RESUMO
In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53(Gag) is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53(Gag) is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53(Gag) or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53(Gag) and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors.
Assuntos
Produtos do Gene gag/metabolismo , Genes gag , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Animais , Baculoviridae , Linhagem Celular , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Produtos do Gene gag/genética , Produtos do Gene gag/ultraestrutura , HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/ultraestrutura , Humanos , Mamíferos , Microscopia Eletrônica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Spodoptera , TransfecçãoRESUMO
The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.
Assuntos
Genes pol , Histidina , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Integrases/metabolismo , Fases de Leitura Aberta , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Vetores Genéticos , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Dados de Sequência Molecular , Peptídeos , Conformação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , DNA Polimerase Dirigida por RNA/químicaRESUMO
The proteases (PR) of retroviruses are expressed as gag-PR fused polyprotein. The active PR is a dimer obtained after the aggregation of the gag and gag-pro precursors, which leads to the formation and the release of the viral particle. Subsequently, in the cell, the PR is present essentially as a monomeric polyprotein. To mimic the antigenic properties of such an intracellular form of the PR, we produced a monomeric form of the HTLV-I (human T-cell leukemia virus, type-I) PR fused to the maltose binding protein (MBP-PR). Monoclonal antibodies (mabs) directed against MBP-PR were developed. Three mabs were obtained that recognized different epitopes. Two were directed against the NH2-terminus, a region that contributes to the dimerization interface. The other was specific to a peptide that lines the substrate binding pocket. This latter epitope is located just downstream of the D-T-G peptide of the catalytic site. The two identified regions contained the amino acids Asp6, Arg10 and Asp36, which were previously shown to be important in the stabilization of the dimer. In view of the localization of the recognized epitopes, these mabs will be useful for inhibition studies of the HTLV-I PR by intracellular immunization.
Assuntos
Anticorpos Monoclonais/imunologia , Endopeptidases/imunologia , Antígenos HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Animais , Western Blotting , Endopeptidases/química , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Antígenos HTLV-I/química , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Hibridomas , Camundongos , Modelos Moleculares , Conformação Proteica , Ensaio de RadioimunoprecipitaçãoRESUMO
The bovine leukemia virus Tax protein transactivates gene expression directed by the viral long terminal repeat (LTR) and contributes to immortalization of primary cells. Theoretical analysis of the protein sequence revealed the presence of a putative zinc finger structure at its amino end. Selected mutations in that region completely abolished transactivation, demonstrating its importance for LTR-directed gene regulation. However, these mutations did not interfere with the ability of tax to bind zinc or to contribute to immortalization of primary cells. Thus, transactivation of bovine leukemia virus LTR and target cell transformation are independent functions of Tax and involve different functional domains of the protein.
Assuntos
Transformação Celular Viral , Produtos do Gene tax/fisiologia , Genes pX , Vírus da Leucemia Bovina/genética , Ativação Transcricional , Animais , Sequência de Bases , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Oligodesoxirribonucleotídeos/química , Ratos , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Zinco/metabolismo , Dedos de ZincoRESUMO
The nucleotide sequences of the env genes of seven bovine leukemia viruses and the encoded peptide sequence were compared, with the objective of (i) determining the genetic distance separating bovine leukemia virus isolates from different geographical regions, (ii) identifying particular amino acids that contribute to the sequential and conformational epitopes, and (iii) relating such epitopes to their projected position in a three-dimensional model of the structure of the gp51 surface glycoprotein. Two bovine leukemia virus subgroups were clearly identified, a Japanese-American subgroup represented by strains lambda BLV-1, VdM, and FLK-BLV and a European subgroup by strains T15-2, LB285, and LB59. It was possible to identify amino acids that were important in determining three of the epitopes (F, G, and H) recognized by neutralizing monoclonal and polyclonal antibodies. On the model, these epitopes were adjacent and located on the exposed region of the molecule. Amino acid sequences contributing to a fourth cryptic epitope were identified; as predicted by the model, they lay on the opposite side to the neutralizable epitopes in a region involved in glycoprotein subunit association. The fact that this region is not normally exposed on the virion surface provides further evidence for the validity of the model.
Assuntos
Genes Virais , Vírus da Leucemia Bovina/genética , Retroviridae/genética , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Viral/genética , Epitopos/análise , Variação Genética , Vírus da Leucemia Bovina/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Mapeamento por Restrição , Proteínas do Envelope Viral/imunologiaRESUMO
The lipid composition of D. radiodurans strain R1 and of two mutant strains has been studied in relation to membrane fluidity and sensitivity to X-ray radiation. No significant difference in the unsaturation degree of fatty acids was found between parental and mutant strains. An important decrease of carbohydrate-containing lipids was observed in the radiosensitive mutant strain. We also observed a higher fluidity in both mutant strains than in the parental one. Modification of membrane lipid fluidity by growing the parental strain at 39 degrees C did not lead to modified radioresistance. These results suggest that a particular chemical composition of the membrane leading to a special lipid phase may be an important parameter in controlling radiosensitivity.
Assuntos
Lipídeos/análise , Carboidratos/análise , Mutação , Especificidade da Espécie , Raios XRESUMO
Fatty acid composition and lipidic carbohydrate to lipidic phosphorus molar ratio of yellow pigmented micrococci are compared to red pigmented ones and may be summarized by three indexes. These bacteria show wide differences in their fatty acid composition: three strains possess saturated branched chain fatty acids and one has unsaturated straight chain ones. A significant increase in 'anteiso/iso indexes' is observed between pink (M. roseus) and yellow colored bacteria (M. lysodeikticus, S. lutea). There is no significant difference (P greater than 0.05) between the 'unsaturation indexes' of the red pigmented parental D. radiodurans strain and its colorless mutant. Radioresistant strains exhibit a higher 'carbohydrate/phosphorus index' than other strains. There seems to be a relationship between a high carbohydrate-containing lipid content and a high resistance to physical and chemical agents, in particular to radiations. These differences observed in the lipid composition have implications in taxonomy and in establishing an evolutionary scheme.
Assuntos
Carboidratos/análise , Ácidos Graxos/análise , Lipídeos de Membrana/análise , Micrococcus/análise , Fósforo/análise , Tolerância a Radiação , EspectrofotometriaRESUMO
The role of retroviruses in murine radioleukemogenesis was reinvestigated using a protocol associating the injection of a non-pathogenic retrovirus (T1223/B virus) and a subleukemogenic dose of X-radiation (2 X 1.75 Gy). Using the Southern blotting technique we studied MuLV proviral organization and RNA expression in thymic lymphomas induced by the combined effect of virus and irradiation or irradiation alone. A recombinant provirus was detected in the chromosomal DNA of every tumor induced by associating virus and radiation whereas it was unconstantly found in radio-induced tumors. In every instance, the provirus was not integrated at a common site. No relationship was observed between viral RNA expression and tumor induction. Trisomy 15 was observed in all metaphases irrespective of the protocol of tumor induction. The G-banding technique revealed an extra-band in several thymic lymphomas induced by irradiation and T1223/B virus injection.
Assuntos
Genes Virais , Leucemia Experimental/genética , Leucemia Induzida por Radiação/genética , Linfoma/etiologia , RNA Viral/genética , Retroviridae/patogenicidade , Neoplasias do Timo/genética , Animais , Aberrações Cromossômicas/genética , Aberrações Cromossômicas/patologia , Transtornos Cromossômicos , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , Regulação da Expressão Gênica , Leucemia Experimental/etiologia , Leucemia Induzida por Radiação/microbiologia , Linfoma/genética , Linfoma/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Poli A/genética , RNA/genética , RNA Mensageiro/genética , Recombinação Genética , Retroviridae/genética , Neoplasias do Timo/microbiologia , Proteínas do Envelope Viral/genética , Raios XRESUMO
The bovine leukemia virus mRNAs expressed in cultured bovine cells of various origins are a 9.0-kilobase genomic RNA, a 5.1-kilobase env RNA, and a newly detected 2.1-kilobase RNA corresponding to the transcription of pX sequences located in between the env gene and the 3' end of the provirus.
Assuntos
Genes Virais , Vírus da Leucemia Bovina/genética , RNA Mensageiro/análise , RNA Viral/análise , Retroviridae/genética , Transcrição Gênica , Sequência de BasesRESUMO
Deinococcus radiodurans, although a gram-positive bacterium, has a complex cell wall with multiple layers and associates to this structural particularity, a quite unusual lipid composition for gram-positive bacteria. The conventional phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol...) are absent. Among the nine polar lipids detected in the R1 Anderson strain, three are glycolipids only one is a phospholipid, the other ones are glycophospholipids. One of the latter compounds contains one free amino group. Analysis by aminoacid autoanalyser enables to identify glucosamine in one glycolipid and in two glycophospholipids. Sugar analysis by gas-liquid chromatography after acid methanolysis and trifluoroacetylation, reveals the occurrence of N-acetyl glucosaminyl residues in one glycolipid and in one phospholipid. The following identification for the two lipids of D. radiodurans is proposed: phosphatidyl glucosaminyl glycerol and phosphatidyl N-acetyl glucosaminyl glycerol.
Assuntos
Bactérias Gram-Positivas/análise , Fosfolipídeos/análise , Acetatos , Ácido Acético , Fenômenos Químicos , Química , Cromatografia Gasosa , Glicolipídeos/análise , Hidrólise , Ácido Periódico , Fosfatidilgliceróis/análiseRESUMO
Micrococcus radiodurans was highly resistant to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) but it was sensitive to the mutagenic action of this chemical. The induction of mutation was not significantly modified by the culture growth phase. This last finding leads to the assumption that the mutation takes place at some distance from the replication fork. Moreover, a low concentration of MNNG induced mutations that were added to those subsequently obtained from a second exposure to a higher concentration of the alkylating agent. Thus, M. radiodurans does not seem to have an inducible error-free repair system for alkylation damage. Furthermore, incubation in the presence of chloramphenicol did not modify the mutation rate, indicating that protein synthesis is not involved in the mutagenic process.
Assuntos
Metilnitronitrosoguanidina/farmacologia , Micrococcus/genética , Mutação/efeitos dos fármacos , Cloranfenicol/farmacologia , Reparo do DNA , Replicação do DNA , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos , Micrococcus/efeitos dos fármacos , Rifampina/farmacologiaRESUMO
Studies were carried out on lipid composition of Micrococcus radiodurans. The polar lipid components were found to be 4 glycolipids, 3 phospholipids and 2 phosphoglycolipids. The major fraction belongs to the last group. Correlation between these components and lipoteichoic acid was not observed. A lipid-polysaccharidic complex was isolated. It was not of this type.
Assuntos
Lipídeos/análise , Lipopolissacarídeos/análise , Micrococcus/análise , Glicolipídeos/análise , Fosfolipídeos/análiseRESUMO
The study on the growth of E. coli cells under low power microwave irradiation the frequency of which can vary between 70 and 75 GHz, demonstrates that in the neighbourhood of 70.5 and 73 GHz a slowdown of growth is most frequently observed. On the contrary, the survival is not altered as long as the power of the irradiation is low. The irradiation does not induce lesions in DNA, whatever the frequency may be.
Assuntos
Escherichia coli , Micro-Ondas , Relação Dose-Resposta à Radiação , Escherichia coli/crescimento & desenvolvimentoRESUMO
The unirradiated bacterial DNA assciated with the membrane is liberated into the cytoplasm after breakage of either a single or a double strand, resulting from X-ray action. During the reincubation period in growth-medium, the DNA is reassociated with the membrane. This phenomenon is very rapid and occurs without increasing the molecular weight of DNA. The study of DNA-membrane complexes shows that the size of the DNA-associated membranous fragment differs according to the lysing technique employed, appearing as a change in the density of the complex. Chloramphenicol decreases reassociation, and iodoacetamide, a radiosensititzing agent, inhibits it completely.