RESUMEN
The structural variability of the external glycoproteins of primate immunodeficiency viruses, has, so far, been investigated exclusively by sequence comparison of the respective proviral genomes. We have examined the structural relationship amount the external glycoproteins from three specific human immunodeficiency viruses (HIF-1, HIV-2), three specific simian immunodeficiency viruses from macaques (SIVmac) and three specific SIV from African green monkeys (SIVagm) by peptide mapping. Differences among glycoproteins were most pronounced between HIV-1 and SIVmac, as well as HIV-2. Two specific glycoproteins from independent SIVagm isolates were closely related to HIV-1, whereas the glycoprotein from a third SIVagm isolate was more similar to those of SIVmac and HIV-2. Our analysis reflects the classification of primate immunodeficiency viruses into three groups, the HIV-2 and SIVmac viruses, the green monkey isolates and HIV-1.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , VIH-1/análisis , VIH-2/análisis , Proteínas de los Retroviridae/química , Virus de la Inmunodeficiencia de los Simios/análisis , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Centrifugación por Gradiente de Densidad , Proteína gp120 de Envoltorio del VIH/ultraestructura , Mapeo Peptídico , Proteínas de los Retroviridae/ultraestructura , Homología de Secuencia de Ácido Nucleico , Síndrome de Inmunodeficiencia Adquirida del Simio/microbiología , Proteínas del Envoltorio Viral/ultraestructuraRESUMEN
We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.
Asunto(s)
Productos del Gen gag/aislamiento & purificación , Virus de la Inmunodeficiencia de los Simios/análisis , Proteínas del Núcleo Viral/aislamiento & purificación , Dicroismo Circular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Productos del Gen gag/química , Productos del Gen gag/genética , Plásmidos , Conformación Proteica , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.
Asunto(s)
ADN Polimerasa Dirigida por ARN/aislamiento & purificación , Virus de la Inmunodeficiencia de los Simios/enzimología , Proteínas Virales/aislamiento & purificación , Animales , Chlorocebus aethiops , ADN/biosíntesis , Nucleótidos de Desoxiadenina/metabolismo , Didesoxinucleótidos , Cinética , Nucleótidos/metabolismo , Péptidos/metabolismo , Virus de la Inmunodeficiencia de los Simios/análisis , Virus de la Inmunodeficiencia de los Simios/metabolismoRESUMEN
The envelope glycoprotein 130 ('130' referring to an Mr of 130,000) of simian immunodeficiency virus from sooty mangabey (Cercocebus atys) (SIVSM) was isolated from the cell-free supernatant of the SIVSM-infected human T-cell line H9, metabolically labelled with D-[6-3H]glucosamine. After digestion with Staphylococcus aureus V8 proteinase, radiolabelled N-glycans were liberated from resulting glycopeptides by sequential treatment with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F and fractionated by h.p.l.c. and gel filtration. Individual oligosaccharide species were characterized by enzymic microsequencing, chromatographic analyses and, in part, by acetolysis. The oligosaccharide structures thus established include oligomannosidic glycans with five to nine mannose residues as well as fucosylated and partially sialylated bi-, tri- and tetra-antennary N-acetyl-lactosaminic oligosaccharide species, the latter of which carry, in part, additional galactose residues or N-acetyl-lactosamine repeats. In comparison with the corresponding envelope glycoprotein 120 from human immunodeficiency virus type 1 (HIV-1), propagated in the same cell line [Geyer, Holschbach, Hunsmann and Schneider (1988) J. Biol. Chem. 263, 11760-11767], carbohydrates of the simian glycoprotein were found to consist of decreased amounts of oligomannosidic glycans and increased quantities of higher-branched N-acetyl-lactosaminic species.
Asunto(s)
Glicoproteínas de Membrana/análisis , Virus de la Inmunodeficiencia de los Simios/análisis , Proteínas del Envoltorio Viral/análisis , Animales , Cercopithecidae , Glicosilación , Humanos , Ácido N-Acetilneuramínico , Oligosacáridos/análisis , Ácidos Siálicos/análisisRESUMEN
Cercopithecus aethiops (African Green monkey), a nonhuman primate species distributed throughout subsaharan Africa, has been shown to have high seroprevalence rates of antibodies to simian immunodeficiency virus (SIV), and therefore, has been proposed as a natural reservoir for immunodeficiency viruses. Our laboratories have isolated SIV-like viruses from two East African subspecies of C. aethiops designated grivet and vervet monkeys. Analysis of the structural proteins based on the molecular weights and immunologic cross-reactivity to the prototypic SIV(MAC), HIV-1, and HIV-2 isolates suggests that these viruses are distinctly different. Heterogeneity was observed in the molecular weights of the gag, pol, and env gene products between SIV isolates from vervets [SIV(AGM(VER))] and grivets [SIV(AGM(GRI))]. Phenotypically, SIV(AGM(VER)) isolates were distinguishable from SIV(AGM(GRI)) isolates by the apparent size difference of the major core antigen p24. All SIV(AGM(GRI)) and SIV(AGM(VER)) isolates were found to encode a transmembrane protein of approximately 40 kD (gp40) in contrast to gp32 of SIV(MAC). Furthermore, the transmembrane protein was shown to be encoded by the entire env open reading frame, unlike gp32 of SIC(MAC) or gp36 of SIV(AGM(TYO-1)). These data indicate that viruses from C. aethiops share common features with SIV(MAC) and HIV-1, but represent diverse SIV-like viruses which may vary according to subspecies and geographic location.
Asunto(s)
Cercopithecus/microbiología , Chlorocebus aethiops/microbiología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas , VIH-1/análisis , VIH-1/inmunología , VIH-2/análisis , VIH-2/inmunología , Datos de Secuencia Molecular , Virus de la Inmunodeficiencia de los Simios/análisis , Virus de la Inmunodeficiencia de los Simios/inmunología , Proteínas Virales/análisisRESUMEN
A striking characteristic of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 2 (HIV-2) is the presence of a nonsense mutation in the env gene resulting in the synthesis of a truncated transmembrane protein lacking the cytoplasmic domain. By mutagenesis of an infectious molecular clone of SIVmac142, we investigated the function of the cytoplasmic domain and the significance of the env nonsense mutation. When the nonsense codon (TAG) was replaced by a glutamine codon (CAG), the virus infected HUT78 cells with markedly delayed kinetics. This negative effect was counterselected in vitro as reversion of the slow phenotype frequently occurred. The sequencing of one revertant revealed the presence of a new stop codon three nucleotides 5' to the original mutation. Deletions or an additional nonsense mutation introduced 3' to the original stop codon did not modify SIV infectivity. In contrast, the same deletions or nonsense mutation introduced in the clone in which the stop codon was replaced by CAG abolished infectivity. These results indicated that the envelope domain located 3' to the stop codon is not necessary for in vitro replication. However, the presence of this domain in SIV transmembrane protein leads to a reduced infectivity. This negative effect might correspond to a function controlling the rate of spread of the virus during in vivo infection.