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1.
BMC Complement Altern Med ; 19(1): 346, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791311

RESUMO

BACKGROUND: Influenza A virus (IAV) is still a major health threat. The clinical manifestations of this infection are related to immune dysregulation, which causes morbidity and mortality. The usage of traditional medication with immunomodulatory properties against influenza infection has been increased recently. Our previous study showed antiviral activity of quercetin-3-O-α-L-rhamnopyranoside (Q3R) isolated from Rapanea melanophloeos (RM) (L.) Mez (family Myrsinaceae) against H1N1 (A/PR/8/34) infection. This study aimed to confirm the wider range of immunomodulatory effect of Q3R on selective pro- and anti-inflammatory cytokines against IAV in vitro, to evaluate the effect of Q3R on apoptosis pathway in combination with H1N1, also to assess the physical interaction of Q3R with virus glycoproteins and RhoA protein using computational docking. METHODS: MDCK cells were exposed to Q3R and 100CCID50/100 µl of H1N1 in combined treatments (co-, pre- and post-penetration treatments). The treatments were tested for the cytokines evaluation at RNA and protein levels by qPCR and ELISA, respectively. In another set of treatment, apoptosis was examined by detecting RhoA GTPase protein and caspase-3 activity. Molecular docking was used as a tool for evaluation of the potential anti-influenza activity of Q3R. RESULTS: The expressions of cytokines in both genome and protein levels were significantly affected by Q3R treatment. It was shown that Q3R was much more effective against influenza when it was applied in co-penetration treatment. Q3R in combination with H1N1 increased caspase-3 activity while decreasing RhoA activation. The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 and H1 of PR/8/1934 and Human RhoA proteins, with docking energy of - 10.81, - 10.47, - 9.52, - 9.24 and - 8.78 Kcal/mol, respectively. CONCLUSIONS: Quercetin-3-O-α-L-rhamnopyranoside from RM was significantly effective against influenza infection by immunomodulatory properties, affecting the apoptosis pathway and binding ability to viral receptors M2 transmembrane and Neuraminidase of 2009 pandemic H1N1 and human RhoA cellular protein. Further research will focus on detecting the detailed specific mechanism of Q3R in virus-host interactions.


Assuntos
Antivirais , Glicosídeos , Vírus da Influenza A Subtipo H1N1 , Myrsine/química , Compostos Fitoquímicos , Quercetina/análogos & derivados , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Cães , Glicosídeos/química , Glicosídeos/metabolismo , Glicosídeos/farmacologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Neuraminidase/química , Neuraminidase/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Compostos Fitoquímicos/farmacologia , Quercetina/química , Quercetina/metabolismo , Quercetina/farmacologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
2.
Biochim Biophys Acta Biomembr ; 1861(8): 1421-1427, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31153909

RESUMO

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61-70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.


Assuntos
Citoplasma/metabolismo , Vírus da Influenza A/metabolismo , Canais Iônicos/metabolismo , Proteínas da Matriz Viral/metabolismo , Membrana Celular/metabolismo , Vírus da Influenza A/fisiologia , Proteínas da Matriz Viral/química , Montagem de Vírus , Liberação de Vírus
3.
Mol Pharmacol ; 96(2): 148-157, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175183

RESUMO

The Food and Drug Administration-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug-binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive toward amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N terminus of transmembrane residues 22-31 that ultimately broadened the drug-binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared with 4 in complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism toward AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high-profile antiviral drug target, as more than 95% of currently circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using a previously developed AM2 S31N channel blocker as a chemical probe, this study was the first to identify a novel resistant mutant, L46P. The L46P mutant is located outside of the drug-binding site. Molecular dynamics simulations showed that L46P causes a dilation of drug-binding site between residues 22 and 31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor amantadine.


Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Vírus da Influenza A/metabolismo , Mutação , Proteínas da Matriz Viral/genética , Regulação Alostérica/efeitos dos fármacos , Motivos de Aminoácidos , Animais , Antivirais/química , Sítios de Ligação , Cães , Farmacorresistência Viral , Feminino , Humanos , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Inoculações Seriadas , Relação Estrutura-Atividade , Proteínas da Matriz Viral/química , Xenopus laevis
4.
PLoS One ; 14(4): e0214448, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30946753

RESUMO

Internal acidification of the influenza virus, mediated by the M2 proton channel, is a key step in its life cycle. The interior M1 protein shell dissolves at pH~5.5 to 6.0, allowing the release of vRNA to the cytoplasm upon fusion of the viral envelope with the endosomal membrane. Previous models have described the mechanisms and rate constants of M2-mediated transport but did not describe the kinetics of pH changes inside the virus or consider exterior pH changes due to endosome maturation. Therefore, we developed a mathematical model of M2-mediated virion acidification. We find that ~32,000 protons are required to acidify a typically-sized virion. Predicted acidification kinetics were consistent with published in vitro experiments following internal acidification. Finally, we applied the model to the in vivo situation. For all rates of endosomal maturation considered, internal acidification lagged ~1 min behind endosomal acidification to pH 6. For slow endosomal maturation requiring several minutes or more, internal and endosomal pH decay together in pseudo-equilibrium to the late endosomal pH~5.0. For fast endosomal maturation (≲2 min), a lag of tens of seconds continued toward the late endosomal pH. Recent experiments suggest in vivo maturation is in this "fast" regime where lag is considerable. We predict that internal pH reaches the threshold for M1 shell solvation just before the external pH triggers membrane fusion mediated by the influenza protein hemagglutinin, critical because outward proton diffusion through a single small fusion pore is faster than the collective M2-mediated transport inward.


Assuntos
Orthomyxoviridae/química , RNA Viral/química , Citoplasma/química , Endossomos/química , Hemaglutininas Virais/química , Humanos , Concentração de Íons de Hidrogênio , Influenza Humana/virologia , Cinética , Modelos Teóricos , Orthomyxoviridae/genética , Orthomyxoviridae/fisiologia , Prótons , Processos Estocásticos , Fatores de Tempo , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Internalização do Vírus
5.
Artigo em Inglês | MEDLINE | ID: mdl-30972303

RESUMO

Influenza A virus (IAV) matrix protein 2 (M2), an ion channel, is crucial for virus infection, and therefore, an important anti-influenza drug target. Adamantanes, also known as M2 channel blockers, are one of the two classes of Food and Drug Administration-approved anti-influenza drugs, although their use was discontinued due to prevalent drug resistance. Fast emergence of resistance to current anti-influenza drugs have raised an urgent need for developing new anti-influenza drugs against resistant forms of circulating viruses. Here we propose a simple theoretical criterion for fast virtual screening of molecular libraries for candidate anti-influenza ion channel inhibitors both for wild type and adamantane-resistant influenza A viruses. After in silico screening of drug space using the EIIP/AQVN filter and further filtering of drugs by ligand based virtual screening and molecular docking we propose the best candidate drugs as potential dual inhibitors of wild type and adamantane-resistant influenza A viruses. Finally, guanethidine, the best ranked drug selected from ligand-based virtual screening, was experimentally tested. The experimental results show measurable anti-influenza activity of guanethidine in cell culture.


Assuntos
Antivirais/isolamento & purificação , Biologia Computacional/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Proteínas da Matriz Viral/antagonistas & inibidores , Antivirais/química , Antivirais/farmacologia , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas da Matriz Viral/química
6.
Emerg Microbes Infect ; 8(1): 45-54, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866759

RESUMO

Current influenza vaccines provide hemagglutinin strain-specific protection, but rarely provide cross-protection against divergent strains. It is, therefore, particularly important to develop a universal vaccine against conserved proteins or conserved regions of the virus. In this study, we used N-terminal extracellular region of the influenza virus M2 protein (M2e) as the target antigen and constructed two optimized M2e DNA vaccines (p-tPA-p3M2e and p-p3M2e) with increased antigenic epitope density and enhanced antigen secretion. Both vaccines induced high M2e-specific humoral and cellular immune responses in the vaccinated mice. These two vaccines also conferred protection against a lethal infection of homo-subtypic H1N1 virus, with p-tPA-p3M2e being the most effective. In addition, p-tPA-p3M2e also showed cross-protection against different subtypes of the influenza virus (H9N2, H6N6, and H10N8) at varying rates (80%, 40%, and 20%, respectively). After passive immunization, M2e DNA vaccine-induced antibodies in the sera provided complete protection against homologous virus challenge. An analysis of the mechanism underlying this immunization-mediated protection indicates that M2e-specific IgG and T-cell immune responses may play critical roles in the prevention of infection and viral clearance. Taken together, our results indicate that this optimized M2e DNA vaccine is a promising candidate for the development of a universal, broad-spectrum influenza virus vaccine.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de DNA/administração & dosagem , Proteínas da Matriz Viral/genética , Animais , Proteção Cruzada , Cães , Células HEK293 , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Passiva , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Vacinas de DNA/imunologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologia
7.
Biochim Biophys Acta Biomembr ; 1861(6): 1123-1134, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902626

RESUMO

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1-protein interactions and multimerization have not been clarified, yet. In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.


Assuntos
Lipídeos de Membrana/metabolismo , Proteínas da Matriz Viral/metabolismo , Lipídeos de Membrana/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas da Matriz Viral/química
8.
Virology ; 531: 48-56, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30852271

RESUMO

Coronavirus membrane (M) protein is the most abundant structural protein playing a critical role in virion assembly. Previous studies show that the N-terminal ectodomain of M protein is modified by glycosylation, but its precise functions are yet to be thoroughly investigated. In this study, we confirm that N-linked glycosylation occurs at two predicted sites in the M protein ectodomain of infectious bronchitis coronavirus (IBV). Dual mutations at the two sites (N3D/N6D) did not affect particle assembly, virus-like particle formation and viral replication in culture cells. However, activation of the ER stress response was significantly reduced in cells infected with rN3D/N6D, correlated with a lower level of apoptosis and reduced production of pro-inflammatory cytokines. Taken together, this study demonstrates that although not essential for replication, glycosylation in the IBV M protein ectodomain plays important roles in activating ER stress, apoptosis and proinflammatory response, and may contribute to the pathogenesis of IBV.


Assuntos
Apoptose , Infecções por Coronavirus/fisiopatologia , Estresse do Retículo Endoplasmático , Vírus da Bronquite Infecciosa/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/genética , Citocinas/imunologia , Glicosilação , Humanos , Vírus da Bronquite Infecciosa/química , Vírus da Bronquite Infecciosa/genética , Domínios Proteicos , Proteínas da Matriz Viral/genética
9.
Phys Chem Chem Phys ; 21(6): 2984-2991, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30672572

RESUMO

The influenza B M2 protein (BM2) forms an acid-activated proton channel that is important for the virus's lifecycle. Despite extensive research efforts, the detailed activation mechanism of the BM2 proton channel is often elusive. Herein a pH-regulated mechanism of the BM2TM domain has been systematically characterized using multiscale computer simulations, including classical molecular dynamics, constant pH molecular dynamics (CpHMD) and quantum mechanics/molecular mechanics (QM/MM) approaches. Our simulations reveal a pH-dependent conformational switch from the C-terminal closed to the C-terminal open conformers, and provide the free energy of conformational activation coupled to the titration of the His19 tetrad. Importantly, our results confirm the coupling titration between the His19 tetrad and His27 tetrad, and identify that the full-cationic state (His2744+) dominates at the low pH (the His19 tetrad at +2, +3 and +4 charge states). Our QM/MM simulations indicate that the second titratable histidine, His27, could further promote the BM2 acid activation and speed up proton dissociation from the HxxxW motif, thus facilitating proton conduction by BM2. Taken together, a unique "activation-promotion mechanism" about the BM2 proton channel is proposed, and these results may be helpful for the understanding of other similar proton channels and the development of BM2 inhibitors.


Assuntos
Vírus da Influenza B/metabolismo , Proteínas da Matriz Viral/metabolismo , Motivos de Aminoácidos , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Teoria Quântica , Termodinâmica , Proteínas da Matriz Viral/química
10.
Virus Genes ; 55(2): 123-126, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30600431

RESUMO

In recent reports about the molecular epidemiology of Schmallenberg virus (SBV), an orthobunyavirus affecting ruminants, it was proposed that the observed sequence variability within the viral M-segment might be higher in sheep than in cattle. However, these analyses are highly biased by the sample material from which the publicly available sequences were generated. While from cattle predominantly blood samples from acutely infected animals were studied, the vast majority of ovine samples originate from malformed fetuses or newborn lambs. Therefore, the observed sequence variability is misinterpreted since the samples from malformed fetuses and lambs do not reflect circulating SBV.


Assuntos
Infecções por Bunyaviridae/virologia , Orthobunyavirus/genética , Proteínas da Matriz Viral/genética , Animais , Animais Recém-Nascidos/virologia , Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/veterinária , Bovinos , Orthobunyavirus/patogenicidade , Ruminantes/virologia , Ovinos/virologia , Proteínas da Matriz Viral/química
11.
J Biomol Struct Dyn ; 37(3): 671-690, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29388479

RESUMO

Influenza A virus, a member of the Orthomyxoviridae family of enveloped viruses, is one of the human and animal top killers, and its structure and components are therefore extensively studied during the last decades. The most abundant component, M1 matrix protein, forms a matrix layer (scaffold) under the viral lipid envelope, and the functional roles as well as structural peculiarities of the M1 protein are still under heavy debate. Despite multiple attempts of crystallization, no high resolution structure is available for the full length M1 of Influenza A virus. The likely reason for the difficulties lies in the intrinsic disorder of the M1 C-terminal part preventing diffraction quality crystals to be grown. Alternative structural methods including synchrotron small-angle X-ray scattering (SAXS), atomic force microscopy, cryo-electron microscopy/tomography are therefore widely applied to understand the structure of M1, its self-association and interactions with the lipid membrane and the viral nucleocapsid. These methods reveal striking similarities in the behavior of M1 and matrix proteins of other enveloped RNA viruses, with the differences accompanied by the specific features of the viral lifecycles, thus suggesting common interaction principles and, possibly, common evolutional ancestors. The structural information on the Influenza A virus M1 protein obtained to the date strongly suggests that the intrinsic disorder in the C-terminal domain has important functional implications.


Assuntos
Vírus da Influenza A/química , Proteínas da Matriz Viral/química , Antivirais/farmacologia , Ligação Proteica , Multimerização Proteica , Vírion/metabolismo
12.
Infect Genet Evol ; 67: 7-16, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30367980

RESUMO

Influenza A virus subtype H3N2 has been a serious health issue across the globe with approximately 36 thousand annual casualties in the United States of America only. Co-circulation in avian, swine and human hosts has led to frequent mutations in the virus genome, due to which development of successful antivirals against the virus has become a formidable challenge. Recently, focussed research is being carried out targeting the matrix proteins of this strain as vaccine candidates. This study is carried out to unravel the key features of the genes encoding the matrix proteins that manoeuvre the codon usage profile in the H3N2 strains. The findings reveal differential codon choice for both matrix protein 1 and matrix protein 2. The overall codon usage bias is less pronounced in both the datasets which is evident from higher value of effective number of codons (>55). Comparison of the codon usage for both the genes under study with that of humans revealed that the viral codon usage is not fully optimized for the human host conditions. Both the genes enrolled in the study showed variation which was reflected in almost all the indices used for codon usage studies. Neutrality analysis revealed a weak role of mutation pressure while selection was the major contributor towards codon usage.


Assuntos
Composição de Bases , Códon , Vírus da Influenza A/genética , Proteínas da Matriz Viral/genética , Evolução Molecular , Regulação Viral da Expressão Gênica , Genoma Viral , Genômica/métodos , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/classificação , Influenza Humana/virologia , Mutação , Seleção Genética , Proteínas da Matriz Viral/química
13.
Traffic ; 20(2): 152-167, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30548142

RESUMO

Morphogenesis of herpesviral virions is initiated in the nucleus but completed in the cytoplasm. Mature virions contain more than 25 tegument proteins many of which perform both nuclear and cytoplasmic functions suggesting they shuttle between these compartments. While nuclear import of herpesviral proteins was shown to be crucial for viral propagation, active nuclear export and its functional impact are still poorly understood. To systematically analyze nuclear export of tegument proteins present in virions of Herpes simplex virus type 1 (HSV1) and Epstein-Barr virus (EBV), the Nuclear EXport Trapped by RAPamycin (NEX-TRAP) was applied. Nine of the 22 investigated HSV1 tegument proteins including pUL4, pUL7, pUL11, pUL13, pUL21, pUL37d11, pUL47, pUL48 and pUS2 as well as 2 out of 6 EBV orthologs harbor nuclear export activity. A functional leucine-rich nuclear export sequence (NES) recognized by the export factor CRM1/Xpo1 was identified in six of them. The comparison between experimental and bioinformatic data indicates that experimental validation of predicted NESs is required. Mutational analysis of the pUL48/VP16 NES revealed its importance for herpesviral propagation. Together our data suggest that nuclear export is an important feature of the herpesviral life cycle required to co-ordinate nuclear and cytoplasmic processes.


Assuntos
Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 4/metabolismo , Sinais de Exportação Nuclear , Proteínas da Matriz Viral/química , Animais , Células HeLa , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 4/fisiologia , Humanos , Células Vero , Proteínas da Matriz Viral/metabolismo , Replicação Viral
14.
Blood ; 133(8): 867-877, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30573634

RESUMO

Cytomegalovirus (CMV) is the most common viral infection in hematopoietic cell transplantation (HCT) recipients. We performed deep phenotyping of CMV-specific T cells to predict CMV outcomes following allogeneic HCT. By using 13-color flow cytometry, we studied ex vivo CD8+ T-cell cytokine production in response to CMV-pp65 peptides in 3 clinically distinct subgroups of CMV-seropositive HCT patients: (1) Elite Controllers (n = 19): did not have evidence of CMV DNAemia on surveillance testing; (2) Spontaneous Controllers (n = 16): spontaneously resolved low-grade CMV DNAemia without antiviral therapy; and (3) Noncontrollers (NC; n = 21): experienced clinically significant CMV. Two CMV-specific CD8+ T-cell functional subsets were strongly associated with risk of CMV: (i) the nonprotective signature (NPS; IL-2-IFN-γ+TNF-α-MIP-1ß+), found at increased levels among NC; and (ii) the protective signature (PS; IL-2+IFN-γ+TNF-α+MIP-1ß+) found at low levels among NC. High levels of the NPS and low levels of PS were associated with an increased 100-day cumulative incidence of clinically significant CMV infection (35% vs 5%; P = .02; and 40% vs 12%; P = .05, respectively). The highest predictive value was observed when these signatures were combined into a composite biomarker consisting of low levels of the PS and high levels of the NPS (67% vs 10%; P < .001). After adjusting for steroid use or donor type, this composite biomarker remained associated with a fivefold increase in the risk of clinically significant CMV infection. CMV-specific CD8+ T-cell cytokine signatures with robust predictive value for risk of CMV reactivation should prove useful in guiding clinical decision making in HCT recipients.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Transplante de Células-Tronco Hematopoéticas , Imunofenotipagem , Ativação Viral/imunologia , Idoso , Aloenxertos , Biomarcadores , Linfócitos T CD8-Positivos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Fosfoproteínas/química , Fatores de Risco , Proteínas da Matriz Viral/química
15.
Virus Genes ; 55(1): 22-32, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30382564

RESUMO

The 23-amino acid ectodomain of influenza virus M2 protein (M2e) is highly conserved among human influenza virus variants and represents an attractive target for developing a universal vaccine. Although this peptide has limited potency and low immunogenicity, the degree of M2e density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. The aim of this study was to design a chimer protein consisting of three tandem repeats of M2e peptide sequence fused to the Leishmania major HSP70 gene and evaluate its characteristics and immunogenicity. The structure of the deduced protein and its stability, aliphatic index, biocomputed half-life and the anticipated immunogenicity were analyzed by bioinformatics software. The oligonucleotides encoding 3M2e and chimer 3M2e-HSP70 were expressed in Escherichia coli and affinity purified. The immunogenicity of the purified recombinant proteins was preliminary examined in mouse model. It was predicted that fusion of HSP70 to the C-terminal of 3M2e peptide led to increased stability, hydropathicity, continuous B cell epitopes and antigenic propensity score of chimer protein. Also, the predominant 3M2e epitopes were not hidden in the chimer protein. The initial in vivo experiment showed that 3M2e-HSP chimer protein stimulates specific immune responses. In conclusion, the results of the current study suggest that 3M2e-HSP chimer protein would be an effective universal subunit vaccine candidate against influenza infection.


Assuntos
Proteínas de Choque Térmico , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Bases de Dados Genéticas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Expressão Gênica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Humanos , Imunogenicidade da Vacina , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética
16.
Viruses ; 10(10)2018 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-30274351

RESUMO

The virus⁻host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1⁻256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for M⁻actin interaction in transporting virion components in the infected cell.


Assuntos
Actinas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas da Matriz Viral/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Citocalasina D/farmacologia , Citoplasma/metabolismo , Deleção de Genes , Humanos , Ligação Proteica/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
17.
Biochemistry ; 57(41): 5957-5968, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30230310

RESUMO

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.


Assuntos
Antivirais , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Canais Iônicos , Mutação de Sentido Incorreto , Proteínas da Matriz Viral , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Escherichia coli , Humanos , Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mutagênese , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
18.
Biochemistry ; 57(41): 5949-5956, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30230312

RESUMO

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.


Assuntos
Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H3N2/química , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Prótons , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/química , Substituição de Aminoácidos , Humanos , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Canais Iônicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Mutação de Sentido Incorreto , Relação Estrutura-Atividade , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
19.
Biochemistry (Mosc) ; 83(8): 982-991, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30208834

RESUMO

Entry of many viral and bacterial pathogens into host cells depends on cholesterol and/or cholesterol-enriched domains (lipid rafts) in the cell membrane. Earlier, we showed that influenza virus A matrix protein M1 contains amphipathic α-helices with exposed cholesterol-recognizing amino acid consensus (CRAC) motifs. In order to test possible functional activity of these motifs, we studied the effects of three synthetic peptides corresponding to the CRAC-containing α-helices of the viral M1 protein on the phagocytic activity of cultured mouse IC-21 macrophages. The following peptides were used: LEVLMEWLKTR (M1 α-helix 3, a.a. 39-49; further referred to as peptide 1), NNMDKAVKLYRKLK (M1 α-helix 6, a.a. 91-105; peptide 2), and GLKNDLLENLQAYQKR (M1 α-helix 13, a.a. 228-243; peptide 3). We found that all three peptides modulated interactions of IC-21 macrophages with non-opsonized 2-µm target particles. The greatest effect was demonstrated by peptide 2: in the presence of 35 µM peptide 2, the phagocytic index of IC-21 macrophages exceeded the control value by 60%; 10-11 mM methyl-ß-cyclodextrin abolished this effect. Peptides 1 and 3 exerted weak inhibitory effect in a narrow concentration range of 5-10 µM. The dose-response curves could be approximated by a sum of two (stimulatory and inhibitory) components with different Hill coefficients, suggesting existence of at least two peptide-binding sites with different affinities on the cell surface. CD spectroscopy confirmed that the peptides exhibit structural flexibility in solutions. Altogether, our data indicate that amphipathic CRAC-containing peptides derived from the viral M1 protein modulate lipid raft-dependent processes in IC-21 macrophages.


Assuntos
Colesterol/metabolismo , Sequência Consenso , Interações Hidrofóbicas e Hidrofílicas , Macrófagos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas da Matriz Viral/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células Cultivadas , Macrófagos/citologia , Camundongos , Fagocitose
20.
Virology ; 523: 110-120, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30119012

RESUMO

Knowledge about the function of varicella-zoster virus glycoprotein M is limited; the requirement of gM for skin and neural tropism are unknown. VZV gM contains two predicted YXXΦ trafficking motifs and a dileucine motif in the carboxyl-terminus. We constructed a recombinant VZV with gM truncated from the first YXXΦ and five additional viruses with YXXΦ tyrosine substitutions, alone and in combination with dileucine substitution. All recombinant viruses grew to high titer but mutation of the membrane-proximal YXXΦ motif reduced plaque size in cultured cells and altered gM localization. C-terminus truncation had a pronounced effect on virion morphogenesis and plaque size, but not on overall replication kinetics in vitro. Mutation of gM trafficking motifs and truncation attenuated replication in human skin xenografts in vivo; gM truncation did not alter neurotropism. Our results demonstrate that the gM C-terminus is dispensable for virus replication in cultured cells but is important for skin pathogenesis.


Assuntos
Gânglios Espinais/virologia , Herpes Zoster/virologia , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidade , Pele/virologia , Proteínas da Matriz Viral/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Gânglios Espinais/patologia , Herpes Zoster/patologia , Herpesvirus Humano 3/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Domínios Proteicos , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/patologia , Carga Viral , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Ensaio de Placa Viral , Tropismo Viral , Virulência , Replicação Viral
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