RESUMEN
In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35â¯kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Ectromelia/inmunología , Virus de la Viruela/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Virus de la Ectromelia/genética , Mapeo Epitopo , Humanos , Pruebas de Neutralización , Biblioteca de Péptidos , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Viruela/inmunología , Viruela/virología , Virus de la Viruela/química , Virus de la Viruela/genética , Virus de la Viruela/fisiología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK.
Asunto(s)
Aciclovir/análogos & derivados , Antivirales/química , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Virus de la Viruela/química , Proteínas Virales/antagonistas & inhibidores , Dominio Catalítico , Cidofovir , Citosina/análogos & derivados , Citosina/química , Diseño de Fármacos , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Nucleósido-Fosfato Quinasa/química , Nucleósido-Fosfato Quinasa/genética , Organofosfonatos/química , Viruela/tratamiento farmacológico , Viruela/virología , Especificidad de la Especie , Relación Estructura-Actividad , Termodinámica , Virus de la Viruela/enzimología , Virus de la Viruela/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a ß-sandwich fold and utilizes its ß-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions.
Asunto(s)
Quimiocina CX3CL1/química , Receptores del Factor de Necrosis Tumoral/química , Virus de la Viruela/química , Proteínas Virales/química , Sustitución de Aminoácidos , Animales , Línea Celular , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/metabolismo , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Mutación Missense , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Relación Estructura-Actividad , Virus de la Viruela/genética , Virus de la Viruela/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Vaccinia virus complement control protein (VCP) is one of the proteins encoded by vaccinia virus to modulate the host inflammatory response. VCP modulates the inflammatory response and protects viral habitat by inhibiting the classical and the alternative pathways of complement activation. The extended structure of VCP, mobility between its sequential domains, charge distribution and type of residues at the binding regions are factors that have been identified to influence its ability to bind to complement proteins. We report that a Lister strain of vaccinia virus encodes a VCP homolog (Lis VCP) that is functional, glycosylated, has two amino acids less than the well-characterized VCP from vaccinia virus WR strain (WR VCP), and the human smallpox inhibitor of complement enzymes (SPICE) from variola virus. The glycosylated VCP of Lister is immunogenic in contrast to the weak immunogenicity of the nonglycosylated VCP. Lis VCP is the only orthopoxviral VCP homolog found to be glycosylated, and we speculate that glycosylation influences its pattern of complement inhibition. We also correlate dimerization of VCP observed only in mammalian and baculovirus expression systems to higher levels of activity than monomers, observed in the yeast expression system.
Asunto(s)
Proteínas Recombinantes/metabolismo , Virus Vaccinia/inmunología , Virus Vaccinia/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Activación de Complemento/efectos de los fármacos , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Dimerización , Expresión Génica , Glicosilación , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Viruela/inmunología , Viruela/metabolismo , Relación Estructura-Actividad , Virus Vaccinia/química , Virus de la Viruela/química , Virus de la Viruela/inmunología , Virus de la Viruela/metabolismo , Proteínas Virales/genética , Proteínas Virales/farmacologíaRESUMEN
En los primeros años del sigo XIX, Juan José Heydeck propone la realización de unos experimentos con el pus de cabras en lugar del pus de vacas, para luchar contra las viruelas naturales. Estos experimentos requerían un número indeterminado de niños donde verificarlos. En España, el pus de cabras sería una alternativa al pus de vaca. En estos territorios era difícil encontrar el pus de vacas. Esta iniciativa del pus hircino no tuvo el éxito esperado
First years of the XIX century, Juan José Heydeck proposes the realization of the experiments with the pus of the goats instead of the pus of the cows, for combat the smallpox. This experiments require more childrens, where to be verified them. In Spain, the pus of the goats will be the one alternative to the p us of the cows. In this country is difficult fall in with the pus of the cows. This iniciative haven´t the hope results
Asunto(s)
Historia del Siglo XIX , Viruela/epidemiología , Viruela/historia , Vacuna contra Viruela/inmunología , Vacuna contra Viruela/uso terapéutico , Medicina Tradicional/historia , Experimentación Humana/historia , Ensayo Patogenético Homeopático/historia , Alternativas al Uso de Animales/métodos , Virus de la Viruela/química , Virus de la Viruela , Virus de la Viruela/patogenicidad , España/epidemiologíaRESUMEN
The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.
Asunto(s)
Complemento C3b/antagonistas & inhibidores , Complemento C4b/antagonistas & inhibidores , Monkeypox virus/química , Monkeypox virus/inmunología , Virus Vaccinia/química , Virus de la Viruela/química , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Enzimas Activadoras de Complemento/metabolismo , Complemento C3b/inmunología , Complemento C3b/metabolismo , Complemento C4b/inmunología , Complemento C4b/metabolismo , Cricetinae , Dimerización , Heparina/metabolismo , Humanos , Datos de Secuencia Molecular , Monkeypox virus/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Virus Vaccinia/inmunología , Virus de la Viruela/inmunologíaRESUMEN
We present immunophysical modeling for VCP, SPICE, and three mutants using MD simulations and Poisson-Boltzmann-type electrostatic calculations. VCP and SPICE are homologous viral proteins that control the complement system by imitating, structurally and functionally, natural regulators of complement activation. VCP and SPICE consist of four CCP modules connected with short flexible loops. MD simulations demonstrate that the rather complex modules of VCP/SPICE and their mutants exhibit a high degree of intermodular spatial mobility, which is affected by surface mutations. Electrostatic calculations using snapshots from the MD trajectories demonstrate variable spatial distribution of the electrostatic potentials, which suggests dynamic binding properties. We use covariance analysis to identify correlated modular oscillations. We also use electrostatic similarity indices to cluster proteins with common electrostatic properties. Our results are compared with experimental data to form correlations between the overall positive electrostatic potential of VCP/SPICE with binding and activity. We show how these correlations can be used to predict binding and activity properties. This work is expected to be useful for understanding the function of native CCP-containing regulators of complement activation and receptors and for the design of antiviral therapeutics and complement inhibitors.
Asunto(s)
Proteínas Inactivadoras de Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Termodinámica , Virus Vaccinia/inmunología , Virus de la Viruela/inmunología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Valor Predictivo de las Pruebas , Estructura Terciaria de Proteína , Especias , Electricidad Estática , Virus Vaccinia/química , Virus de la Viruela/químicaRESUMEN
Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.
Asunto(s)
Anticuerpos Monoclonales/química , Virus de la Ectromelia/química , Epítopos/química , Proteínas Recombinantes de Fusión/química , Virus de la Viruela/química , Proteínas Virales/química , Animales , Anticuerpos Monoclonales/inmunología , Virus de la Ectromelia/genética , Virus de la Ectromelia/inmunología , Epítopos/genética , Epítopos/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Especificidad de la Especie , Virus de la Viruela/genética , Virus de la Viruela/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Although eradicated from nature more than two decades ago, the threat of smallpox has reemerged because of concerns over its use as a biological weapon. We present the structure of the poxvirus L1 protein, a molecule that is conserved throughout the poxvirus family and is nearly identical in vaccinia virus and in variola virus, which causes smallpox. L1 is a myristoylated envelope protein that is a potent target for neutralizing antibodies and an important component of current experimental vaccines. The L1 structure reveals a hydrophobic cavity located adjacent to its N terminus. The cavity would be capable of shielding the myristate moiety, which is essential for virion assembly. The structure of L1 is a step in the elucidation of molecular mechanisms common to all poxviruses that may stimulate the design of safer vaccines and new antipoxvirus drugs.
Asunto(s)
Poxviridae/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales , Secuencia de Bases , Cristalografía por Rayos X , ADN Viral/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Disulfuros/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Mirísticos/química , Pruebas de Neutralización , Poxviridae/genética , Poxviridae/inmunología , Conformación Proteica , Homología de Secuencia de Aminoácido , Vacuna contra Viruela/química , Vacuna contra Viruela/genética , Vacuna contra Viruela/inmunología , Electricidad Estática , Virus Vaccinia/química , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus de la Viruela/química , Virus de la Viruela/genética , Virus de la Viruela/inmunología , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.
Asunto(s)
Poxviridae/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Mutagénesis , Filogenia , Mutación Puntual , Proteínas Recombinantes de Fusión , Proteínas Recombinantes , Alineación de Secuencia , Relación Estructura-Actividad , Transfección , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Virus de la Viruela/química , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Variola, the causative agent of smallpox, is a highly infectious double-stranded DNA virus of the orthopox genus that replicates within the cytoplasm of infected cells. For unknown reasons prominent skin manifestations, including "pox," mark the course of this systemic human disease. Here we characterized smallpox growth factor (SPGF), a protein containing an epidermal growth factor (EGF)-like domain that is conserved among orthopox viral genomes, and investigated its possible mechanistic link. We show that after recombinant expression, refolding, and purification, the EGF domain of SPGF binds exclusively to the broadly expressed cellular receptor, erb-B1 (EGF receptor), with subnanomolar affinity, stimulating the growth of primary human keratinocytes and fibroblasts. High affinity monoclonal antibodies specific for SPGF reveal in vivo immunoprotection in a murine vaccinia pneumonia model by a mechanism distinct from viral neutralization. These findings suggest that blockade of pathogenic factor actions, in general, may be advantageous to the infected host.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sustancias de Crecimiento/fisiología , Virus de la Viruela/química , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Células Cultivadas , Secuencia Conservada , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/química , Humanos , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/biosíntesis , Datos de Secuencia Molecular , Péptidos/metabolismo , Proteínas Virales/análisis , Proteínas Virales/químicaRESUMEN
Molluscum contagiosum virus (MCV) commonly causes asymptomatic cutaneous neoplasms in children and sexually active adults as well as persistent opportunistic acquired immunodeficiency syndrome (AIDS)-associated disease. Sequencing the 190-kilobase pair genome of MCV has now revealed that the virus potentially encodes 163 proteins, of which 103 have homologs in the smallpox virus. MCV lacks counterparts to 83 genes of the smallpox virus, including those important in suppression of host responses to infection, nucleotide biosynthesis, and cell proliferation. MCV possesses 59 genes that are predicted to encode previously uncharacterized proteins, including major histocompatibility complex class I, chemokine, and glutathione peroxidase homologs, which suggests that there are MCV-specific strategies for coexistence with the human host.
Asunto(s)
Genoma Viral , Virus del Molusco Contagioso/genética , Proteínas Virales/química , Secuencia de Aminoácidos , Composición de Base , Quimiocinas/química , Quimiocinas/genética , ADN Viral/genética , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Datos de Secuencia Molecular , Virus del Molusco Contagioso/química , Virus del Molusco Contagioso/patogenicidad , Sistemas de Lectura Abierta , Orthopoxvirus/química , Orthopoxvirus/genética , Alineación de Secuencia , Virus de la Viruela/química , Virus de la Viruela/genética , Proteínas Virales/genéticaRESUMEN
Computer analysis of full coding sequences of variola major virus strain India-1967 genome and vaccinia virus strain Copenhagen genome have been carried out. A wide set of proteins containing ankyrin-like repeats have been identified for both viruses. Only three proteins of this family of the studied viruses are highly homologous. The rest of the proteins are different. The possible role of such proteins in determination of virus tissue tropism is discussed.
Asunto(s)
Ancirinas/química , Virus Vaccinia/química , Virus de la Viruela/química , Proteínas Virales/química , Secuencia de Aminoácidos , Secuencia de Consenso , Genes Virales , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Virus Vaccinia/genética , Virus de la Viruela/genética , Proteínas Virales/genéticaRESUMEN
Analysis of variola virus nucleotide sequence revealed proteins belonging to several families which provide the virus with the possibility of overcoming the barriers of specific and non-specific host defence against viral infection. The complement-binding proteins, lymphokine-binding proteins, and serine protease inhibitors can be assigned to this type, as can the proteins providing the orthopoxviruses with resistance to interferon. The revealed differences between the genes (proteins) of variola and vaccinia viruses under study are discussed.