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1.
J Chem Inf Model ; 64(13): 5262-5272, 2024 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-38869471

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.


Asunto(s)
Unión Competitiva , Simulación de Dinámica Molecular , Señales de Localización Nuclear , alfa Carioferinas , alfa Carioferinas/metabolismo , alfa Carioferinas/química , alfa Carioferinas/antagonistas & inhibidores , Ligandos , Señales de Localización Nuclear/química , Virus de la Encefalitis Equina Venezolana/metabolismo , Virus de la Encefalitis Equina Venezolana/química , Unión Proteica , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Secuencia de Aminoácidos
2.
Nature ; 598(7882): 677-681, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34646021

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is an enveloped RNA virus that causes encephalitis and potentially mortality in infected humans and equines1. At present, no vaccines or drugs are available that prevent or cure diseases caused by VEEV. Low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3) was recently identified as a receptor for the entry of VEEV into host cells2. Here we present the cryo-electron microscopy structure of the LDLRAD3 extracellular domain 1 (LDLRAD3-D1) in complex with VEEV virus-like particles at a resolution of 3.0 Å. LDLRAD3-D1 has a cork-like structure and is inserted into clefts formed between adjacent VEEV E2-E1 heterodimers in the viral-surface trimer spikes through hydrophobic and polar contacts. Mutagenesis studies of LDLRAD3-D1 identified residues that are involved in the key interactions with VEEV. Of note, some of the LDLRAD3-D1 mutants showed a significantly increased binding affinity for VEEV, suggesting that LDLRAD3-D1 may serve as a potential scaffold for the development of inhibitors of VEEV entry. Our structures provide insights into alphavirus assembly and the binding of receptors to alphaviruses, which may guide the development of therapeutic countermeasures against alphaviruses.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/química , Receptores de LDL/química , Receptores Virales/química , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Internalización del Virus
3.
Nature ; 598(7882): 672-676, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34646020

RESUMEN

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus-receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/química , Receptores de LDL/química , Receptores Virales/química , Secuencia de Aminoácidos , Animales , Línea Celular , Microscopía por Crioelectrón , Humanos , Ratones , Modelos Moleculares , Estructura Secundaria de Proteína , Alineación de Secuencia , Internalización del Virus
4.
Antiviral Res ; 182: 104905, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32800880

RESUMEN

There is a pressing need for new vaccines against alphaviruses, which can cause fatal encephalitis (Venezuelan equine encephalitis virus (VEEV) and others) and severe arthralgia (e.g. Chikungunya virus, CHIKV). These positive-strand RNA viruses are diverse and evolve rapidly, meaning that the sequence of any vaccine should cover multiple strains that may be quite different from any previous isolate. Here, consensus proteins were produced to represent the common physicochemical properties (PCPs) of the epitope rich, B domain of the E2 envelope protein. PCP-consensus proteins were based on multiple strains of VEEV (VEEVcon) and CHIKV (CHIKVcon) or the conserved PCPs of 24 different alphaviruses (AllAVcon). The AllAVcon was altered to include binding sites for neutralizing antibodies of both VEEV and CHIKV strains (Mosaikcon). All four designed proteins were produced solubly in E. coli and purified. They formed the ß-strand core expected from experimental structures of this region of the wild type E2 proteins as indicated by circular dichroism (CD) spectra. Furthermore, the CHIKVcon protein bound to a structure dependent, CHIKV neutralizing monoclonal antibody. The AllAVcon and Mosaikcon proteins bound to polyclonal antibodies generated during natural infection with either VEEV or CHIKV, indicating they contained epitopes of both serotypes. The Mosaikcon antigen induced antibodies in rabbit sera that recognized both the VEEVcon and CHIKVcon spike proteins. These PCP-consensus antigens are promising starting points for novel, broad-spectrum alphavirus vaccines.


Asunto(s)
Alphavirus/química , Alphavirus/inmunología , Anticuerpos Antivirales/sangre , Proteínas Virales/química , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Animales , Antígenos Virales/química , Antígenos Virales/inmunología , Virus Chikungunya/química , Virus Chikungunya/inmunología , Dicroismo Circular , Consenso , Diseño de Fármacos , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/inmunología , Epítopos/inmunología , Adyuvante de Freund/administración & dosificación , Masculino , Espectrometría de Masas , Conejos , Vacunas Virales/administración & dosificación
5.
Nucleic Acids Res ; 46(7): 3657-3670, 2018 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-29361131

RESUMEN

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , ARN/genética , Virus Sindbis/genética , Replicación Viral/genética , Alphavirus/química , Alphavirus/genética , Alphavirus/patogenicidad , Animales , Encefalitis/genética , Encefalitis/virología , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/patogenicidad , Genoma Viral/genética , Caballos/virología , Humanos , Conformación de Ácido Nucleico , ARN/química , Virus Sindbis/química , Virus Sindbis/patogenicidad
6.
Antiviral Res ; 131: 49-60, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27105836

RESUMEN

The mosquito-borne New World alphavirus, Venezuelan equine encephalitis virus (VEEV) is a Category B select agent with no approved vaccines or therapies to treat infected humans. Therefore it is imperative to identify novel targets that can be targeted for effective therapeutic intervention. We aimed to identify and validate interactions of VEEV nonstructural protein 3 (nsP3) with host proteins and determine the consequences of these interactions to viral multiplication. We used a HA tagged nsP3 infectious clone (rTC-83-nsP3-HA) to identify and validate two RNA helicases: DDX1 and DDX3 that interacted with VEEV-nsP3. In addition, DDX1 and DDX3 knockdown resulted in a decrease in infectious viral titers. Furthermore, we propose a functional model where the nsP3:DDX3 complex interacts with the host translational machinery and is essential in the viral life cycle. This study will lead to future investigations in understanding the importance of VEEV-nsP3 to viral multiplication and apply the information for the discovery of novel host targets as therapeutic options.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Virus de la Encefalitis Equina Venezolana/metabolismo , Interacciones Huésped-Patógeno , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Virus de la Encefalitis Equina Venezolana/química , Técnicas de Silenciamiento del Gen , Humanos , ARN Helicasas/metabolismo , Células Vero , Carga Viral , Replicación Viral
7.
J Virol ; 88(17): 9616-23, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24920796

RESUMEN

UNLABELLED: Alphaviruses are serious, sometimes lethal human pathogens that belong to the family Togaviridae. The structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy and homology modeling. We characterize these monoclonal antibody Fab fragments, which are known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both of these antibody Fab fragments cross-link the surface E2 glycoproteins and therefore probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain. IMPORTANCE: Alphaviral infections are transmitted mainly by mosquitoes. Venezuelan equine encephalitis virus (VEEV) is an alphavirus with a wide distribution across the globe. No effective vaccines exist for alphaviral infections. Therefore, a better understanding of VEEV and its associated neutralizing antibodies will help with the development of effective drugs and vaccines.


Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Virus de la Encefalitis Equina Venezolana/química , Sustancias Macromoleculares/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Simulación por Computador , Microscopía por Crioelectrón , Virus de la Encefalitis Equina Venezolana/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Unión Proteica , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología
8.
Appl Microbiol Biotechnol ; 97(14): 6359-72, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23512478

RESUMEN

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108-119 (domain A), a subtype I/IV-specific epitope at amino acids 211-226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231-246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131-146 and 241-256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.


Asunto(s)
Virus de la Encefalitis Equina del Este/inmunología , Encefalomielitis Equina/virología , Epítopos de Linfocito B/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Equina del Este/química , Virus de la Encefalitis Equina del Este/clasificación , Virus de la Encefalitis Equina del Este/genética , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/clasificación , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Humanos , Ratones , Especificidad de la Especie , Spodoptera , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética
9.
J Vis Exp ; (70): e4429, 2012 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-23222916

RESUMEN

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.


Asunto(s)
Sistema Nervioso Central/virología , Virus de la Encefalitis Equina Venezolana/fisiología , Mediciones Luminiscentes/métodos , Animales , Encéfalo/virología , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/inmunología , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/química , Luciferasas de Luciérnaga/genética , Ratones , Vacunas Atenuadas/química , Vacunas Atenuadas/inmunología , Vacunas Virales/química , Vacunas Virales/inmunología , Replicación Viral
10.
J Mol Model ; 18(1): 39-51, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21445710

RESUMEN

To date, no suitable vaccine or specific antiviral drug is available to treat Chikungunya viral (CHIKV) fever. Hence, it is essential to identify drug candidates that could potentially impede CHIKV infection. Here, we present the development of a homology model of nsP2 protein based on the crystal structure of the nsP2 protein of Venezuelan equine encephalitis virus (VEEV). The protein modeled was optimized using molecular dynamics simulation; the junction peptides of a nonstructural protein complex were then docked in order to investigate the possible protein-protein interactions between nsP2 and the proteins cleaved by nsP2. The modeling studies conducted shed light on the binding modes, and the critical interactions with the peptides provide insight into the chemical features needed to inhibit the CHIK virus infection. Energy-optimized pharmacophore mapping was performed using the junction peptides. Based on the results, we propose the pharmacophore features that must be present in an inhibitor of nsP2 protease. The resulting pharmacophore model contained an aromatic ring, a hydrophobic and three hydrogen-bond donor sites. Using these pharmacophore features, we screened a large public library of compounds (Asinex, Maybridge, TOSLab, Binding Database) to find a potential ligand that could inhibit the nsP2 protein. The compounds that yielded a fitness score of more than 1.0 were further subjected to Glide HTVS and Glide XP. Here, we report the best four compounds based on their docking scores; these compounds have IDs of 27943, 21362, ASN 01107557 and ASN 01541696. We propose that these compounds could bind to the active site of nsP2 protease and inhibit this enzyme. Furthermore, the backbone structural scaffolds of these four lead compounds could serve as building blocks when designing drug-like molecules for the treatment of Chikungunya viral fever.


Asunto(s)
Virus Chikungunya/química , Virus Chikungunya/enzimología , Cisteína Endopeptidasas/química , Simulación de Dinámica Molecular , Infecciones por Alphavirus/tratamiento farmacológico , Secuencia de Aminoácidos , Sitios de Unión , Fiebre Chikungunya , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/enzimología , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Alineación de Secuencia
11.
J Mol Graph Model ; 29(3): 347-53, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21036084

RESUMEN

The outbreaks of chikungunya (CHIKV) and venezuelan equine encephalitis (VEEV) viral infections in humans have emerged or re-emerged in various countries of "Africa and southeast Asia", and "central and south America", respectively. At present, no drug or vaccine is available for the treatment and therapy of both viral infections, but the non-structural protein, nsP3, is a potential target for the design of potent inhibitors that fit at the adenosine-binding site of its macro domain. Here, so as to understand the fundamental basis of the particular interactions between the ADP-ribose bound to the nsP3 amino acid residues at the binding site, molecular dynamics simulations were applied. The results show that these two nsP3 domains share a similar binding pattern for accommodating the ADP-ribose. The ADP-ribose phosphate unit showed the highest degree of stabilization through hydrogen bond interactions with the nsP3 V33 residue and the consequent amino acid residues 110-114. The adenine base of ADP-ribose was specifically recognized by the conserved nsP3 residue D10. Additionally, the ribose and the diphosphate units were found to play more important roles in the CHIKV nsP3-ADP-ribose complex, while the ter-ribose was more important in the VEEV complex. The slightly higher binding affinity of ADP-ribose toward the nsP3 macro domain of VEEV, as predicted by the simulation results, is in good agreement with previous experimental data. These simulation results provide useful information to further assist in drug design and development for these two important viruses.


Asunto(s)
Adenosina Difosfato Ribosa/química , Adenosina Difosfato Ribosa/metabolismo , Virus Chikungunya/química , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Animales , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Termodinámica , Proteínas no Estructurales Virales/genética
12.
Virology ; 406(2): 261-9, 2010 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-20701942

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/virología , Ensamble de Virus , Alphavirus/química , Alphavirus/aislamiento & purificación , Alphavirus/fisiología , Alphavirus/ultraestructura , Animales , Cricetinae , Virus de la Encefalitis Equina Venezolana/aislamiento & purificación , Virus de la Encefalitis Equina Venezolana/ultraestructura , Humanos , Modelos Moleculares , Nucleocápside/química , Nucleocápside/aislamiento & purificación , Nucleocápside/ultraestructura
13.
J Virol ; 83(13): 6534-45, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19386706

RESUMEN

Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1''-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1''-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.


Asunto(s)
Virus Chikungunya/química , Virus de la Encefalitis Equina Venezolana/química , Proteínas no Estructurales Virales/química , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Virus Chikungunya/genética , Secuencia Conservada , Virus de la Encefalitis Equina Venezolana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , ARN Viral/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética
14.
J Mol Model ; 12(6): 921-9, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16607494

RESUMEN

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1-E1 and E1-E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/química , Proteínas del Envoltorio Viral/química , Aminoácidos , Animales , Sitios de Unión , Dimerización , Virus de la Encefalitis Equina Venezolana/patogenicidad , Caballos , Complejos Multiproteicos/química
15.
Bioorg Khim ; 25(4): 253-6, 1999 Apr.
Artículo en Ruso | MEDLINE | ID: mdl-10422590

RESUMEN

Homogeneous (according to PAGE) capsid and surface viral proteins were isolated from concentrated purified suspensions of tick-borne encephalitis and Venezuelan equine encephalomyelitis viruses by one-stage reversed-phase HPLC. The amino acid composition and the sequences of their N-terminal parts were determined.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Transmitidos por Garrapatas/química , Proteínas Estructurales Virales/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Proteínas Estructurales Virales/química
16.
Anal Chem ; 70(18): 3863-7, 1998 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-9751026

RESUMEN

Matrix-assisted laser desorption/ionization mass spectrometry has enabled viral coat proteins to be characterized directly from the virus. This analysis, demonstrated here with tobacco mosaic virus U2, a bacteriophage MS2, and equine encephalitis TRD, is achieved with a combination of organic acid, UV-absorbing matrix, and high-energy desorption with a nitrogen laser. The molecular weights of these proteins are determined with sufficient accuracy to allow differentiation among viral species and strains. The abundant hydrophobic MS2 coat protein was analyzed in aliquots of culture medium and of the tobacco mosaic virus coat protein in infected leaves. This method provides rapid detection of coat protein in the low-femtomole range, as estimated by titering plaque-forming units of MS2.


Asunto(s)
Proteínas de la Cápside , Cápside/análisis , Virus/química , Virus de la Encefalitis Equina Venezolana/química , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Virus del Mosaico del Tabaco/química , Proteínas Virales/análisis
17.
Virology ; 219(1): 314-20, 1996 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-8623548

RESUMEN

The complete nuleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC-P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97--100% identity) and more distantly related to VEE strains of other antigenic varieties (83--93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94--95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1--329), which is highly conserved among all alphaviruses, was 93--94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330--563) was only 59--61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts.


Asunto(s)
Secuencia Conservada , Virus de la Encefalitis Equina Venezolana/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Virus de la Encefalitis Equina Venezolana/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/química , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética
18.
Vopr Virusol ; 38(4): 162-7, 1993.
Artículo en Ruso | MEDLINE | ID: mdl-7694427

RESUMEN

Nine peptides were synthesized for detailed mapping of VEE virus E2-2 and E2-6 sites responsible for the formation of protective antibodies that neutralize the virus and block hemagglutination. The sequence of the peptides over-lapped the regions of amino acid residues 30-75 and 202-250 of VEE virus E2 protein in which antigenic mutations caused by monoclonal antibodies to E2-2 and E2-6 sites had been mapped. None of the synthesized peptides reacted in the enzyme immunoassay with a panel of 17 Mabs to VEE virus E2 protein. However, eight peptides reacted with polyclonal antiviral serum and two of them elicited antiviral antibody production. The E2-2 site might be associated with amino acid residues 30-45, and the region of E2 residues 57-62 in which antigenic mutations are observed is not a linear type antigenic determinant, but participates in the formation of antigenic determinants of the conformational type. The mapping of residues 202-250 demonstrated that all the peptides in this region were well recognized by polyclonal antiviral serum. The residues 235-240 were shown to form a linear epitope which provided a crossover between VEE and EEE viruses and was not recognized by 19 types of monoclonal antibodies cross-reacting with VEE and EEE viruses.


Asunto(s)
Sitios de Unión de Anticuerpos/inmunología , Virus de la Encefalitis Equina Venezolana/química , Mapeo Peptídico/métodos , Proteínas del Envoltorio Viral/análisis , Proteínas Virales de Fusión/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos Virales/análisis , Antígenos Virales/inmunología , Virus de la Encefalitis Equina Venezolana/inmunología , Epítopos/análisis , Epítopos/inmunología , Hibridomas/inmunología , Inmunización , Técnicas Inmunológicas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación/inmunología , Péptidos/síntesis química , Ratas , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales de Fusión/inmunología
19.
J Gen Virol ; 74 ( Pt 3): 519-23, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8445371

RESUMEN

Enzootic strains of Venezuelan equine encephalitis (VEE) virus occur in the United States (Florida), Mexico, Central America and South America. Epizootic VEE first occurred in North and Central America in a widespread outbreak between 1969 and 1972. To investigate the likelihood that this epizootic VEE virus, identified as VEE antigenic subtype I-AB, evolved from enzootic viruses extant in the region, we cloned and sequenced the 26S mRNA region of the genomes of the Florida VEE subtype II virus, strain Everglades Fe3-7c, and the Middle American subtype I-E virus, strain Mena II. This region of the genome encodes the viral structural proteins. The sequences of the 26S mRNA regions of the Everglades and Mena virus genomes differed from that of the reference epizootic VEE subtype I-AB virus, Trinidad donkey strain, by 453 and 887 nucleotides and by 66 and 131 amino acids, respectively. These data confirm previous reports demonstrating significant antigenic and genetic distance between VEE I-AB virus and viruses of subtypes I-E and II. It is unlikely that the epizootic VEE I-AB virus responsible for the 1969 outbreak originated from mutation of enzootic VEE viruses in North or Middle America.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , ARN Mensajero/genética , ARN Viral/genética , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Evolución Biológica , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/clasificación , Genoma Viral , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
20.
J Gen Virol ; 72 ( Pt 10): 2431-5, 1991 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-1919525

RESUMEN

Four monoclonal antibody-resistant variants (MARVs) of Venezuelan equine encephalitis (VEE) virus were used to study mosquito-virus interactions. In vitro experiments using an Aedes albopictus cell line, C6/36, demonstrated that an amino acid change in the glycoprotein E2h epitope (MARV 1A3B-7) decreased virus growth when compared with the wild-type, Trinidad donkey virus, and its vaccine derivative, TC-83. The MARVs replicated as efficiently as the parent virus when inoculated into Aedes aegypti mosquitoes, but MARV 1A3B-7 was restricted in its ability to infect and disseminate from the midgut following oral infection. These results demonstrate that a single amino acid change in the E2 glycoprotein can affect the ability of VEE virus to replicate and disseminate in Ae. aegypti mosquitoes.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Proteínas del Envoltorio Viral/genética , Replicación Viral , Aedes/microbiología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Cricetinae , Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/fisiología , Insectos Vectores/microbiología , Cinética , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología
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