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1.
Talanta ; 206: 120201, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514868

RESUMO

Human immunodeficiency virus (HIV) is a lentivirus that leads to acquired immunodeficiency syndrome (AIDS). With increasing awareness of AIDS emerging as a global public health threat, different HIV testing kits have been developed to detect antibodies (Ab) directed toward different parts of HIV. A great limitation of these tests is that they can not detect HIV antibodies during early virus infection. Therefore, to overcome this challenge, a wide range of biosensors have been developed for early diagnosis of HIV infection. A significant amount of these studies have been focused on the application of nanomaterials for improving the sensitivity and accuracy of the sensing methods. Following an introduction into this field, a first section of this review covers the synthesis and applicability of such nanomaterials as metal nanoparticles (NPs), quantum dots (QDs), carbon-based nanomaterials and metal nanoclusters (NCs). A second larger section covers the latest developments concerning nanomaterial-based biosensors for HIV diagnosis, with paying a special attention to the determination of CD4+ cells as a hall mark of HIV infection, HIV gene, HIV p24 core protein, HIV p17 peptide, HIV-1 virus-like particles (VLPs) and HIV related enzymes, particularly those that are passed on from the virus to the CD4+ T lymphocytes and are necessary for viral reproduction within the host cell. These studies are described in detail along with their diverse principles/mechanisms (e.g. electrochemistry, fluorescence, electromagnetic-piezoelectric, surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS) and colorimetry). Despite the significant progress in HIV biosensing in the last years, there is a great need for the development of point-of-care (POC) technologies which are affordable, robust, easy to use, portable, and possessing sufficient quantitative accuracy to enable clinical decision making. In the final section, the focus is on the portable sensing devices as a new standard of POC and personalized diagnostics.


Assuntos
Técnicas Biossensoriais/métodos , Infecções por HIV/diagnóstico , HIV , Nanoestruturas/química , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Biomarcadores/análise , DNA Viral/análise , Diagnóstico Precoce , HIV/química , HIV/genética , HIV/imunologia , Humanos , Testes Imediatos , RNA Viral/análise , Proteínas Virais/análise
3.
Talanta ; 203: 83-89, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31202353

RESUMO

An integrated immunodetection platform employing a simple, reusable, centrifugal microchannel array chip and a smartphone as detection unit was developed. The applicability of the platform to the detection of HIV p24 antigen was demonstrated. The microchip was made of polycarbonate and contained 4 × 8 zigzag microchannels. After the monoclonal antibody of HIV p24 was adsorbed onto the channel surfaces, HIV p24 was introduced into the microchannel to react with the antibody. A biotin linked polyclonal antibody was then brought in to react with HIV p24, and SP80 (containing streptavidin and horseradish peroxidase) was introduced to react with the biotin. Finally, a solution containing 3,3',5,5'-tetramethylbenzidine and other reagents was passed through the above channels, horseradish peroxidase catalyzed the oxidation of tetramethylbenzidine (to 3,3',5,5'- tetramethylbenzidine diamine) forming a dark color. The color intensity, indicating HIV p24 antigen quantity, was then photographed via a smartphone, and the color of each microchannel was processed via a computer to determine the HIV p24 antigen concentration. Under the optimized conditions, limits of detection (LODs) of 0.17 ng/ml and 0.11 ng/ml were obtained for p24 antigen in a buffer solution and human serum, respectively. Channel washing/rinsing was implemented via a centrifugal force. An economic portable centrifugal device that could accommodate up to 4 microchips was assembled, and multi-step solution loading and rinsing involved in this sandwich immunoassay were performed conveniently. The microchip could be reused after a simple regeneration process. The low-cost polycarbonate microchip and centrifugal device together with the simple but efficient operation make the method a promising tool for HIV screening in resource limited areas.


Assuntos
Proteína do Núcleo p24 do HIV/análise , Dispositivos Lab-On-A-Chip , Smartphone , Animais , Anticorpos Monoclonais Murinos/imunologia , Armoracia/enzimologia , Centrifugação , Ensaio de Imunoadsorção Enzimática/métodos , Desenho de Equipamento , Reutilização de Equipamento , HIV/química , Proteína do Núcleo p24 do HIV/imunologia , Peroxidase do Rábano Silvestre/química , Humanos , Limite de Detecção , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Estudo de Prova de Conceito , Coelhos
4.
Structure ; 27(1): 134-139.e3, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30344107

RESUMO

Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins that is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup, and refinement of glycan-containing structures. We benchmarked this approach using 12 crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.


Assuntos
Glicoproteínas/química , Simulação de Dinâmica Molecular/normas , Proteínas Virais/química , Coronavirus Humano NL63/química , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , HIV/química , Software
5.
Anal Chem ; 90(22): 13373-13377, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30345744

RESUMO

Early diagnosis of HIV biomarkers or genes is the key to reducing acquired immunodeficiency syndrome (AIDS) mortality. In our work, we developed a novel polymerase chain reaction-dynamic light scattering (PCR-DLS) assay for one-step sensitive detection of HIV DNA based on the average-diameter change of gold nanoparticles (AuNPs). This is the first PCR assay that makes use of the DLS technique as a signal read-out, with the particle size measured by DLS increasing with the concentration of target DNA. With the help of the AuNP probes, this PCR-DLS assay can effectively improve the specificity of PCR reactions, which can greatly increase the detection sensitivity, with a detection limit of 1.8 aM (S/N = 3). In addition, the proposed strategy was successfully used to analyze target DNA in human serum samples, indicating that the PCR-DLS assay has a promising potential application for rapid and early clinical diagnosis of HIV infection.


Assuntos
DNA Viral/sangue , Difusão Dinâmica da Luz/métodos , Reação em Cadeia da Polimerase/métodos , Ouro/química , HIV/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Tamanho da Partícula
6.
Nano Lett ; 18(10): 6318-6325, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30234311

RESUMO

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.


Assuntos
HIV/ultraestrutura , Nanotecnologia , Conformação de Ácido Nucleico , RNA Viral/ultraestrutura , HIV/química , Humanos , Microscopia de Força Atômica , Pinças Ópticas , RNA Viral/química , Imagem Individual de Molécula
7.
Angew Chem Int Ed Engl ; 57(43): 14032-14036, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30063096

RESUMO

We report a strategy to construct peptidyl virus-like particles (pVLPs) by mimicking the human immunodeficiency virus and simian virus 40. We designed two viral peptides with cell/nucleus-targeting capabilities that can co-assemble in their active conformations into well-defined nanoparticles. The self-assembled nanoparticles can encapsulate the DNA of clustered regularly interspaced short palindromic repeat associated proteins 9 (CRISPR/Cas9) to form biodegradable pVLPs with excellent cell-targeting ability and biocompatibility. The pVLPs can penetrate the cellular membrane and deliver genetic cargos into the nucleus through the viral entry route. The results provide a promising pathway for engineering artificial viruses with desired functions.


Assuntos
Técnicas de Transferência de Genes , Peptídeos/química , Vírion/química , Sistemas CRISPR-Cas , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , HIV/química , HIV/fisiologia , Humanos , Fusão de Membrana , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Vírus 40 dos Símios/química , Vírus 40 dos Símios/fisiologia
8.
Angew Chem Int Ed Engl ; 57(30): 9305-9309, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29870126

RESUMO

Reaction of the Au-C N chelate [Au(bnpy)Cl2 ] with the full-length zinc finger (ZnF; ZnCys3 His) of HIV nucleocapsid protein NCp7 results in C-S aryl transfer from the AuIII organometallic species to a cysteine of the ZnF. The reaction is general and occurs even for finger 3 of the transcription factor Sp1, containing a ZnCys2 His2 coordination sphere. This reaction is the first demonstration of group transfer from a coordination compound to biologically important zinc fingers, and is especially noteworthy for the ZnCys2 His2 transcription factors. The work expands the corpus of organometallic species which can efficiently modify biomolecules through C-atom transfer. The electronic features of the gold compound leading to this unexpected reaction were explored by X-ray absorption spectroscopy.


Assuntos
Dedos de Zinco CYS2-HIS2 , Carbono/química , Ouro/química , HIV/química , Proteínas do Nucleocapsídeo/química , Enxofre/química , Catálise , Estrutura Molecular
9.
PLoS Comput Biol ; 14(4): e1006093, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29677181

RESUMO

Mounting evidence suggests that glycans, rather than merely serving as a "shield", contribute critically to antigenicity of the HIV envelope (Env) glycoprotein, representing critical antigenic determinants for many broadly neutralizing antibodies (bNAbs). While many studies have focused on defining the role of individual glycans or groups of proximal glycans in bNAb binding, little is known about the effects of changes in the overall glycan landscape in modulating antibody access and Env antigenicity. Here we developed a systems glycobiology approach to reverse engineer the complexity of HIV glycan heterogeneity to guide antigenicity-based de novo glycoprotein design. bNAb binding was assessed against a panel of 94 recombinant gp120 monomers exhibiting defined glycan site occupancies. Using a Bayesian machine learning algorithm, bNAb-specific glycan footprints were identified and used to design antigens that selectively alter bNAb antigenicity as a proof-of concept. Our approach provides a new design strategy to predictively modulate antigenicity via the alteration of glycan topography, thereby focusing the humoral immune response on sites of viral vulnerability for HIV.


Assuntos
Antígenos HIV/química , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Polissacarídeos/química , Polissacarídeos/imunologia , Algoritmos , Sequência de Aminoácidos , Anticorpos Neutralizantes , Teorema de Bayes , Sítios de Ligação , Biologia Computacional , Epitopos/química , Epitopos/genética , Glicosilação , HIV/química , HIV/imunologia , Anticorpos Anti-HIV , Antígenos HIV/genética , Proteína gp120 do Envelope de HIV/genética , Humanos , Aprendizado de Máquina , Modelos Moleculares , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Biologia de Sistemas
10.
Int J Mol Sci ; 18(10)2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28972547

RESUMO

Human immunodeficiency virus (HIV) hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-ß-D-J-ß of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR). This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS). Quantitative enzyme-linked immunoadsorption assays (ELISA) demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10-8 to 10-9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.


Assuntos
Síndrome de Imunodeficiência Adquirida/imunologia , HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Síndrome de Imunodeficiência Adquirida/virologia , Animais , Autoimunidade , Reações Cruzadas , HIV/química , Proteínas do Vírus da Imunodeficiência Humana/química , Humanos , Microbiota , Mimetismo Molecular , Proteômica , Receptores de Antígenos de Linfócitos T/química
11.
J Chem Inf Model ; 57(5): 1166-1178, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28448138

RESUMO

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions do not have a fixed tertiary structure but play key roles in signal regulation, molecule recognition, and drug targeting. However, it is difficult to study the structure and function of IDPs by traditional experimental methods because of their diverse conformations. Limitations of current generic protein force fields and solvent models were reported in the previous simulations of IDPs. We have also explored overcoming these limitations by developing the ff99IDPs and ff14IDPs force fields to correct the dihedral distribution for eight disorder-promoting residues often observed in IDPs and found encouraging improvements. Here we extend our correction of backbone dihedral terms to all 20 naturally occurring amino acids in the IDP-specific force field ff14IDPSFF to further improve the quality of the modeling of IDPs. Extensive tests of seven IDPs and 14 unstructured short peptides show that the simulated Cα chemical shifts obtained with the ff14IDPSFF force field are in quantitative agreement with those from NMR experiments and are more accurate than those obtained with the base generic force field and also our previous ff14IDPs that only corrects the eight disorder-promoting amino acids. The influence of the solvent model was also investigated and found to be less important. Finally, our explicit-solvent MD simulations further show that ff14IDPSFF can still be used to model structural and dynamical properties of two tested folded proteins, with a slightly better agreement in the loop regions for both structural and dynamical properties. These findings confirm that the newly developed IDP-specific force field ff14IDPSFF can improve the conformer sampling of intrinsically disordered proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Modelos Biológicos , Simulação de Dinâmica Molecular , Simulação por Computador , HIV/química , Dobramento de Proteína , Solventes/química
12.
Biosens Bioelectron ; 94: 471-477, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28342375

RESUMO

Since HCV and HIV share a common transmission path, high sensitive detection of HIV and HCV gene is of significant importance to improve diagnosis accuracy and cure rate at early stage for HIV virus-infected patients. In our investigation, a novel nanozyme-based bio-barcode fluorescence amplified assay is successfully developed for simultaneous detection of HIV and HCV DNAs with excellent sensitivity in an enzyme-free and label-free condition. Here, bimetallic nanoparticles, PtAuNPs, present outstanding peroxidase-like activity and act as barcode to catalyze oxidation of nonfluorescent substrate of amplex red (AR) into fluorescent resorufin generating stable and sensitive "Turn On" fluorescent output signal, which is for the first time to be integrated with bio-barcode strategy for fluorescence detection DNA. Furthermore, the provided strategy presents excellent specificity and can distinguish single-base mismatched mutant from target DNA. What interesting is that cascaded INHIBIT-OR logic gate is integrated with biosensors for the first time to distinguish individual target DNA from each other under logic function control, which presents great application in development of rapid and intelligent detection.


Assuntos
Técnicas Biossensoriais , DNA Viral/isolamento & purificação , HIV/isolamento & purificação , Hepacivirus/isolamento & purificação , DNA Viral/química , HIV/química , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , Hepacivirus/química , Hepatite C/diagnóstico , Hepatite C/virologia , Humanos , Nanopartículas/química , Espectrometria de Fluorescência
13.
PLoS One ; 12(3): e0174386, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28350835

RESUMO

Protein classification is one of the critical problems in bioinformatics. Early studies used geometric distances and polygenetic-tree to classify proteins. These methods use binary trees to present protein classification. In this paper, we propose a new protein classification method, whereby theories of information and networks are used to classify the multivariate relationships of proteins. In this study, protein universe is modeled as an undirected network, where proteins are classified according to their connections. Our method is unsupervised, multivariate, and alignment-free. It can be applied to the classification of both protein sequences and structures. Nine examples are used to demonstrate the efficiency of our new method.


Assuntos
Algoritmos , Proteínas/classificação , Proteômica/métodos , Animais , HIV/química , Infecções por HIV/virologia , Humanos , Vírus da Influenza A/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/classificação , Análise Multivariada , Infecções por Orthomyxoviridae/virologia , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/classificação , Proteínas/química , Proteínas Virais/química , Proteínas Virais/classificação , Globinas beta/química , Globinas beta/classificação
14.
Proc Natl Acad Sci U S A ; 114(9): 2265-2270, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28196882

RESUMO

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.


Assuntos
Produtos do Gene tat/química , Ensaios de Triagem em Larga Escala , Fosfotransferases/química , beta-Lactamases/química , Substituição de Aminoácidos , Aspergillus niger/química , Aspergillus niger/enzimologia , Sítios de Ligação , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Produtos do Gene tat/metabolismo , HIV/química , HIV/metabolismo , Modelos Moleculares , Mutação , Biblioteca de Peptídeos , Fosfotransferases/genética , Fosfotransferases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Solubilidade , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-Híbrido , beta-Lactamases/genética , beta-Lactamases/metabolismo
15.
Acta Crystallogr D Struct Biol ; 72(Pt 10): 1137-1148, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27710935

RESUMO

The structures of protein assemblies are important for elucidating cellular processes at the molecular level. Three-dimensional electron microscopy (3DEM) is a powerful method to identify the structures of assemblies, especially those that are challenging to study by crystallography. Here, a new approach, PRISM-EM, is reported to computationally generate plausible structural models using a procedure that combines crystallographic structures and density maps obtained from 3DEM. The predictions are validated against seven available structurally different crystallographic complexes. The models display mean deviations in the backbone of <5 Å. PRISM-EM was further tested on different benchmark sets; the accuracy was evaluated with respect to the structure of the complex, and the correlation with EM density maps and interface predictions were evaluated and compared with those obtained using other methods. PRISM-EM was then used to predict the structure of the ternary complex of the HIV-1 envelope glycoprotein trimer, the ligand CD4 and the neutralizing protein m36.


Assuntos
Microscopia Crioeletrônica/métodos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Animais , Antígenos CD4/química , Antígenos CD4/metabolismo , Bases de Dados de Proteínas , HIV/química , HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas/química
16.
Biochemistry ; 55(44): 6100-6114, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27731975

RESUMO

We recently reported the discovery of a recombinant chimera, denoted DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively cause specific and potent lytic inactivation of both pseudotyped and fully infectious human immunodeficiency virus (HIV-1) virions. The chimera is composed of the lectin cyanovirin-N (CVN) fused to the 20-residue membrane-proximal external region (MPER) of HIV-1 gp41. Because the Env gp120-binding CVN domain on its own is not lytic, we sought here to determine how the MPER(DAVEI) domain is able to endow the chimera with virolytic activity. We used a protein engineering strategy to identify molecular determinants of MPER(DAVEI) that are important for function. Recombinant mutagenesis and truncation demonstrated that the MPER(DAVEI) domain could be significantly minimized without loss of function. The dependence of lysis on specific MPER sequences of DAVEI, determination of minimal linker length, and competition by a simplified MPER surrogate peptide suggested that the MPER domain of DAVEI interacts with the Env spike trimer, likely with the gp41 region. This conclusion was further supported by observations from binding of the biotinylated MPER surrogate peptide to Env protein expressed on cells, monoclonal antibody competition, a direct binding enzyme-linked immunosorbent assay on viruses with varying numbers of trimeric spikes on their surfaces, and comparison of maximal interdomain spacing in DAVEI to that in high-resolution structures of Env. The finding that MPER(DAVEI) in CVN-MPER linker sequences can be minimized without loss of virolytic function provides an improved experimental path for constructing size-minimized DAVEI chimeras and molecular tools for determining how simultaneous engagement of gp120 and gp41 by these chimeras can disrupt the metastable virus Env spike.


Assuntos
Biopolímeros/química , Proteína gp120 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/química , HIV/patogenicidade , Inativação de Vírus , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Células HEK293 , HIV/química , Humanos , Virulência
17.
Crit Rev Biochem Mol Biol ; 51(5): 379-394, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27685368

RESUMO

Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.


Assuntos
Multimerização Proteica , Proteínas Virais/química , Viroses/virologia , Vírus/química , Animais , Capsídeo/química , Capsídeo/imunologia , Capsídeo/metabolismo , Ebolavirus/química , Ebolavirus/imunologia , Ebolavirus/metabolismo , Flavivirus/química , Flavivirus/imunologia , Flavivirus/metabolismo , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , HIV/química , HIV/imunologia , HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Viroses/imunologia , Viroses/metabolismo , Replicação Viral , Vírus/imunologia , Vírus/metabolismo
18.
Bioorg Med Chem ; 24(20): 4826-4834, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27364608

RESUMO

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNAPhe and tRNAinit, Escherichia coli tRNAinit and HIV tRNALys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem.2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg2+. Results showed a stabilizing effect associated with the presence of the modifications and Mg2+ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNAinit and limitations were observed in the conformation of E. coli tRNAinit, the presence of the modified nucleotides and of Mg2+ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.


Assuntos
Magnésio/química , RNA de Transferência/química , Ribonucleotídeos/química , Escherichia coli/química , HIV/química , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/química
19.
J Struct Biol ; 195(2): 216-226, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27291071

RESUMO

The active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pH-dependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25' and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding.


Assuntos
Ácido Aspártico/química , Catálise , Inibidores da Protease de HIV/química , Protease de HIV/química , HIV/química , Sítios de Ligação , Domínio Catalítico , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
20.
J Phys Chem B ; 120(26): 6298-305, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27128962

RESUMO

Human immunodeficiency virus (HIV) capsid proteins spontaneously assemble around the genome into a protective protein shell called the capsid, which can take on a variety of shapes broadly classified as conical, cylindrical, and irregular. The majority of capsids seen in in vivo studies are conical in shape, while in vitro experiments have shown a preference for cylindrical capsids. The factors involved in the selection of the unique shape of HIV capsids are not well understood, and in particular the impact of RNA on the formation of the capsid is not known. In this work, we study the role of the genome and its interaction with the capsid protein by modeling the genomic RNA through a mean-field theory. Our results show that the confinement free energy for a homopolymeric model genome confined in a conical capsid is lower than that in a cylindrical capsid, at least when the genome does not interact with the capsid, which seems to be the case in in vivo experiments. Conversely, the confinement free energy for the cylinder is lower than that for a conical capsid if the genome is attracted to the capsid proteins as the in vitro experiments. Understanding the factors that contribute to the formation of conical capsids may shed light on the infectivity of HIV particles.


Assuntos
Capsídeo/metabolismo , Genoma Viral , Modelos Biológicos , RNA Viral , Capsídeo/química , Capsídeo/ultraestrutura , Simulação por Computador , HIV/química , HIV/genética , HIV/ultraestrutura
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