal , RNA Mensageiro/metabolismo , Coelhos , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/prevenção & controleRESUMO
Post-translational modification plays a critical role in viral replication. Previously we reported that neddylation of PB2 of influenza A virus (IAV) can inhibit viral replication. However, we found that NEDD8 overexpression can still inhibit the replication of PB2 K699R mutant viruses, implying that other viral protein(s) can be neddylated. In this study, we revealed that M1 of IAV can also be modified by NEDD8. We found that the E3 ligase HDM2 significantly promotes M1 neddylation. Furthermore, we identified M1 K187 as the major neddylation site. We generated an IAV M1 K187R mutant (WSN-M1 K187R) and compared the growth of wild-type and mutant viruses in Madin-Darby canine kidney (MDCK) cells. The data showed that the replication of WSN-M1 K187R was more efficient than that of wild-type WSN. More importantly, we observed that overexpression of NEDD8 inhibited the replication of the wild-type WSN more effectively than that of WSN-M1 K187R. In addition, we found that the neddylation-deficient M1 mutant (M1 K187R) had a longer half-life than that of wild-type M1, indicating that the neddylation of M1 reduces stability. Then we performed a viral infection assay and found that WSN-M1 K187R exhibited greater virulence in mice than wild-type WSN, suggesting that the neddylation of M1 reduced IAV replication in vivo. In conclusion, we uncovered that neddylation of M1 by HDM2 negatively regulates the stability of M1, which in turn inhibits viral replication.
Assuntos
Vírus da Influenza A/fisiologia , Proteína NEDD8/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Feminino , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Lisina/genética , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo , Proteínas da Matriz Viral/genética , VirulênciaRESUMO
Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here, we used a yeast two-hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 suppressed influenza virus replication. Transient, stable, or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Moreover, SLD5 partially rescued M1 expression- or influenza virus infection-induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared with wild-type mice. Therefore, influenza virus M1 blocks host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus-host interaction.
Assuntos
Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Infecções por Orthomyxoviridae/genética , Proteínas da Matriz Viral/genética , Células A549 , Animais , Proteínas Cromossômicas não Histona/metabolismo , Cães , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Cultura Primária de Células , Ligação Proteica , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Proteínas da Matriz Viral/metabolismo , Replicação Viral/genéticaRESUMO
Objective: To investigate the effect of hypoxia-inducible factor 2α (HIF-2α) gene on the expression of Forkhead box M1 (FoxM1) protein in the proliferation of hypoxic rat pulmonary artery smooth muscle cells (PASMC). Methods: HIF-2α overexpression lentiviral vector (LV-HIF-2α) and silencing RNA (siRNA) were constructed and transfected into rat PASMC under normoxia and hypoxia, respectively. The PASMC under normoxia were classified into normoxic control group, normoxia + LV-HIF-2α empty group, normoxia + LV-HIF-2α group; the PASMC under hypoxia were classified into hypoxic control group, hypoxia + siRNA-HIF-2α empty group, hypoxia + siRNA-HIF-2α group. The expression of HIF-2α and its downstream proteins FoxM1, cyclin D1 and Aurora A expressions were detected by Western blot. 5-Ethyny-2'-deoxyuridine (EdU) cell proliferation assay was used to evaluate the effect of overexpression and inhibition of HIF-2α expression on the proliferation of rat PASMC. Results: The expression of HIF-2α in normoxia + LV-HIF-2α group was significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.17±0.02 vs 0.09±0.01 and 0.07±0.00), while the expression of HIF-2α in PASMC of hypoxia + siRNA-HIF-2α group was significantly lower than that of hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.28±0.01 vs 0.35±0.02 and 0.30±0.01) (all P<0.05); the expression of FoxM1 protein, cyclinD1 and cell proliferation-related Aurora A protein in normoxia+LV-HIF-2α group were significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.40±0.03 vs 0.24±0.01 and 0.30±0.01, 0.22±0.02 vs 0.09±0.01 and 0.08±0.02, 0.29±0.02 vs 0.04±0.01 and 0.07±0.01, respectively) (all P<0.05); the expressions of FoxM1 protein, cyclinD1 and Aurora A protein in hypoxia + siRNA-HIF-2α group were significantly lower than those in hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.23±0.01 vs 0.36±0.02 and 0.32±0.01, 0.15±0.01 vs 0.31±0.01 and 0.28±0.03, 0.14±0.02 vs 0.33±0.03 and 0.27±0.02, respectively) (all P<0.05); the positive expression rate of EdU in the normoxic control group was significantly lower than that in the normoxia+LV-HIF-2α group [(30.77±2.43)% vs (55.56±3.01)%], while the hypoxic control group was significantly higher than the hypoxic+siRNA-HIF-2α group [(65.28±3.21)% vs (44.64±2.78)%] (both P<0.05). Conclusion: HIF-2α up-regulates the expression of FoxM1 and promotes the proliferation of pulmonary artery smooth muscle cells in hypoxic rats.
Assuntos
Artéria Pulmonar , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Proteína Forkhead Box M1 , Hipóxia , Miócitos de Músculo Liso , RatosRESUMO
BACKGROUND: The influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection. N-domain and C-domain are connected by a loop region, which consists of conserved RQMV motif. METHODS: The function of the highly conserve RQMV motif in the influenza virus life cycle was investigated by site-directed mutagenesis and by rescuing mutant viruses by reverse genetics. Co-localization of M1 with nucleoprotein (NP), clustered mitochondria homolog protein (CLUH), chromosome region maintenance 1 protein (CRM1), or plasma membrane were studied by confocal microscopy. RESULTS: Mutant viruses containing an alanine substitution of R163, Q164 and V166 result in the production of the virus indistinguishable from the wild type phenotype. Single M165A substitution was lethal for rescuing infection virus and had a striking effect on the distribution of M1 and NP proteins. We have observed statistically significant reduction in distribution of both M165A (p<0,05) and NP (p<0,001) proteins to the nucleus in the cells transfected with the reverse -genetic system with mutated M1. M165A protein was co-localized with CLUH protein in the cytoplasm and around the nucleus but transport of M165-CLUH complex through the nuclear membrane was restricted. CONCLUSIONS: Our finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.
Assuntos
Membrana Celular/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/genética , Carioferinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas da Matriz Viral/genética , Replicação Viral/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Membrana Nuclear/metabolismo , Nucleoproteínas/metabolismo , Domínios Proteicos/genética , Montagem de Vírus/fisiologia , Proteína Exportina 1RESUMO
Large-scale sequence analysis of Matrix (M) gene and its coding proteins M1 and M2 was performed for 274 highly pathogenic avian influenza viruses H5N1 circulated in Egypt from 2006 to 2016. The aim is to study the amantadine-resistant markers distribution and to estimate the evolutionary rate. 246 viruses were obtained from the Global Initiative on Sharing All Influenza Data base, and 28 additional viruses were sequenced. Maximum clade credibility (MCC) phylogenetic tree revealed that the M gene has evolved into two different lineages. Estimated Evolutionary analysis showed that the M2 protein possessed higher evolutionary rates (3.45 × 10-3) than the M1 protein (2.73 × 10-3). M gene encoding proteins revealed significant markers described to be associated with host tropism and increase in virulence: V15I, N30D, and T121A in M1 and L55F in M2 protein. Site analysis focusing attention on the temporal and host distribution of the amantadine-resistant markers was carried out and showed that vast majority of the M2 amantadine-resistant variants of clade 2.2.1.1 (n = 90) is N31 marker, in addition to G27 (n = 7), A27 (n = 5), I27 (n = 1), and S30 (n = 1). In 2010-2011, amantadine resistant frequency increased considerably resembling more than half of the resistant variants. Notably, all viruses of clade 2.2.1.1 possessed amantadine-resistant marker. However, almost all current circulating viruses in Egypt of clade 2.2.1.2 from 2014 to 2016 did not carry any amantadine-resistant markers.
Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/epidemiologia , Influenza Humana/virologia , Proteínas da Matriz Viral/genética , Animais , Galinhas , Egito/epidemiologia , Genótipo , História do Século XX , História do Século XXI , Humanos , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Influenza Humana/história , Modelos Moleculares , Filogenia , Conformação Proteica , Proteínas da Matriz Viral/químicaRESUMO
RNA interference (RNAi) represents a form of post-transcriptional gene silencing mediated by small interfering RNAs (siRNA) and provides a powerful tool to specifically inhibit viral infection. To investigate therapeutic capacity of siRNAs targeting M gene, six vectors with U1-short hairpin RNA (shRNA) expression system were prepared and tested in infected cells and animals. In infected cells, three of six shRNAs targeting M1 gene significantly (P <0,01) reduced the virus titer to 66%, 45% or 21%, respectively. Replication of IAV and levels of M1 RNAs were significantly reduced in the cells transfected with shRNAs, which decreased the virus titer. IFN-α/ß altered in shRNAs-treated cells. The level of IFN-λ (type III interferon) mRNA was significantly increased in the infected cells treated with shM22, shM349, shM522, and (type I interferon) as well as IP-10 (type II interferon) mRNAs were not significantly their mixtures. The increased level of IFN-λ mRNA corresponded to significantly increased level of RIG-1 mRNA. shRNAs inhibited influenza virus infection in a gene-specific manner in co-operation with IFN-λ. Some constructs targeting the M1 transcript prolonged the survival of infected mice.
Assuntos
Vírus da Influenza A/genética , Influenza Humana/genética , Interleucinas/genética , RNA Interferente Pequeno/genética , Proteínas da Matriz Viral/genética , Animais , Feminino , Humanos , Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Influenza Humana/virologia , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/metabolismo , Proteínas da Matriz Viral/metabolismoRESUMO
Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14-15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Humanos , Hemaglutininas , Peptídeos/química , Vírus da Influenza A/metabolismo , Concentração de Íons de HidrogênioRESUMO
Cold atmospheric plasma (CAP) has been demonstrated to be a successful antiseptic for chronic and infected wounds. Although experimental work has focused on elucidation of the curative power of CAP for wound healing, the molecular mechanisms behind this ability are less understood. To date, the direct effect of CAP on the activity of microbial virulence factors has not been investigated. In the present study, we therefore examined whether CAP can modulate the detrimental activity of M1 protein, one of the most studied Streptococcus pyogenes virulence determinant. Our results show that CAP abolishes the ability of M1 protein to trigger inflammatory host responses. Subsequent mass spectrometric analysis revealed that this effect was caused by oxidation of Met81 and Trp128 located at the sub-N-terminal region of M1 protein provoking a conformational change. Notably, our results also show that CAP has an insignificant effect on the host immune system, supporting the benefits of using CAP to combat infections. Considering the growing number of antibiotic-resistant bacteria, novel antimicrobial therapeutic approaches are urgently needed that do not bear the risk of inducing additional resistance. Our study therefore may open new research avenues for the development of novel approaches for the treatment of skin and wound infections caused by S. pyogenes.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Gases em Plasma/química , Streptococcus pyogenes , Fatores de Virulência/imunologia , Linhagem Celular Transformada , Humanos , Oxirredução , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidadeRESUMO
BACKGROUND: Streptococcus pyogenes is a Gram positive bacterial species commonly involved in sepsis. Invasive strains express virulence factors such as the M1 protein. M1 protein forms complexes with fibrinogen leading to a cytokine storm in plasma contributing to the development of septic shock and organ failure. In experimental animals M1 protein causes vascular nitric oxide production and hyporesponsiveness to pressors, but it is not known whether it affects the human vascular wall. METHODS: Human omental arteries obtained during surgery were incubated in vitro with M1 protein or lipopolysaccharide (LPS) as positive control, with or without plasma. After 48 h, contractile response to noradrenaline was measured, and levels of nitrite/nitrate and the cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α in the incubation medium were measured. A second set of arteries were incubated with or without main components of plasma (immunoglobulin G, albumin or fibrinogen), in the presence of M1 protein followed by cytokine measurement. RESULTS: Artery segments incubated with M1 protein and plasma contracted weaker in response to noradrenaline, and levels of IL-6 and IL-8 were significantly higher compared to after incubation with M1 protein alone. Incubation with M1 protein and fibrinogen resulted in elevated levels of IL-6 and IL-8, while incubation with M1 protein and albumin or immunoglobulin G did not affect the levels. Neither any of the other cytokines nor nitrite/nitrate was detected in the medium in any of the incubation conditions. CONCLUSIONS: The study shows that M1 protein of Streptococcus pyogenes has a direct effect on the human vascular wall in the presence of plasma, demonstrated both as a diminished contractile response to noradrenaline and increased cytokine production. The effect of plasma was attributed to fibrinogen. The findings suggest that M1 protein contributes to the development of septic shock through impairment of the contractility of the vascular wall.
Assuntos
Antígenos de Bactérias/farmacologia , Artérias/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Proteínas de Transporte/farmacologia , Citocinas/metabolismo , Fibrinogênio/metabolismo , Choque Séptico/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Artérias/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Omento/irrigação sanguínea , Choque Séptico/microbiologia , Choque Séptico/patologia , Infecções Estreptocócicas/patologiaRESUMO
A previous report demonstrated that influenza virus infection induces accumulation of EGFP-tagged M1 protein (EGFP-M1) in the sub-nuclear domain ND10. Here, we show that the transfection of four viral protein (NP, PB2, PB1, PA) expression vectors and eight RNA segment expression vectors induced the formation of nuclear dots of EGFP-M1 as seen in virus infections. Omission of the segment 7 RNA expression vector, however, abolished the nuclear dots of EGFP-M1. This result suggests an essential role for authentic M1 protein and/or M2 protein, both of which are encoded in segment 7, in the formation of nuclear dots of EGFP-M1. Co-expression of M1 protein but not M2 protein with EGFP-M1 induced the formation of nuclear dots of EGFP-M1. The dots co-localized with PML protein, which is an indicator of ND10. When only M1 protein was expressed, immunostaining of M1 protein clearly revealed the nuclear dots and their colocalization with PML protein. These results demonstrate that the accumulation in ND10 is an intrinsic characteristic of M1 protein and EGFP addition abolishes this characteristic. The addition of EGFP to M1 protein induced a defect in M1 protein.
Assuntos
Proteínas de Fluorescência Verde/genética , Proteínas Nucleares/genética , Proteínas da Matriz Viral/genética , Linhagem Celular , Núcleo Celular , Fluorescência , Proteínas de Fluorescência Verde/química , Humanos , Transfecção , Proteínas da Matriz Viral/química , Replicação ViralRESUMO
The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions.
Assuntos
Colesterol/metabolismo , Vírus da Influenza A/química , Vírus da Influenza A/ultraestrutura , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Animais , Sítios de Ligação , Análise Mutacional de DNA , Vírus da Influenza A/genética , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Genética Reversa , Proteínas da Matriz Viral/genética , Vírion/ultraestruturaRESUMO
Avian influenza viruses represent a growing threat for an influenza pandemic. The currently licensed influenza vaccines have inherent drawbacks which has led many research groups to explore different approaches of vaccine development among which Virus Like particles (VLPs) seem like a promising alternative in the near future. Although it is known that the Matrix 1 protein (M1) of influenza plays an essential role in VLP formation and it is documented that M1 is able to form dimers, it is not clear if M1 is capable of forming higher order structures without the interference of other influenza proteins or cell derived envelope. Here, for the first time we have demonstrated that expression of M1 alone is enough to form a Capsid Like Particle (CLP) without the requirement of any other external factor.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Influenza Humana/prevenção & controle , Proteínas da Matriz Viral/imunologia , Animais , Aves , Capsídeo/imunologia , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Humanos , Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Influenza Humana/virologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas da Matriz Viral/metabolismoRESUMO
A novel influenza virus of H7N9 subtype circulated throughout China in 2013. The high fatality rate, appearance of several family clusters, and transmission in animal models observed during this outbreak accelerated efforts to identify effective strategies to prevent the spread of this influenza subtype. In this study, the recombinant protein NP-M1-HSP60, a fusion of the nucleoprotein and M1 matrix protein of the A/PR/8/34 (H1N1) influenza virus strain and HSP60, was effectively expressed in Escherichia coli and purified as a candidate component for an influenza vaccine. Intranasal immunization of female BALB/c mice with NP-M1-HSP60 in combination with an oil-in-water adjuvant twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses. Moreover, this immunization strategy completely protected mice from lethal influenza H7N9 virus challenge and significantly inhibited viral replication in the challenged mouse lung. These data suggest that this vaccine construct has great potential for the basic development of an influenza H7N9 vaccine.
Assuntos
Chaperonina 60/administração & dosagem , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Proteínas de Ligação a RNA/administração & dosagem , Proteínas do Core Viral/administração & dosagem , Proteínas da Matriz Viral/administração & dosagem , Animais , Anticorpos Antivirais/imunologia , Chaperonina 60/genética , Chaperonina 60/imunologia , Feminino , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Vacinação , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
Objective Exosomes secreted from mouse bone marrow mesenchymal stem cells (BMSC) overexpressing the cardiomyocyte transcription factor GATA-4 (BMSCGATA-4-exosome) may play a key role in repairing myocardial injury. This study aimed to investigate the molecular regulatory network of BMSCGATA-4-exosome for inhibiting the apoptosis of cardiomyocytes. Methods Exosomes extracted from GATA-4-overexpressing BMSCs of the mouse cultured with miR-330-3p-mimic were cultured with myocardial cells under hypoxic and serum-free conditions for 24 hours (the experimental group), the overexpressed GATA-4, empty vector and BMSCs were taken as the confounding factor control (CFC), the myocardial cells cultured under hypoxic and serum-free conditions for 24 hours were used as the positive control, and those cultured under the normal condition for 24 hours as the negative control. The apoptosis rates of myocardial cells in different groups were measured by flow cytometry, the expression levels of miR-330-3p in the myocardial cells determined by RT-PCR, and those of the corresponding miR-330-3p target gene Ap2m1 and transcriptional protein Cnot4 detected by Western blot. Results CD29 was expressed in 99.71% of the mouse BMSCs, CD44 in 97.28%, SCA-1 in 99.40%, and CD11b overexpressed in only 0.1%. The early apoptosis rate of myocardial cells was significantly higher in the experimental than in the negative control group ([7.90 ± 0.34]% vs [2.30 ± 0.09]%, P < 0.05) but lower than in the positive control ([51.48 ± 0.40]%), BMSC ([18.32 ± 3.03]%), empty vector ([16.99 ± 2.93]%) and overexpressed GATA-4 groups ([10.22 ± 0.35]%) (P < 0.05). The expression of miR-330-3p in the myocardial cells was markedly higher in the experimental ([396.10 ± 1.02]%) than in the negative control ([1.37 ± 0.33]%), positive control ([0.26±0.32]%), BMSC ([1.40 ± 0.42]%), empty vector ([1.41 ± 0.27]%) and overexpressed GATA-4 groups ([3.80 ± 0.62]%) (P < 0.05). The expressions of Ap2m1 and Cont4 in the myocardial cells were remarkably decreased in the experimental group compared with those in the other five groups (P < 0.05). Conclusion Overexpressed BMSCGATA-4-exosomes suppress the apoptosis of myocardial cells by inhibiting the expression of the Ap2m1 protein via miR-330-3p.
RESUMO
@#Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/(M*s)]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.