RESUMO
The nonapeptide Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln has been reported as a model substrate for an aspartyl protease produced by the human immunodeficiency virus (HIV-1). Cleavage of this peptide at the Tyr-Pro linkage to produce tetra- and pentapeptide fragments is the basis of high-performance liquid chromatographic assays to detect HIV-1 protease activity. Confirmation of the cleavage site has been proved by using microbore liquid chromatography coupled to a dynamic fast atom bombardment interface. Comparison with fortified control incubates indicates that an approximate stoichiometric amount of the tetrapeptide was formed from the nonapeptide, confirming that the cleavage of the substrate by HIV-1 protease is both specific and quantitative.
Assuntos
Protease de HIV , Peptídeos/análise , Prolina/análise , Tirosina/análise , Sequência de Aminoácidos , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Protease de HIV/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria UltravioletaRESUMO
A plasmid vector was used to express the HIV-1 pol open reading frame under the regulation of the bacterial trp promoter in Escherichia coli. This expression system has been used as a source of recombinant viral protease. The self-processed active enzyme was recovered from a soluble fraction of a bacterial cell lysate and purified by a procedure involving four steps of chromatography. The protocol yielded 0.3 mg of protease for each liter of bacterial culture. The protease formed tetragonal bipyramidal crystals which have been used in high-resolution X-ray diffraction studies.
Assuntos
Endopeptidases/genética , Escherichia coli/genética , Expressão Gênica , Genes Virais/genética , HIV-1/enzimologia , Síndrome da Imunodeficiência Adquirida/imunologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Cromatografia , Cristalização , Eletroforese em Gel de Poliacrilamida , Endopeptidases/isolamento & purificação , Protease de HIV , HIV-1/genética , Humanos , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Transformação Bacteriana , Difração de Raios XRESUMO
Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS. The conserved Asp-Thr/Ser-Gly sequence in retroviral proteinases suggests that they exist as dimers similar to the ancestor proposed for the pepsins. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases, these structures have overall folds that are similar to each other only where they are also similar to the pepsins. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 A resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded beta-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by alpha-aminobuytric acid. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.
Assuntos
Endopeptidases , HIV-1/enzimologia , Cristalografia , Protease de HIV , Substâncias Macromoleculares , Conformação Proteica , Proteínas Recombinantes , Solubilidade , Difração de Raios XRESUMO
Undegraded mRNA transcripts were isolated from human parainfluenza virus type 1 (hPIV-1)-infected LLC-MK2 cells and their size was determined through denaturing agarose electrophoresis. The two predominantly represented mRNA species (1.65 and 1.87 kb) are similar in size to other paramyxoviral mRNAs that encode their respective glycoproteins. The cDNA transcripts corresponding to these two mRNAs were used to construct two size-restricted cDNA libraries. A cDNA clone, containing a 1.87-kb insert, was identified as encoding the hPIV-1 fusion protein by positively hybridizing with a synthetic oligonucleotide mix whose sequence was derived from the conserved sequences of other paramyxoviral F0 genes. The nucleotide sequence of the cDNA insert was determined and found to contain a single, large open reading frame encoding a putative protein of 60,795 Da consisting of 556 amino acids. Comparison of the amino acid sequence with the fusion proteins of other paramyxoviruses enabled the identification of the highly conserved amino acids of the F1 N-terminus. In addition, the positions of the hydrophobic signal and transmembrane regions, cysteine, and proline residues are all conserved. These analyses confirm that the cDNA sequence is that of the F0 protein. The 5' end of the fusion protein mRNA was determined by primer extension to lie 155 bases beyond the 5' end of the cDNA insert.
