RESUMO
The complete nucleotide sequence of an HIV-2 isolate derived from a German AIDS patient with predominantly neurological symptoms is reported. The HIV-2BEN sequence is highly divergent from those of previously described HIV-2 and SIV strains. Evolutionary tree analysis of eight HIV-2 sequences reveals the existence of three HIV-2 groups. HIV-2BEN belongs to a group with two isolates from Ghana and The Gambia. Based on a comparison of HIV-2BEN with six HIV-2 isolates, SIVsmm and SIVmac, the variability of the structural env and gag proteins is similar within the HIV-2/SIVsmm/mac and HIV-1 groups. In contrast, the regulatory HIV-1 proteins are more highly conserved than those from HIV-2 strains. Multiple sequence alignments reveal that some domains of the envelope and regulatory proteins are well conserved among HIV-1, HIV-2/SIVsmm/mac, SIVagm and SIVmnd. The identification of conserved domains within the external glycoprotein could help to develop broadly active vaccines.
Assuntos
Síndrome da Imunodeficiência Adquirida/microbiologia , Variação Genética , HIV-2/genética , Sequência de Aminoácidos , Sequência de Bases , Produtos do Gene env/química , Produtos do Gene env/genética , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene nef/química , Produtos do Gene nef/genética , Produtos do Gene pol/química , Produtos do Gene pol/genética , HIV-2/classificação , Humanos , Mali , Dados de Sequência Molecular , Filogenia , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Vírus da Imunodeficiência Símia/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Three chondroitin sulphates and five chondroitin polysulphates, with molecular weights ranging from 3000 to 30,000 daltons, were evaluated applying the MT-4 cell-culture assay for inhibition of HIV-1 replication. These results were compared with those obtained with compounds of known in vitro antiretroviral activity, namely, dermatan sulphate, heparin, dextran sulphate, pentosan polysulphate, zidovudine (AZT) and suramin. Chondroitin polysulphate with a molecular weight (MW) of 9000 daltons (CPS 9000) was the most effective polyanionic compound studied. In contrast with zidovudine, this CPS 9000 was not toxic for MT-4 cells up to a concentration of 500 micrograms/ml. Moreover, CPS 9000 is highly specific for inhibition of HIV-1 reverse transcriptase.
Assuntos
Sulfatos de Condroitina/farmacologia , Condroitina/análogos & derivados , HIV/efeitos dos fármacos , Células Cultivadas , Humanos , Peso Molecular , Inibidores da Transcriptase ReversaRESUMO
A HIV-2 strain named HIV-2ben was isolated from peripheral blood lymphocytes of a patient who, since 1984, had developed neurological symptoms such as Raynaud's syndrome, followed by paresthesia of extremities and ataxia, and finally paraparesis of the legs and incontinence. This new isolate could be distinguished from HIV-2rod by antibody-binding epitopes, peptide maps of core p24 and p18 polypeptides and restriction endonuclease cleavage pattern.
Assuntos
HIV-2/isolamento & purificação , Doenças do Sistema Nervoso/microbiologia , Animais , Produtos do Gene gag/análise , Proteína do Núcleo p24 do HIV , HIV-2/imunologia , Humanos , Mapeamento de Peptídeos , Coelhos , Proteínas do Core Viral/análiseRESUMO
Ten rhesus (Macaca mulatta) and six fascicularis (Macaca fascicularis) macaques were inoculated with HIV-2ben using three different virus preparations and two routes of inoculation. Thirteen of the 16 inoculated macaques seroconverted 2-6 weeks after infection. Three M. mulatta remained seronegative. The seroconverted animals developed antibody titres from 80 to 40,000. Their antibodies reacted with gp160 and gp130 and, in varying degrees, with gp32 and core proteins. Virus could be re-isolated from 11 of the 16 macaques. M. mulatta were transiently viraemic 6-14 weeks after infection whereas all M. fascicularis were persistently viraemic 2-7 weeks after infection onwards. In the 6-18 months after infection one M. mulatta lost 20% of its body weight and two M. fascicularis showed transient lymphadenopathy and splenomegaly; the other animals remained clinically normal. A re-isolated virus from a M. mulatta was indistinguishable from the inoculated HIV-2ben by genomic restriction enzyme analysis. M. mulatta and M. fascicularis are infectable by a single intravenous injection of cell-free HIV-2ben. Persistent viraemia in M. fascicularis represents a valuable and reliable parameter for studies on antivirals and vaccines.
Assuntos
Infecções por HIV/etiologia , HIV-2 , Animais , Modelos Animais de Doenças , Feminino , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/imunologia , Infecções por HIV/microbiologia , HIV-2/isolamento & purificação , HIV-2/patogenicidade , Ativação Linfocitária , Macaca fascicularis , Macaca mulatta , Masculino , Especificidade da Espécie , Linfócitos T/imunologia , Viremia/etiologiaRESUMO
The products of the endogenous reverse transcriptase reaction of SIVagmTYO-7 were characterized after the reaction conditions had been optimized. The major reaction product in the presence of actinomycin D and oligo(dT) was a DNA with a size of 300 bases. Without actinomycin D two additional reaction products with 600 or 700 bases appeared. The 300 base product was identified as the (-)strong-stop DNA, whereas the 700 base product is the (+)strong-stop DNA. The 600 base product appeared only after oligo(dT) priming. The (-)strong-stop DNA hybridized specifically with a 9 kb RNA found in virus particles and three RNA species of 1.8, 4.8 and 9 kb isolated from SIVagmTYO-7 infected cells.
Assuntos
DNA Polimerase Dirigida por RNA/isolamento & purificação , Vírus da Imunodeficiência Símia/enzimologia , Animais , Northern Blotting , Linhagem Celular , Chlorocebus aethiops , Sondas de Oligonucleotídeos , RNA Viral/química , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/isolamento & purificaçãoRESUMO
The genetic diversity of HIV-1 is well documented. Except for the HIV-1 isolate LAV-1BRU, no nucleic acid sequence of a European isolate of HIV-1 has been published to date. To further investigate the extent of the genetic variability and the evolution of HIV-1, we have isolated, cloned, and subsequently sequenced HIV-1 from a German patient with AIDS-related complex. Comparative studies of the nucleic acid sequence revealed that this isolate, designated HAN2, is highly divergent from the North American and African subtypes of HIV-1 and may represent a European subtype of HIV-1. Furthermore, a full-length molecular clone was derived from this isolate which was infectious in human T-cell lines. Therefore this new isolate will be particularly useful for studies on the genetic evolution and biology of HIV-1 as well as for testing antiviral substances and for developing vaccines.
Assuntos
HIV-1/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Viral/genética , Alemanha Ocidental , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/genéticaRESUMO
The reverse transcriptase (RT) inhibition and the specificity of 15 aminonaphthalenesulfonic acid derivatives were examined with RT of a simian immunodeficiency virus derived from an African green monkey (SIVagmTYO-7). The two compounds with the strongest RT inhibition (NF415) or the highest specificity (NF345), together with suramin, were evaluated against polymerase alpha-primase complex from calf thymus. We have also compared the kinetics of inhibition of the viral and the cellular polymerase by these three compounds. While RT inhibition followed a mixed competitive and non-competitive mechanism, inhibition of the DNA polymerase alpha was competitive for suramin and non-competitive for NF415 and NF345. Certain structural characteristics appeared to be common for specific RT inhibitors.
Assuntos
DNA Polimerase II/efeitos dos fármacos , Naftalenossulfonatos/farmacologia , RNA Nucleotidiltransferases/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Vírus da Imunodeficiência Símia/enzimologia , Suramina/análogos & derivados , Suramina/farmacologia , Animais , Transporte Biológico , Células Cultivadas , Chlorocebus aethiops , DNA Polimerase II/isolamento & purificação , DNA Primase , Relação Dose-Resposta a Droga , Humanos , Cinética , Naftalenossulfonatos/toxicidade , Permeabilidade , RNA Nucleotidiltransferases/isolamento & purificação , DNA Polimerase Dirigida por RNA/isolamento & purificação , Inibidores da Transcriptase Reversa , Vírus da Imunodeficiência Símia/metabolismo , Relação Estrutura-Atividade , Suramina/toxicidadeRESUMO
A micromethod for the detection of human immunodeficiency virus (HIV) and other retroviruses in cell culture supernatants is described which applies a DEAE ion exchanger for recovery of polynucleotides synthesized in vitro by the retroviral reverse transcriptase. Cell culture, sample preparation, and test performance including the washing step are adapted to microtitre plates. Compared to the standard method this technique produced less non-specific reactions, resulting in a more than 3-fold higher sensitivity, a higher reproducibility due to lower intrarun variations and allowed an increase in the daily sample accomplishment per person 3- to 4-fold at lower costs per sample.
Assuntos
HIV/enzimologia , DNA Polimerase Dirigida por RNA/análise , Retroviridae/enzimologia , DEAE-Celulose , HIV/isolamento & purificação , Humanos , Troca Iônica , Retroviridae/isolamento & purificaçãoRESUMO
The HIV-1 protease is essential for maturation of virus particles and is, therefore, an attractive target for antiviral drugs. The function of this protease depends on the dimerization of two identical subunits. Commonly used protease inhibitors are directed mainly against the active site of the enzyme which often leads to viral resistance. To determine the inhibitory effect of peptides interfering with the dimerization site of the HIV-1 protease, a recombinant bacterial screening assay was established. Escherichia coli was co-transformed with two different plasmids, expressing the 'interface' peptide and an active HIV-1 protease toxic for the bacteria. Co-expression of inhibitory peptides overcomes the incomplete membrane transmission of supplemented inhibitors and leads to a direct interaction of the inhibitory peptide and the HIV-1 protease. The inhibitory effect of co-expressed peptides was measured by an increased growth of co-transformed bacteria, compared with a slowly growing E. coli control culture only expressing the HIV-1 protease. Using this assay several penta- and hexa-peptides were screened for their ability to inhibit HIV-1 protease activity. One of these peptides showed a significant inhibitory effect on co-expressed recombinant HIV-1 protease.
Assuntos
Escherichia coli/enzimologia , Inibidores da Protease de HIV/metabolismo , Protease de HIV/metabolismo , HIV-1/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , GTP Fosfo-Hidrolases/farmacologia , Vetores Genéticos/genética , Protease de HIV/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Transformação BacterianaRESUMO
Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV-I). Of these compounds, 57 inhibited the poly(rC).oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty-four substances were superior to suramin in our RT inhibition assay. The RT-inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.
Assuntos
Deltaretrovirus/enzimologia , Inibidores da Transcriptase Reversa , Suramina/análogos & derivados , Suramina/farmacologia , Linhagem Celular , Cinética , Relação Estrutura-Atividade , Suramina/síntese químicaRESUMO
High producer cell lines were established by infecting Jurkat cells with either HTLV-IIIB from H9 cells or with STLV-IIImac from HUT-78 cells. The Jurkat cells produced both viruses at least 10 times more efficiently then the original cell lines; the pelleted virus from Jurkat cells was also less contaminated with non-virion proteins. Accordingly, a higher specificity was attained in an ELISA for antibodies to HTLV-III if the antigenic activity was derived from the virus from Jurkat cells, as opposed to that from H9 cells.
Assuntos
Linhagem Celular , HIV/crescimento & desenvolvimento , Linfócitos T/microbiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Animais , Especificidade de Anticorpos , Antígenos Virais/imunologia , Humanos , Leucemia Linfoide , Macaca mulatta/microbiologia , DNA Polimerase Dirigida por RNA/análise , Proteínas Virais/análise , Replicação ViralRESUMO
We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I. Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material. In addition, RNA polymerase can be isolated as a by-product. We have applied this method to purify DNA polymerase both from wild type E. coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5632-5636). This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold.
Assuntos
DNA Polimerase I/isolamento & purificação , DNA Polimerase Dirigida por DNA/isolamento & purificação , Escherichia coli/enzimologia , Colífagos/enzimologia , RNA Polimerases Dirigidas por DNA/isolamento & purificação , LisogeniaRESUMO
The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.
Assuntos
Cercopithecus/microbiologia , Chlorocebus aethiops/microbiologia , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Imunodeficiência Símia/enzimologia , Animais , Centrifugação com Gradiente de Concentração , Cinética , Peso Molecular , Polinucleotídeos , DNA Polimerase Dirigida por RNA/isolamento & purificação , Vírus da Imunodeficiência Símia/isolamento & purificação , Relação Estrutura-Atividade , Moldes GenéticosRESUMO
To investigate the accuracy of retroviral in vitro DNA replication we have examined with two fidelity assays the reverse transcriptases (RTs) from SIVagm, HIV-1, MoMLV as well for comparison the Klenow fragment from E. coli and DNA polymerase a from calf-thymus. These forward mutation assays measured the loss of bacteriophage M13 lacZa gene function by mutations. In the EnvlacZa assay frameshift mutations occurring during polymerisation of a 176 b long simian immunodeficiency virus (SIV) envelope (env) sequence were phenotypically detected by blue/white-plaque screening. To measure in addition substitutions, a 116 b long M13 lacZa gene DNA template was used as the mutational target (LacZa assay). With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs. Nevertheless, the SIVagm RT was slightly more accurate than the other RTs and nearly all frameshifts were observed at two homopolymeric runs of its homologous template. Measuring also substitution errors at the lacZa template the mutation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.
Assuntos
DNA Polimerase III/metabolismo , Genes env , Óperon Lac , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Imunodeficiência Símia/enzimologia , Animais , Bacteriófago M13/genética , Sequência de Bases , Bovinos , DNA Polimerase I , DNA Viral , Mutação da Fase de Leitura , Transcriptase Reversa do HIV/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Vírus da Imunodeficiência Símia/genética , Moldes GenéticosRESUMO
To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli-expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.
Assuntos
Baculoviridae/genética , Vetores Genéticos , HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Western Blotting , Células Cultivadas , Quimotripsina/metabolismo , Primers do DNA , Expressão Gênica , Protease de HIV/metabolismo , Transcriptase Reversa do HIV , Humanos , Insetos , Cinética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMO
The antibody response to structural and regulatory viral proteins was studied in 14 rhesus (Macaca mulatta) and 6 cynomolgus (Macaca fascicularis) macaques experimentally infected with HIV-2 or SIVMAC. To investigate the humoral antibody response to the negative regulatory factor (nef), the recombinant protein was expressed to high levels with recombinant vaccinia virus (VV). nef-specific antibodies were detected in 14 of 20 infected macaques (70%). In sera of all infected monkeys antibodies directed to the structural proteins gp120, p56, and p24 appeared 2 to 6 weeks postinfection. In contrast, the extent and the appearance of nef-specific antibodies during the course of infection varied considerably between individual animals. However, only in sera of four animals (20%) were nef-specific antibodies detectable as early as those against the core proteins p24 and p56. In SIVMAC-infected rhesus macaques at different clinical stages, the antibody response towards nef neither correlated with the development of viral latency nor to disease progression or viremia. Our data indicate that in macaques experimentally infected with SIV or HIV-2 antibody formation against nef is not a useful diagnostic marker either for early detection of viral infection or of disease progression.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Produtos do Gene nef/imunologia , Anticorpos Anti-HIV/biossíntese , HIV-2/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Proteínas Estruturais Virais/imunologia , Viremia/imunologia , Complexo Relacionado com a AIDS/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Proteína gp120 do Envelope de HIV/imunologia , Soropositividade para HIV , Contagem de Leucócitos , Macaca fascicularis , Macaca mulatta , Proteínas Recombinantes/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/microbiologia , Trombocitopenia/imunologia , Viremia/complicações , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.
Assuntos
Mutação da Fase de Leitura , Produtos do Gene gag/genética , Produtos do Gene pol/genética , HIV-1/genética , Processamento de Proteína Pós-Traducional , Transfecção , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Expressão Gênica , Produtos do Gene gag/metabolismo , Produtos do Gene pol/metabolismo , Protease de HIV/metabolismo , Humanos , Hidrólise , Camundongos , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
An outbreak of classical herpetic infection causing vesicoulcerative stomatitis in a family group (eight animals) of Callithrix jacchus is described. In all eight infected animals, human herpesvirus 1 (HHV-1) was identified as the causative agent. This was confirmed by histologic, immunohistologic, and molecular biologic investigations, as well as by virus isolation. The clinical picture, the macroscopic appearance, and the histologic results indicated a herpes infection as the cause of mortality. Alterations of the oral mucous membranes were erosive to ulcerative with typical intranuclear inclusions. Immunohistologic and molecular biologic techniques clearly identified the HHV-1 virus and excluded other possible primate herpesviruses such as B-virus, SA8, HVP-2, and Herpes tamarinus. The significance of this herpesvirus infection for colony management is discussed.
Assuntos
Callithrix/virologia , Herpes Simples/patologia , Herpes Simples/veterinária , Doenças dos Macacos/patologia , Animais , Surtos de Doenças/veterinária , Feminino , Herpes Simples/mortalidade , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Masculino , Doenças dos Macacos/mortalidade , Doenças dos Macacos/virologia , Mucosa/patologia , Língua/patologia , Língua/virologiaRESUMO
The macronuclear rRNA genes of Tetrahymena thermophila contain an 0.4 kb intervening sequence in the 26S rRNA coding region. The sequence is represented within the primary transcription product. We demonstrate in this paper that the enzyme activities necessary for the endonucleolytic cleavage as well as for the ligation of the transcript are associated with the isolated. The intervening sequence is excised as an unique molecule, which is stable in vitro. About 50% of the in vitro synthesized RNA is processed. Faithful in vitro transcription occurs in the presence of the divalent ions Mg2+, Mn2+ and Co2+ while processing takes place only in the presence of Mg2+. The absolute requirement for Mg2+ in the excision reaction enables us to synthesize labelled pre-rRNA in the presence of Mn2+ or Co2+. The synthesized RNA can be used as a substrate in studies of th processing enzymes in vitro.
Assuntos
Nucléolo Celular/metabolismo , Precursores de Ácido Nucleico/metabolismo , RNA Ribossômico/metabolismo , Tetrahymena/metabolismo , Animais , DNA/genética , Genes , Técnicas In Vitro , Transcrição GênicaRESUMO
Infectious molecular clones of the human immunodeficiency virus type 2 (HIV-2) will be valuable tools for the study of regulatory gene functions and the development of an animal model for the human acquired immunodeficiency syndrome (AIDS). To this end, we have cloned and sequenced a novel HIV-2 isolate, HIV-2BEN. One clone, designated MK6, is infectious for various human T-cell lines and for human and macaque peripheral blood lymphocytes (PBL), allowing molecular studies of HIV-2 infection and replication. Since MK6 is highly cytopathic in MT-2 and Molt-4 clone 8 cells, antiviral agents and neutralizing sera may be tested. Cluster analysis of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) env and gag genes revealed that HIV-2BEN yielded the earliest node of phylogenetic divergence for all reported HIV-2 sequences. Noise analysis showed that, with the current data, no specification of any branching order can be made among the four groups of primate lentiviruses, HIV-1, HIV-2/SIVSMM/MAC, SIVAGM, and SIVMND.