RESUMEN

Recombinant polypeptide containing the 260-466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E(260-466)) was constructed. Immunochemical similarity between the E(260-466) and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E(260-466). Polypeptide E(260-466) induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E(260-466) with one of the proposed cell receptors of WNV that average E(260-466)-alphaVbeta3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and alphaVbeta3 integrin) during virus entry.

Asunto(s)

Integrina alfa5/química , Proteínas Recombinantes de Fusión/química , Proteínas del Envoltorio Viral/química , Virus del Nilo Occidental/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Epítopos/química , Epítopos/inmunología , Humanos , Integrina alfa5/metabolismo , Microscopía de Fuerza Atómica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Proteínas del Envoltorio Viral/inmunología , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Mapeo Epitopo , Epítopos/química , Inmunoensayo , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Virus del Nilo Occidental/química , Virus del Nilo Occidental/genética

Asunto(s)

Biología Computacional/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regiones Promotoras Genéticas/genética , Proteínas Ribosómicas/genética , Secuencia de Bases , Técnicas Biosensibles , Escherichia coli/efectos de los fármacos , Peróxido de Hidrógeno/farmacología

RESUMEN

The level of laminin-binding protein (LBP) expression on cellular membranes was studied in three cell lines including 293 cells transformed by plasmide with human LBP gene. Vero cells show a high level of LBP on the cell surfaces and demonstrate a high level of the Venezuelan equine encephalomyelitis (VEE) virus replication. The inhibition of VEE virus replication was more than 200 times as much after treatment of Vero cell surfaces with monoclonal antibodies to human LBP. 293 cells have more low level of LBP on their surfaces but being transformed by plasmide with LBP human gene these cells showed an increase in the level of cellular LBP. The VEE virus replication in transformed cells (9S2) was more than 2000 times higher compared to 293 cells. The results obtained demonstrate a principal role of cellular LBP in VEE virus entry into mammalian cells. It can be proposed that LBP is a key cellular protein at the early stage of VEE virus replication in cells. So, LBP might be a target protein for development of some new generation of antiviral drugs that would be able to inhibit (enhance) the alphavirus replication in human cells.

Asunto(s)

Virus de la Encefalitis Equina Venezolana/fisiología , Receptores de Laminina/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Transfección , Células Vero , Replicación Viral

RESUMEN

The paper describes the structure and functions of Ebola virus properties. It also presents information on the role of structural (NP, VP40, VP35, GP, VP30, VP24, and L) and secreted (sGP, delta-peptide, GP1, GP(1,2delta), ssGP) proteins in the viral replication cycle and in the pathogenesis of Ebola hemorrhagic fever.

Asunto(s)

Ebolavirus/química , Proteínas Virales/fisiología , Ebolavirus/genética , Ebolavirus/metabolismo , Genoma Viral , Glicoproteínas/fisiología , Peso Molecular , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/fisiología

Asunto(s)

ARN Interferente Pequeño/genética , Replicación Viral , Virus del Nilo Occidental/genética , Animales , Antígenos/química , Células Cultivadas , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/metabolismo , Humanos , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Viral/química , Factores de Tiempo , Células Vero

RESUMEN

Fragments cDNA (nt 935-1475, 1091-1310, 935-1193) encoding N-terminal part of protein E of West Nile virus (WNV), strain LEIV-Vlg99-27889-human were obtained and cloned. Recombinant polypeptides of glycoprotein E (E1-86, E53-126, E1-180) of the WNV with corresponding amino acid sequence to the cloned fragments of cDNA and modeling the epitopes of domains I and II of surface glycoprotein E were purified by affinity chromatography. Twelve types of monoclonal antibodies (MAbs) created in our laboratory against recombinant polypeptide E1-180 interact with glycoprotein E of the WNV as results of Western blot and ELISA that is demonstrating an similarity of chemical structure of short recombinant polypeptides and corresponding amino acid sequence regions of WNV protein E. Analysis of interactions of MAbs with short recombinant polypeptides and protein E of tick-borne encephalitis virus let us reveal no less than six epitopes within domains I and II of glycoprotein E of the WNV. No less than seven types of MAbs to 86-126 aa region of the domain II were found where located peptide providing fusion of virus--cell membranes (98-110 aa). The epitope for anti-receptor MAbs 10H10 within 53-86 aa region of domain II of protein E of the WNV was mapped and it shows that the fusion peptide and co-receptor of protein E for cellular laminin-binding protein (LBP) are spatial nearness. X-ray model of protein E let us suppose that bc-loop (73-89 aa) of domain II interacts with LBP and together with cd-loop (fusion peptide) determines an initial stages of penetration virions into cell.

Asunto(s)

Glicoproteínas/inmunología , Epítopos Inmunodominantes/inmunología , Péptidos/inmunología , Proteínas Virales/inmunología , Virus del Nilo Occidental/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Células Cultivadas , Clonación Molecular , Mapeo Epitopo , Glicoproteínas/biosíntesis , Glicoproteínas/química , Humanos , Inmunoquímica , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Virales/biosíntesis , Proteínas Virales/química , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/metabolismo

RESUMEN

The occurrence rate of HGV/GBV-C RNA, genotypic variety of isolates and various risk factors of infection with HGV/GBV-C were evaluated in 500 patients of the narcological dispensary of Novosibirsk. The occurrence rate of HGV/GBV-C RNA among all examined blood sera was 33.6%. At the same time in blood sera with HCV markers the occurrence rate of HGV/ GBV-C was 42.9% and in sera with negative results for markers HCV--25%. For gene typing of obtained isolates the direct sequencing of the amplification products of fragment NS3B and the phylogenetic analysis of the sequences thus obtained were used. Almost all isolates subjected to gene typing belonged to genotype 2, widespread in Europe, and only 1 isolate was classified with genotype 4. Statistically significant (p<0.05) risk of HGV/GBV-C infection among the examined subjects was linked with the intravenous use of drugs (OR 2.15), risky sexual behavior (OR 1.8) and the presence of virus hepatitis C (OR 2.26).

Asunto(s)

Atención Ambulatoria , Infecciones por Flavivirus/virología , Virus GB-C/genética , Hepatitis Viral Humana/virología , Narcóticos , ARN Viral/genética , Abuso de Sustancias por Vía Intravenosa/virología , Adolescente , Adulto , Comorbilidad , Femenino , Infecciones por Flavivirus/sangre , Infecciones por Flavivirus/epidemiología , Virus GB-C/aislamiento & purificación , Genotipo , Hepatitis C/epidemiología , Hepatitis C/virología , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Factores de Riesgo , Siberia/epidemiología , Abuso de Sustancias por Vía Intravenosa/sangre , Abuso de Sustancias por Vía Intravenosa/epidemiología

RESUMEN

It was demonstrated by subtractive hybridization that the infection of a human embryonic kidney cell line with tick-borne encephalitis virus causes an approximately tenfold transcription activation of the RIG-1 gene, which encodes a protein of the DExH/D-box-containing RNA helicase family. A possible involvement of the protein in antiviral cell systems is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Asunto(s)

Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , ARN Helicasas/genética , Secuencia de Bases , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box , Cartilla de ADN , Humanos , Hibridación de Ácido Nucleico , Receptores Inmunológicos , Técnica de Sustracción

RESUMEN

The interaction of the VEE virus virions with human LBP was investigated. The affinely purified 43 kDa recombinant LBP (rLBP) of man was found to interact effectively with the VEE virus virions purified in immune enzyme assay. The affinity constant of 43 kDa rLBP with virions was equal to 4.3-4.8 x 10(7) M-1. The rabbit antiviral polyclonal antibodies blocked the interaction of rLBP with the VEE virus virions. According to Western blot, rLBP is capable of interacting with the E1 glycoprotein of the VEE virus, which suggests the presence of a specific epitope of binding with LBP in the surface of the E1-E2 heterodimer of the VEE virus. The results confirm that human LBP could be a receptor for the VEE virus.

Asunto(s)

Virus de la Encefalitis Equina Venezolana/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Laminina/metabolismo , Virión/metabolismo , Animales , Western Blotting , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/virología , Epítopos , Glicoproteínas/metabolismo , Enfermedades de los Caballos/virología , Caballos , Humanos , Precursores de Proteínas/genética , Conejos , Receptores de Laminina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virión/inmunología

RESUMEN

Site-directed mutagenesis was used to construct human adenovirus serotype 5 (Ad5) variants defective in E1A or E1B. Mutant Adel3 with deletion from E1A was markedly attenuated in permissive cell cultures regardless of the p53 status, and replicated efficiently only in cells of the complementing 293 line. Mutant Adel2 with deletion from E1B55K infected the 293 line cells and p53-deficient human tumor cells (A431, SW480, HEp2) with efficiencies similar to those of Ad5, whereas its replication in normal p53-positive cells was substantially limited. Thus, Adel2 proved to be capable of selective infection and lysis of p53-deficient human tumor cells in vitro. On intratumor injection, Adel2 dramatically suppressed the growth of human epidermoid carcinoma (A431) in nude mice. Adel2 is thus a promising model for designing therapeutic agents against p53-deficient human tumors.

Asunto(s)

Adenoviridae/genética , Eliminación de Gen , Genes Inmediatos-Precoces , Genes p53 , Replicación Viral/genética , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Células Tumorales Cultivadas

RESUMEN

The results of polymerase chain reaction and of DNA sequencing of the Adel2 mutant variant of adenovirus serotype 5, passaged 10 times and capable of selectively infecting and lysing the p53-deficient human tumor cells, are indicative of a high stability of its genotype and of the phenotypic properties acquired by it in successive passage on 293 cells. The absence of admixtures of wild-type adenovirus was clearly shown in the cultivation and passage processes. It was revealed in an experimental analysis of virus-productive properties of the studied continuous cell culture 293 by using the method of multilayer cultivation, that the maximal Adel2 yield is obtained at the 50% cytopathic effect. Virus doses, that are effective for cell-culture contamination, are within a range of 100-10 TCPE50 per cell. In order to spare the viral material, the infecting dose of 10 TCPE50 per cell was chosen to infect a cell monolayer.

Asunto(s)

Adenoviridae/crecimiento & desarrollo , Mutación , Adenoviridae/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa

RESUMEN

Five types of anti-VP35 monoclonal antibodies (MAbs), four immune sera against Marburg virus (MBGV), and 11 overlapping recombinant VP35 fragments were used to map the epitopes for VP35 of MBGV. The purified full-size recombinant VP35 was highly immunogenic and retained the B-cell epitopes that were identical to those of the viral VP35. Two major sites on VP35 and a set of truncated VP35 fragments were found by use of an enzyme immunoassay and immunoblot. Site I was located in a region between amino acids 1 and 174 of the VP35 sequence, and only polyclonal antibodies (PAbs) against MBGV recognized epitopes at this site. Site II was mapped by use of anti-VP35 MAbs to the region between amino acid residues 167 and 278 of VP35. Amino acids 252-278 of VP35 might be involved in the formation of the epitopes for MAbs. B-cell epitopes were not found on the C-terminus of VP35 by use of PAbs or MAbs.

Asunto(s)

Antígenos Virales/inmunología , Marburgvirus/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Humanos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Reguladoras y Accesorias Virales

Asunto(s)

Genoma Viral , Marburgvirus/genética , Transcripción Genética , Secuencia de Bases , Datos de Secuencia Molecular , ARN Catalítico/genética , Proteínas Virales/genética

RESUMEN

The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.

Asunto(s)

Marburgvirus/metabolismo , Nucleoproteínas/metabolismo , Proteínas de Unión al ARN , Ribonucleoproteínas , Proteínas Virales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Recombinante , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Escherichia coli/genética , Humanos , Microscopía Electrónica/métodos , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo

RESUMEN

The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.

Asunto(s)

Receptores de Laminina/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Riñón/química , Unión Proteica , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Antigenic structure of Marburg virus (MBG) VP35 was compared with that of the recombinant VP35 (f35) expressed in a prokaryotic system. For this purpose, a gene encoding the full-length VP35 was cloned in vector pQE31(QIAGEN) and expressed at about 70 mg/liter culture fluid in Escherichia coli JM103 as a recombinant fusion protein f35. BALB/c mice were immunized with soluble f35 or purified inactivated virions of MBG. Antibodies cross-reacting with VP35 and f35 antigens were detected by ELISA and Western Blot analysis in immune sera. Serum from a convalescent after Marburg disease and polyclonal antibodies from animals immunized with MBG recognized f35 and the MBG VP35. VP35 and its recombinant analog induced the production of specific antiviral antibodies in mice, which cross-reacted with the studied antigens. Competitive EIA showed that VP35 and f35 cross-inhibit the antigenic reactivity with polyclonal antibodies of immune sera. Antigenic structure of f35 protein corresponded to antigenic structure of MBG VP35 protein.

Asunto(s)

Antígenos Virales/inmunología , Marburgvirus/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Clonación Molecular , Reacciones Cruzadas , ADN Recombinante , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Reguladoras y Accesorias Virales