RESUMO
Some genomic elements of the multicopy HERV-W endogenous retroviral family have been previously identified in databases. One of them, located on chromosome 7, contains a single complete open reading frame (ORF) putatively encoding an envelope protein. We have experimentally investigated the genomic complexity and coding capacity of the HERV-W family. The human haploid genome contains at least 70, 100, and 30 HERV-W-related gag, pro, and env regions, respectively, widely and heterogeneously dispersed among chromosomes. Using in vitro transcription-translation procedures, three putative HERV-W gag, pro, and env ORFs were detected on chromosomes 3, 6, and 7, respectively, and their sequences analyzed. A 363 amino acid gag ORF containing matrix and carboxy-terminal truncated capsid domains encoded a putative 45-kDa protein. No gag-pro ORF was found, but a pro sequence containing a DTG active site was detected. Finally, the previously described 538 amino acid HERV-W env ORF, located on chromosome 7, was shown to be unique and encoded a putative 80-kDa glycosylated protein. Proteins of molecular mass identical to the one obtained by an in vitro transcription-translation procedure were detected in human placenta, using anti HERV-W Gag- and Env-specific antibodies. The absence of an HERV-W replication-competent provirus versus the existence of HERV-W-related Gag and Env proteins in healthy human placenta is discussed with respect to particle formation, physiology, and pathology.
Assuntos
Mapeamento Cromossômico , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Sequência de Aminoácidos , Animais , Southern Blotting , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 7/genética , Endopeptidases/genética , Produtos do Gene env/química , Produtos do Gene env/genética , Genes Virais , Genes env/genética , Genes gag/genética , Humanos , Dados de Sequência Molecular , Placenta/metabolismo , Reação em Cadeia da PolimeraseRESUMO
A novel human endogenous retrovirus, HERV-W, has been characterized on the basis of multiple sclerosis-associated retrovirus (MSRV) probes. We have analyzed the phylogenetic distribution of HERV-W in humans and other primate species. As HERV-W presents a C/D chimeric nature and is largely composed of deleted elements, Southern blots were performed using gag, pol, env, and LTR probes. The relative complexities observed for gag, pol, env, and LTR regions were similar in humans, apes, and Old World monkeys, the minimal number of bands observed after Southern blot analysis being 25, 50, 10, and at least 100, respectively. The HERV-W family entered the genome of catarrhines more than 25 million years ago.
Assuntos
Retrovirus Endógenos/classificação , Filogenia , Primatas/virologia , Animais , Sequência de Bases , Southern Blotting/veterinária , Sondas de DNA/genética , Retrovirus Endógenos/genética , Produtos do Gene env/genética , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Genoma Viral , Humanos , Dados de Sequência Molecular , RNA Viral/análise , Sequências Repetidas Terminais/genéticaRESUMO
The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.
Assuntos
Polidesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , DNA Polimerase Dirigida por RNA/análise , Retroviridae , Animais , Meios de Cultura , DNA , Vírus da Leucemia Murina/enzimologia , Camundongos , DNA Polimerase Dirigida por RNA/genética , Sensibilidade e EspecificidadeAssuntos
Artefatos , DNA Recombinante/análise , Retrovirus Endógenos/genética , Genes pol , Técnicas de Amplificação de Ácido Nucleico , Plasmídeos/química , RNA Mensageiro/análise , RNA Viral/análise , Transcrição Gênica , Colorimetria , Retrovirus Endógenos/isolamento & purificação , Produtos do Gene pol/genética , Humanos , Plasmídeos/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Recombinantes de Fusão/genética , Sensibilidade e EspecificidadeRESUMO
The adsorption of BSA and RNA onto hydrophilic and thermosensitive poly(N-isopropyl-acrylamide) (NIPAM) latex particles was described as a function of pH, ionic strength and temperature. The hydrogel poly(NIPAM) latex was synthesized by precipitation polymerization in the presence of a cationic amino-containing monomer. The latex obtained was characterized in terms of particle size, and electrophoretic mobility as a function of pertinent variables: pH, temperature and ionic strength. The adsorption of BSA onto the latex was investigated to identify the conditions at which the adsorbed amount of BSA was negligible. The adsorption of RNA was studied to establish the conditions which give rise to maximal adsorption of RNA. In order to favor the desorption of RNA, desorption was investigated by changing the pH, ionic strength, and temperature. The adsorption of BSA was found to be lower at 20 than at 40 degrees C. However, the adsorption of RNA is drastically affected by the pH and the ionic strength of the medium. Maximal adsorbed amounts were obtained at acidic pH, 20 degrees C, and low ionic strength. The adsorption is shown to decrease when the pH, temperature and ionic strength increase, implying that the adsorption was mainly governed by electrostatic interactions. Maximal release of RNA molecules was obtained at high ionic strength and basic pH.
Assuntos
Bioquímica/métodos , Látex/química , Ácidos Nucleicos/isolamento & purificação , Adsorção , Cátions , Eletroforese , Hidrogéis , Concentração de Íons de Hidrogênio , Microesferas , Ácidos Nucleicos/metabolismo , Concentração Osmolar , Reação em Cadeia da Polimerase/métodos , Proteínas/isolamento & purificação , Proteínas/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Sensibilidade e Especificidade , Soroalbumina Bovina/isolamento & purificação , Soroalbumina Bovina/metabolismo , TemperaturaRESUMO
The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein-Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.