RESUMO
Nonenveloped virus-like particles (VLPs) are self-assembled oligomeric structures composed of one or more proteins that originate from diverse viruses. Because these VLPs have similar antigenicity to the parental virus, they are successfully used as vaccines against cognate virus infection. Furthermore, after foreign antigenic sequences are inserted in their protein components (chimVLPs), some VLPs are also amenable to producing vaccines against pathogens other than the virus it originates from (these VLPs are named platform or epitope carrier). Designing chimVLP vaccines is challenging because the immunogenic response must be oriented against a given antigen without altering stimulant properties inherent to the VLP. An important step in this process is choosing the location of the sequence modifications because this must be performed without compromising the assembly and stability of the original VLP. Currently, many immunogenic data and computational tools can help guide the design of chimVLPs, thus reducing experimental costs and work. In this study, we analyze the structure of a novel VLP that originate from an insect virus and describe the putative regions of its three structural proteins amenable to insertion. For this purpose, we employed molecular dynamics (MD) simulations to assess chimVLP stability by comparing mutated and wild-type (WT) VLP protein trajectories. We applied this procedure to design a chimVLP that can serve as a prophylactic vaccine against the SARS-CoV-2 virus. The methodology described in this work is generally applicable for VLP-based vaccine development.
Assuntos
Epitopos , Vacinas de Partículas Semelhantes a Vírus , Vacinas de Partículas Semelhantes a Vírus/imunologia , Epitopos/imunologia , Epitopos/genética , Humanos , SARS-CoV-2/imunologia , Simulação de Dinâmica Molecular , COVID-19/prevenção & controle , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Biologia Computacional/métodosRESUMO
Research on vaccines against trypanosomatids, a family of protozoa that cause neglected tropical diseases, such as Chagas disease, leishmaniasis, and sleeping sickness, is a current need. Today, according to modern vaccinology, virus-like particle (VLP) technology is involved in many vaccines, including those undergoing studies related to COVID-19. The potential use of VLPs as vaccine adjuvants opens an opportunity for the use of protozoan antigens for the development of vaccines against diseases caused by Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei. In this context, it is important to consider the evasion mechanisms of these protozoa in the host and the antigens involved in the mechanisms of the parasite-host interaction. Thus, the immunostimulatory properties of VLPs can be part of an important strategy for the development and evaluation of new vaccines. This work aims to highlight the potential of VLPs as vaccine adjuvants for the development of immunity in complex diseases, specifically in the context of tropical diseases caused by trypanosomatids.
RESUMO
Small icosahedral viruses have a compact capsid that apparently lacks holes through which solvents can be exchanged with the external milieu. However, due to the steric hindrance of amino acids, upon folding, capsid proteins form narrow cavities in which water and ions can be trapped. These occluded solvent molecules can form lines of water, called water wires, representing an arrangement with special features for proton conduction. In this chapter, we review the physico-chemical principles that permit proton conduction through protein cavities. We also describe how a combination of these elements found in an insect viral capsid can allow the virus to sense alkaline environments. Through this analysis, we stress the need to combine experimental and theoretical techniques when modeling complex biological systems.
Assuntos
Capsídeo , Vírus de Insetos , Prótons , Capsídeo/química , Concentração de Íons de Hidrogênio , Vírus de Insetos/fisiologia , Solventes , ÁguaRESUMO
In this work, we assess a previously advanced hypothesis that predicts the existence of ion channels in the capsid of small and non-enveloped icosahedral viruses. With this purpose we examine Triatoma Virus (TrV) as a case study. This virus has a stable capsid under highly acidic conditions but disassembles and releases the genome in alkaline environments. Our calculations range from a subtle sub-atomic proton interchange to the dismantling of a large-scale system representing several million of atoms. Our results provide structure-based explanations for the three roles played by the capsid to enable genome release. First, we observe, for the first time, the formation of a hydrophobic gate in the cavity along the five-fold axis of the wild-type virus capsid, which can be disrupted by an ion located in the pore. Second, the channel enables protons to permeate the capsid through a unidirectional Grotthuss-like mechanism, which is the most likely process through which the capsid senses pH. Finally, assuming that the proton leak promotes a charge imbalance in the interior of the capsid, we model an internal pressure that forces shell cracking using coarse-grained simulations. Although qualitatively, this last step could represent the mechanism of capsid opening that allows RNA release. All of our calculations are in agreement with current experimental data obtained using TrV and describe a cascade of events that could explain the destabilization and disassembly of similar icosahedral viruses.
Assuntos
Dicistroviridae/fisiologia , Dicistroviridae/ultraestrutura , Canais Iônicos/metabolismo , Animais , Capsídeo/fisiologia , Capsídeo/ultraestrutura , Biologia Computacional , Dicistroviridae/genética , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Prótons , Eletricidade Estática , Montagem de Vírus/fisiologiaRESUMO
Vectors of Chagas disease are currently controlled by employing several chemical insecticides though there is a continuing search for alternative ecological methods against disease causing vectors. An effective method includes the use of specific pathogens as biological control agents. The aim of this work was to describe a complete experimental inoculation protocol in triatomines. The intrahaemocoelic inoculation technique can be applied to inoculate different kinds of microorganisms such as viruses, fungi, bacteria and protozoa; so it could be considered a useful tool in infective bioassays. This article includes results from evaluations of Triatoma virus (TrV, Dicistroviridae: Triatovirus) infectivity in several triatomine species. The protocol, also suitable for any other kind of insects, describes the materials and steps required to safely inoculate the insects, preventing any damage and/or contamination.
RESUMO
Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.
Assuntos
Inibidor da Ligação a Diazepam/química , 1,2-Dipalmitoilfosfatidilcolina/química , Acil Coenzima A/metabolismo , Animais , Tatus , Simulação por Computador , Membranas Artificiais , Modelos Moleculares , Fosfatidilserinas/química , Conformação Proteica , Eletricidade EstáticaRESUMO
Triatoma virus (TrV) is the only entomopathogenic virus found in triatomines. TrV replicates in cells of the midgut epithelium of triatomines, causing a high mortality rate and delayed development of the infected insect. In this work, we report an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of TrV infection. For antiserum production, rabbits and hens where inoculated with purified TrV. Antiserum reactivity was checked by immunodiffusion, and its specificity was confirmed by western blot and AC-ELISA. Totally 90 fecal samples from T. infestans were analysed. AC-ELISA and RT-PCR results correlated well with transmission electron microscopy (EM) observations, which are considered the gold standard, with Kappa values of 0.73 for AC-ELISA and 0.93 for RT-PCR when compared with EM. Applications and complementary uses of the two techniques reported in this work are discussed.
Assuntos
Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática/métodos , Infecções por Picornaviridae/diagnóstico , Picornaviridae/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Triatoma/virologia , Animais , Anticorpos Antivirais/imunologia , Bovinos , Hemípteros/virologia , Imunoensaio , Vírus de Insetos/isolamento & purificação , Vírus de Insetos/patogenicidade , Microscopia Eletrônica de Transmissão , Picornaviridae/isolamento & purificação , Picornaviridae/fisiologia , Infecções por Picornaviridae/imunologia , CoelhosRESUMO
The X-ray structure of the tetragonal form of apo acyl-CoA-binding protein (ACBP) from the Harderian gland of the South American armadillo Chaetophractus villosus has been solved. ACBP is a carrier for activated long-chain fatty acids and has been associated with many aspects of lipid metabolism. Its secondary structure is highly similar to that of the corresponding form of bovine ACBP and exhibits the unique flattened alpha-helical bundle (up-down-down-up) motif reported for animal, yeast and insect ACBPs. Conformational differences are located in loops and turns, although these structural differences do not suffice to account for features that could be related to the unusual biochemistry and lipid metabolism of the Harderian gland.
Assuntos
Tatus/metabolismo , Inibidor da Ligação a Diazepam/química , Animais , Bovinos , Cristalização , Cristalografia por Raios X , Glândula de Harder/química , Glândula de Harder/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Triatoma virus (TrV) is a viral pathogen of the blood-sucking reduviid bug Triatoma infestans, the most important vector of American human trypanosomiasis (Chagas' disease). TrV has been putatively classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work describes the purification of TrV particles from infected T. infestans and their crystallization and preliminary crystallographic analyses. Two different crystal forms, rhombohedral and orthorhombic, were obtained at room temperature by the hanging-drop vapour-diffusion technique using polyethylene glycol and polyethylene glycol monomethylether as precipitants. The rhombohedral crystals have unit-cell parameters a = b = 306.6, c = 788.4 A (hexagonal setting), diffract to 3.2 A resolution and contain one-third of the viral particle per asymmetric unit. The orthorhombic crystals have cell parameters a = 336, b = 351, c = 332 A, diffract to about 2.5 A resolution, and contain one-half of a virus particle in the asymmetric unit. A complete diffraction data set has been collected to 3.2 A resolution, using synchrotron radiation, from a single rhombohedral crystal under cryogenic conditions.
Assuntos
Vírus de Insetos/química , Triatoma/virologia , Animais , Proteínas do Capsídeo/química , Cristalização , Cristalografia por Raios X , Interpretação Estatística de Dados , RNA Viral/químicaRESUMO
Previous authors demonstrated that Triatoma virus (TrV) is able to infect several species of triatomines when injected with viral inoculum obtained from its original host, T. infestans. Both vertical (transovarian) and horizontal (faecal-oral) mechanisms of viral transmission were also described. In this paper we report the experimental TrV infection of a wild species from southern Argentina, T. patagonica. The inoculum consisted of clarified gut contents of infected T. infestans rubbed on the chicken skin whereupon T. patagonica individuals were fed. The results demonstrate that this is another potential host for the virus, and that the oral route is also effective for experimental interspecific infections.