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1.
Viruses ; 13(7)2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202573

RESUMO

Adenovirus vector-based genetic vaccines have emerged as a powerful strategy against the SARS-CoV-2 health crisis. This success is not unexpected because adenoviruses combine many desirable features of a genetic vaccine. They are highly immunogenic and have a low and well characterized pathogenic profile paired with technological approachability. Ongoing efforts to improve adenovirus-vaccine vectors include the use of rare serotypes and non-human adenoviruses. In this review, we focus on the viral capsid and how the choice of genotypes influences the uptake and subsequent subcellular sorting. We describe how understanding capsid properties, such as stability during the entry process, can change the fate of the entering particles and how this translates into differences in immunity outcomes. We discuss in detail how mutating the membrane lytic capsid protein VI affects species C viruses' post-entry sorting and briefly discuss if such approaches could have a wider implication in vaccine and/or vector development.


Assuntos
Adenovírus Humanos/imunologia , Adenovírus Humanos/fisiologia , Capsídeo/metabolismo , Vetores Genéticos , Vacinas Virais/imunologia , Internalização do Vírus , Imunidade Adaptativa , Adenovírus Humanos/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Ensaios Clínicos como Assunto , Humanos , Imunidade Inata , Camundongos , SARS-CoV-2/imunologia
2.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200973

RESUMO

Nucleocapsid proteins (NCp) are zinc finger (ZF) proteins, and they play a central role in HIV virus replication, mainly by interacting with nucleic acids. Therefore, they are potential targets for anti-HIV therapy. Natural products have been shown to be able to inhibit HIV, such as turmeric and licorice, which is widely used in traditional Chinese medicine. Liquiritin (LQ), isoliquiritin (ILQ), glycyrrhizic acid (GL), glycyrrhetinic acid (GA) and curcumin (CUR), which were the major active components, were herein chosen to study their interactions with HIV-NCp7 C-terminal zinc finger, aiming to find the potential active compounds and reveal the mechanism involved. The stacking interaction between NCp7 tryptophan and natural compounds was evaluated by fluorescence. To elucidate the binding mode, mass spectrometry was used to characterize the reaction mixture between zinc finger proteins and active compounds. Subsequently, circular dichroism (CD) spectroscopy and molecular docking were used to validate and reveal the binding mode from a structural perspective. The results showed that ILQ has the strongest binding ability among the tested compounds, followed by curcumin, and the interaction between ILQ and the NCp7 zinc finger peptide was mediated by a noncovalent interaction. This study provided a scientific basis for the antiviral activity of turmeric and licorice.


Assuntos
Fármacos Anti-HIV/farmacologia , Produtos Biológicos/farmacologia , Curcuma/química , Glycyrrhiza/química , HIV-1/efeitos dos fármacos , Dedos de Zinco/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos Biológicos/química , Proteínas do Capsídeo/metabolismo , HIV-1/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Nat Commun ; 12(1): 4176, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234134

RESUMO

Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor µ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase µ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.


Assuntos
Proteínas do Capsídeo/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Orthoreovirus/ultraestrutura , RNA Viral/metabolismo , Fatores de Transcrição/metabolismo , Regulação Alostérica , Animais , Proteínas do Capsídeo/ultraestrutura , Linhagem Celular , Microscopia Crioeletrônica , Regulação Viral da Expressão Gênica , Genoma Viral , Macaca mulatta , Nucleosídeo-Trifosfatase/ultraestrutura , Orthoreovirus/genética , Orthoreovirus/metabolismo , Multimerização Proteica , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Mensageiro/metabolismo , RNA Viral/ultraestrutura , RNA Polimerase Dependente de RNA/metabolismo , Fatores de Transcrição/ultraestrutura , Ativação Transcricional , Montagem de Vírus/genética
4.
Molecules ; 26(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206893

RESUMO

PF74 is a capsid-targeting inhibitor of HIV replication that effectively perturbs the highly sensitive viral uncoating process. A lack of information regarding the optical purity (enantiomeric excess) of the single stereogenic centre of PF74 has resulted in ambiguity as to the potency of different samples of this compound. Herein is described the synthesis of enantiomerically enriched (S)- and (R)-PF74 and further enrichment of the samples (≥98%) using chiral HPLC resolution. The biological activities of each enantiomer were then evaluated, which determined (S)-PF74 (IC50 1.5 µM) to be significantly more active than (R)-PF74 (IC50 19 µM). Computational docking studies were then conducted to rationalise this large discrepancy in activity, which indicated different binding conformations for each enantiomer. The binding energy of the conformation adopted by the more active (S)-PF74 (ΔG = -73.8 kcal/mol) was calculated to be more favourable than the conformation adopted by the less active (R)-enantiomer (ΔG = -55.8 kcal/mol) in agreement with experimental observations.


Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Indóis/farmacologia , Fenilalanina/análogos & derivados , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Capsídeo/química , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Indóis/síntese química , Indóis/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Fenilalanina/síntese química , Fenilalanina/química , Fenilalanina/farmacologia , Estereoisomerismo
5.
Ann Diagn Pathol ; 53: 151744, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33991784

RESUMO

OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.


Assuntos
COVID-19/patologia , Imuno-Histoquímica/métodos , Doenças Pulmonares Intersticiais/patologia , Pulmão/patologia , Síndrome de Ativação Macrofágica/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , COVID-19/complicações , COVID-19/diagnóstico , COVID-19/virologia , Proteínas do Capsídeo/metabolismo , Comorbidade , Feminino , Humanos , Pulmão/metabolismo , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/virologia , Linfócitos/metabolismo , Linfócitos/patologia , Síndrome de Ativação Macrofágica/etiologia , Síndrome de Ativação Macrofágica/virologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/patologia , SARS-CoV-2/genética , Licença Médica
6.
Nat Commun ; 12(1): 3226, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050170

RESUMO

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/ultraestrutura , Ácido Fítico/metabolismo , Vírus do Sarcoma de Rous/ultraestrutura , Montagem de Vírus , Capsídeo/metabolismo , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Multimerização Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Vírus do Sarcoma de Rous/patogenicidade , Vírus do Sarcoma de Rous/fisiologia , Imagem Individual de Molécula , Transfecção , Liberação de Vírus
7.
Nucleic Acids Res ; 49(10): 5832-5844, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037793

RESUMO

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5'-3' panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5'-3' panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/metabolismo , Chaperonas Moleculares/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Recombinação Genética/genética , Replicação Viral/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Códon de Iniciação , Ciclização/genética , Vírus da Dengue/genética , Cinética , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier
8.
Nat Commun ; 12(1): 2766, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986255

RESUMO

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.


Assuntos
Proteínas do Capsídeo/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Zika virus/metabolismo , Células A549 , Aedes/virologia , Animais , Capsídeo/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína com Valosina/genética , Replicação Viral/fisiologia , Zika virus/genética , Infecção por Zika virus/patologia
9.
Nat Commun ; 12(1): 2903, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006828

RESUMO

Molecular communication across physical barriers requires pores to connect the environments on either side and discriminate between the diffusants. Here we use porous virus-like particles (VLPs) derived from bacteriophage P22 to investigate the range of molecule sizes able to gain access to its interior. Although there are cryo-EM models of the VLP, they may not accurately depict the parameters of the molecules able to pass across the pores due to the dynamic nature of the P22 particles in the solution. After encapsulating the enzyme AdhD within the P22 VLPs, we use a redox reaction involving PAMAM dendrimer modified NADH/NAD+ to examine the size and charge limitations of molecules entering P22. Utilizing the three different accessible morphologies of the P22 particles, we determine the effective pore sizes of each and demonstrate that negatively charged substrates diffuse across more readily when compared to those that are neutral, despite the negatively charge exterior of the particles.


Assuntos
Bacteriófago P22/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Vírion/metabolismo , Algoritmos , Bacteriófago P22/genética , Bacteriófago P22/ultraestrutura , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Dendrímeros/química , Dendrímeros/metabolismo , Difusão , Microscopia Eletrônica de Transmissão , Modelos Teóricos , Mutação , NAD/química , NAD/metabolismo , Tamanho da Partícula , Porosidade , Eletricidade Estática , Vírion/genética , Vírion/ultraestrutura
10.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33789995

RESUMO

Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and, furthermore, is incorporated into the viral capsid, accounting for most of the "endogenous kinase" activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs in the HBc NTD known as cyclin docking motifs (CDMs), which mediate the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection.IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.


Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Vírus da Hepatite B/fisiologia , Domínios e Motivos de Interação entre Proteínas , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Replicação Viral , Capsídeo/enzimologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Ciclinas/metabolismo , Células Hep G2 , Vírus da Hepatite B/química , Humanos , Nucleocapsídeo/metabolismo , Fosforilação , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/genética , RNA Viral/metabolismo , Proteínas do Core Viral/genética , Montagem de Vírus
11.
Biochemistry (Mosc) ; 86(2): 230-240, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33832421

RESUMO

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen.


Assuntos
Proteínas do Capsídeo/metabolismo , Potyvirus/ultraestrutura , Vírion/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Dicroísmo Circular , Microscopia Eletrônica de Transmissão , Potyvirus/metabolismo , Espalhamento a Baixo Ângulo , Vírion/metabolismo , Difração de Raios X
12.
J Med Chem ; 64(9): 5500-5518, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33887912

RESUMO

Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.


Assuntos
Antivirais/química , Proteínas do Capsídeo/metabolismo , Vírus da Hepatite B/fisiologia , Pirimidinas/química , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Sítios de Ligação , Proteínas do Capsídeo/química , DNA Viral/sangue , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Meia-Vida , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Simulação de Acoplamento Molecular , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Relação Estrutura-Atividade , Tenofovir/metabolismo , Tenofovir/farmacologia , Montagem de Vírus/efeitos dos fármacos
13.
Methods Mol Biol ; 2300: 99-106, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792875

RESUMO

RiboNucleoProtein particles (RNPs), which are composed of RNAs and proteins, play essential roles in many biological processes. The isolation of these molecular machines is a critical step to better understand their mechanisms of action. In this chapter, we describe the MS2-MBP affinity chromatography used to purify the protein content of the RNPs formed with an RNA of interest in a nuclear extract. Substrate RNAs are furnished with a tag consisting of three stem-loops that provide specific binding sites for the phage MS2 protein. Here, we successfully applied this method to isolate RNPs formed with subfragments of the long noncoding RNA ANRIL (Antisense Noncoding RNA in the INK4 Locus).


Assuntos
Proteínas do Capsídeo/metabolismo , RNA Longo não Codificante/metabolismo , Ribonucleoproteínas/isolamento & purificação , Sítios de Ligação , Cromatografia de Afinidade , Humanos , Levivirus/metabolismo , Ribonucleoproteínas/genética
14.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803568

RESUMO

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein-cargo and protein-protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.


Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Vírion/metabolismo , Bromovirus/ultraestrutura , Modelos Moleculares , Tamanho da Partícula , RNA de Transferência/metabolismo , Temperatura , Vírion/ultraestrutura
15.
Eur J Med Chem ; 217: 113380, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33751981

RESUMO

A capsid is the protein shell of a virus, encircling its genetic material. The HIV capsid is erected from a single protein, known as capsid protein. The capsid of HIV-1 significantly involved in many processes of the virus life cycle, which makes it as a novel target for the new inhibitors. Recently many novel HIV-1 inhibitors binding to capsid proteins have been reported successfully. Most of these inhibitors can inhibit or accelerate the disassembly or assembly of the capsid, and some of them can inhibit reverse transcription. Unfortunately, none of them are currently approved by U.S. FDA. However, GS-6207, an inhibitor binds to the NTD-CTD interface with potent antiviral activity and the long metabolic cycle, is expected to be the first approved drug targeting HIV-1 capsid. Herein, we provide a concise report focusing on the recent prospective of HIV-1 capsid inhibitors in medicinal chemistry in order to enlighten drug design.


Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/química , Proteínas do Capsídeo/metabolismo , Relação Dose-Resposta a Droga , HIV-1/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
16.
Nat Commun ; 12(1): 1642, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712599

RESUMO

Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Dependovirus/metabolismo , Animais , Dependovirus/genética , Células HEK293 , Humanos , Sorogrupo , Células Sf9 , Proteínas Virais/metabolismo , Montagem de Vírus
17.
Nat Commun ; 12(1): 1576, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707432

RESUMO

We apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qß (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.


Assuntos
Allolevivirus/genética , Proteínas do Capsídeo/metabolismo , Escherichia coli/virologia , Levivirus/genética , RNA/metabolismo , Sítios de Ligação Microbiológicos/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Escherichia coli/genética , Biblioteca Gênica , Humanos , Aprendizado de Máquina , Plasmídeos/genética
18.
Arch Biochem Biophys ; 702: 108822, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33722536

RESUMO

Capsids of several RNA viruses are reported to have unconventional roles attributed to their subcellular trafficking property. The capsid of CHIKV is also found to localize in the nucleus, but the rationale is not yet clear. To understand the role of the nuclear-localized capsid, we examined the nucleic acid binding and cargo delivery activity of the CHIKV capsid. We used bacterially purified capsid protein to probe the binding affinity with CHIKV genome-specific and non-specific nucleic acids. We found that the capsid was able to bind non-specifically to different forms of nucleic acids. The successful transfection of GFP-tagged plasmid DNA by CHIKV capsid protein shows the DNA delivery ability of the protein. Further, we selected and investigated the DNA binding and cargo delivery activity of commercially synthesized Nuclear Localization Signal sequences (NLS 1 and NLS2) of capsid protein. Both peptides showed comparable DNA binding affinity, however, only the NLS1 peptide was capable of delivering plasmid DNA inside the cell. Furthermore, the cellular uptake study using the FITC-labelled NLS1 peptide was performed to highlight the membrane penetrating ability. Structural analysis was performed using circular dichroism and NMR spectroscopy to elucidate the transfection ability of the NLS1 peptides. Our findings suggest that the capsid of CHIKV might influence cellular trafficking in the infected cell via non-specific interactions. Our study also indicates the significance of NLS sequences in the multifunctionality of CHIKV capsid protein.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus Chikungunya/metabolismo , DNA/metabolismo , Sinais de Localização Nuclear , Sequência de Aminoácidos , Transporte Biológico , Modelos Moleculares , Domínios Proteicos
19.
J Med Chem ; 64(7): 3747-3766, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33750123

RESUMO

Poor metabolic stability of the human immunodeficiency virus type-1 (HIV-1) capsid (CA) inhibitor PF-74 is a major concern in its development toward clinical use. To improve on the metabolic stability, we employed a novel multistep computationally driven workflow, which facilitated the rapid design of improved PF-74 analogs in an efficient manner. Using this workflow, we designed three compounds that interact specifically with the CA interprotomer pocket, inhibit HIV-1 infection, and demonstrate enantiomeric preference. Moreover, using this workflow, we were able to increase the metabolic stability 204-fold in comparison to PF-74 in only three analog steps. These results demonstrate our ability to rapidly design CA compounds using a novel computational workflow that has improved metabolic stability over the parental compound. This workflow can be further applied to the redesign of PF-74 and other promising inhibitors with a stability shortfall.


Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Indóis/farmacologia , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Indóis/química , Indóis/metabolismo , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estabilidade Proteica , Estereoisomerismo , Fluxo de Trabalho
20.
J Vet Sci ; 22(1): e8, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33522160

RESUMO

BACKGROUND: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. OBJECTIVES: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. METHODS: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. RESULTS: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. CONCLUSIONS: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.


Assuntos
Proteínas do Capsídeo/genética , Circovirus/imunologia , Dependovirus/genética , Imunidade Celular , Imunidade Humoral , Vacinas Virais/imunologia , Animais , Proteínas do Capsídeo/metabolismo , Circovirus/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C
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