RESUMO
Previously different authors described various flavivirus mutants with high affinity to cell glycosaminoglycans and low neuroinvasiveness in mice that were obtained consequently passages in cell cultures or in ticks. In present study the analysis of TBEV isolates has shown existence of GAG-binding variants in natural virus population. Affinity to GAG has been evaluated by sorption on heparin-Sepharose. GAG-binding phenotype corresponds to such virus properties, like small plaque phenotype in PEK cells, absence of hemagglutination at pH 6.4, and low neuroinvasiveness in mice. Mutations increasing charge of E protein were necessary but not sufficient for acquisition of GAG-binding phenotype. Molecular modeling and molecular dynamics simulation have shown that the flexibility of E protein molecule could bear influence on the phenotypic manifestation of substitutions increasing charge of the virions.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Produtos do Gene gag/metabolismo , Animais , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/virologia , Variação Genética/genética , Testes de Hemaglutinação , Imunoeletroforese , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Fenótipo , Sefarose/análogos & derivados , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
A model for virion RNA of the poliomyelitis virus, which does not pass the stage of DNA copies during biogenesis, demonstrates that Taq DNA polymerase is capable of synthesizing 960-bp cDNA with the specific primer. When comparing the nucleotide sequence of the starting virion RNA and recombinant DNAs, isolated from several independent clones, copying and amplification of virion RNA appear accurate (one substitution per 960 bp). A comparison of Taq and Tth DNA polymerases in RT/PCR indicates that the sensitivity of Taq polymerase seems to be two orders of magnitude higher than that of Tth polymerase. The RNA detection level under the chosen conditions approached 10(4) RNA copies per test. The present investigation indicates the great versatility of Taq polymerase, which promoted the reverse transcription reaction of RNA, cDNA amplification, screening of recombinant clones as well as sequencing of recombinant DNA. Thus application of Taq polymerase is rather promising not only to detect nucleic acids in biological samples, but also for isolating and cloning individual genes, encoded on DNA and/or on RNA templates.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Poliovirus/genética , RNA Viral/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Primers do DNA , DNA Viral , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , Taq Polimerase , Transcrição GênicaRESUMO
The rate of the synthesis of tick-borne encephalitis (TBE) virus-specified proteins at high multiplicity of infection (MOI of 100 PFU per cell) was the highest by 8-14 hr post-infection (p.i.) as compared to the lower MOI of 4 PFU per cell (14 to 17 hr p.i.). The first virus-specific proteins appeared in cells from 2 to 5 hr p.i.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Proteínas Virais/biossíntese , Animais , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Camundongos , Proteínas Virais/análise , Replicação ViralRESUMO
The time course of tick-borne encephalitis virus cell-free protein synthesis was studied by using either [35S]-methionine or formyl[35S]methionyl-tRNAMet/f as substrates, and the [35S]methionine-labelled products were compared by fingerprinting tryptic peptides. An intermediate in the protein processing, the polypeptide doublet p36/33, was characterized and a tentative model for flavivirus structural protein synthesis and processing was proposed.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , RNA de Transferência de Metionina , Proteínas Virais/biossíntese , Sequência de Aminoácidos , Modelos Biológicos , Aminoacil-RNA de Transferência/metabolismo , Radioisótopos de Enxofre , Proteínas Estruturais ViraisRESUMO
Tick-borne encephalitis virus (TBEV) RNA was translated in extracts from Krebs-2 cells and in rabbit reticulocyte lysates. In the former system, two polypeptides, p53 and p13, corresponding to envelope (E) and core (C) proteins of the virion, respectively, were synthesized preferentially. In contrast, the major product in reticulocyte lysates was represented by a heterogeneous set of high-molecular-weight polypeptides which did not appear to include p53 or p13. The reticulocyte lysates, however, acquired the ability to produce structural proteins (p53 at least) after addition of purified membranes isolated from the rough endoplasmic reticulum of Krebs-2 cells. On the other hand, the ability of Krebs-2 extracts to generate identifiable viral structural proteins was lost after degradation of membranes by the nonionic detergent Triton X-100. These findings strongly suggest that membrane-dependent processing of protein precursors is involved in the formation of TBEV structural proteins. Evidence has been obtained that only nascent precursor polypeptides can be processed efficiently into structural proteins in the membrane-dependent reaction.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Biossíntese de Proteínas , RNA Viral/genética , Proteínas Virais/genética , Animais , Carcinoma Krebs 2/metabolismo , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Camundongos , Peso Molecular , Coelhos , Reticulócitos/metabolismo , Proteínas Virais/isolamento & purificaçãoAssuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Biossíntese de Proteínas , RNA Viral/genética , Proteínas Virais/biossíntese , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Biossíntese Peptídica , Precursores de Proteínas/biossíntese , Proteínas Virais/genéticaRESUMO
Purified virions of tick-borne encephalitis (TBE) virus contain 3 proteins, V1, V2 and V3, with molecular weights of 8 000, 13 000 and 53 000 daltons, respectively. Seven virus-specific polypeptides were revealed in TBE-virus infected continuous pig embryo kidney cells, namely p93-96, p79, p69, p53, p47, p16 and p13 (pN designating polypeptide with a mol wt of N X 1 000 daltons). Proteins p93-96, p69, p53, p47 and p13 corresponded by their mol wt to proteins NV5, NV4, V3 and V2 (NV1 1/2) of mosquito-borne flaviviruses. Protein p79, designated NV4 1/2, and protein p16 (the only virus-specific protein inhibited by hypertonic NaCl concentrations), had no analogues among proteins of mosquito-borne flaviviruses. The possibility of a cellular origin of protein p47 (NV3) is discussed.