High‐level expression of enzymatically active bovine leukemia virus proteinase in <i>E. coli</i>

Andreánsky, Martin; Hrušková−Heidingsfeldová, Olga; Sedláčeka, Juraj; Konvalinka, Jan; Bláha, Ivo; Ječmen, P.; Hořejší, M.; Štrop, Petr; Fábry, Milan

doi:10.1016/0014-5793(91)80032-x

Cited by 21 publications

(5 citation statements)

References 14 publications

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“…Since the cleavage site at the amino terminus of the capsid (CA) protein is always a type 1 site having Pro at the PI' position, a specific function has been suggested (Oroszlan et al, 1978;Oroszlan & Gilden, 1979;Pettit et al, 1991). It is of interest to note that in homologous systems of EIAV (Table II) HIV-1 and HIV-2 (Tozser et al, 1991a), BLV (Andreansky et al, 1991;Blaha et al, 1992), and MMTV (Menendes-Arias et al, 1992) the peptides representing these cleavage sites are usually among the best substrates. However, neither the location in the (Roberts et al, 1991).…”

Section: Resultsmentioning

confidence: 99%

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

Tőzsér¹,

Friedman²,

Weber³

et al. 1993

Biochemistry

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The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.

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Section: Resultsmentioning

confidence: 99%

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

Tőzsér¹,

Friedman²,

Weber³

et al. 1993

Biochemistry

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show abstract

“…Proteinases are synthesized from gene sequences that can be inserted into a wide array of expression plasmids. Several proteinases have been synthesized in this manner (5,7,10,16,17,52,122,123,126,195). After purity has been assessed biochemically, analysis of the catalytic activity typically has been performed with peptides (8 to 16 amino acids in length) as substrates.…”

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confidence: 99%

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

Dougherty¹,

Semler²

1993

Microbiological Reviews

207

172

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“…A Different means to obtain the BLV PR, chemical synthesis of the long sequence [13], expression in E. coli [14], extraction from the virus [3,15], will allow comparative studies of their specific activities. These experiments, together with crystallographic studies would shed a…”

Section: Resultsmentioning

confidence: 99%

Modelling, synthesis and biological activity of a BLV proteinase, made of (only) 116 amino acids

et al. 1993

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Bovine leukaemia virus (BLV) is the aetiological agent of Leukosis enzootica bovis [Viral Oncology (1980), G. Klein (Ed.) Raven Press, New York, pp. 231 238], a widely spread disease in cattle. BLV is reported as the animal model of human T-cell leukaemia virus (HLTV) which is the causative agent of adult T-cell leukaemia and tropical spastic paraparesis. Like the viruses themselves, the two retroviral proteinases (PR) are very closely related [Virology 142 (1985) 357-377]. BLV and HTLV-I PR are reported as putative proteins made of 126 [J. Virol. 57 (1986) 826 832] and 125 [FEBS Lett. 293 (1991) 106-110] amino acids, respectively (long sequences), belonging to the aspartyl proteinase family [Nature 329 (1987) 351-354], with the aid of molecular modelling, we show that BLV and HLTV-I proteinases made of only 116 and 115 amino acids, respectively (short sequences), display three-dimensional structures similar to that observed for other retroviral aspartyl proteinases. The models are based on three-dimensional structures of the Rous sarcoma virus (RSV PR) and the human immunodeficiency virus (HIV-1 PR). We used solid phase peptide synthesis to produce the putative proteolytic enzyme of BLV (116 amino acids). In this study, we show that the folded synthetic protease accurately hydrolyzes a decapeptide corresponding to the sequence of the Matrice-Capside (MA/CA) cleavage site of the gag polyprotein. In addition, the proteolytic activity is inhibited by astatine ((4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid) containing an analogous sequence.BLV protease; Modelling; Solid phase synthesis; Protease activity and inhibition

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High‐level expression of enzymatically active bovine leukemia virus proteinase in E. coli

Cited by 21 publications

References 14 publications

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

Modelling, synthesis and biological activity of a BLV proteinase, made of (only) 116 amino acids

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