Expression and Processing of the AIDS Virus Reverse Transcriptase in
            <i>Escherichia coli</i>

Farmerie, W G; Loeb, DD; Casavant, N. Carol; Hutchison, C.A.; Edgell, MH; Swanstrom, Ronald

doi:10.1126/science.2436298

Cited by 201 publications

(114 citation statements)

References 24 publications

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“…Several laboratories have reported making fully active 66-kDa forms of HIV-1 RT in bacteria (Hansen et al, 1987;Larder et a/., 1987;LeGrice et a/., 1987Tanese eta/., 1986;Farmerie et al, 1987;Mous eta/., 1988). The bacterial expression systems were also used to express truncated proteins similar to the 51 -kDa form found in virions.…”

Section: Introductionmentioning

confidence: 99%

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

Ferris¹,

Hizi

Showalter

et al. 1990

Virology

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HIV-1 virions contain two reverse transcriptase polypeptides that have apparent molecular weights of 66 and 51 kDa. The 51 -kDa form lacks the carboxy-terminal sequences found in the 66-kDa form, and is believed to be a proteolytic digestion product. We have treated purified 66-kDa reverse transcriptase with viral and nonviral proteases. The digestion products were characterized by their ability to react with monoclonal antibodies known to recognize particular segments of the HIV-1 reverse transcriptase.The approximate location of the segments recognized by the monoclonal antibodies was determined by testing the ability of the antibodies to recognize a series of amino-and carboxy-terminaldeleted forms of HIV-1 reverse transcriptase.The segments recognized are not uniformly distributed along the primary amino acid sequence of HIV-1 reverse transcriptase.We suggest that these segments are probably on the surface of the properly folded form of reverse transcriptase.Of the tested proteases, only the viral protease was able to cleave the 66-kDa form to the 51-kDa form without producing additional cleavage products, suggesting that the viral protease cleaves the 66-kDa protein to the 51 -kDa form in virions.

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Section: Introductionmentioning

confidence: 99%

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

Ferris¹,

Hizi

Showalter

et al. 1990

Virology

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“…First we investigated the influence of different pH values in the range 7.0-9.0 because the pH optimum of the HIV-1 enzyme differed from the pH normally chosen when measuring reverse transcriptases [23,24]. From the results illustrated in Fig.…”

Section: Optimum Reaction Conditionsfor D N a Synthesis B Y Sip' mentioning

confidence: 99%

Simian immunodeficiency virus reverse transcriptase

Kraus

Behr

Baier

et al. 1990

European Journal of Biochemistry

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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, p H 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.

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“…Mapping by monoclonal antibodies of the p66 subunit of HIV-1 with C-terminally truncated mutants revealed that p51 was derived from the p66 polypeptide and that p66 and p51 possess identical amino termini (Lightfoote et al, 1986). The tightly associated p66/p51 heterodimer is generated from a HIV-1 p66/p66 homodimer (Farmerie et al, 1987;Mous et al, 1988) in the presence of the HIV-1 PR (diMarzo Veronese et al, 1986;Lightfoote et al, 1986;McHenry, 1989), thus generating an additional polypeptide of 15 kDa from the C-terminus. Once the heterodimer is formed, it is not susceptible to further cleavage.…”

Section: Expression Of Hiv-1 Reverse Transcriptasementioning

confidence: 99%

Review

1996

Biological Chemistry Hoppe-Seyler

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Proteolytic enzymes have many physiological functions in plants, bacteria, viruses, protozoa and mammals. They play a role in processes such as food digestion, complement activation or blood coagulation. The action of proteolytic enzymes is biologically controlled by proteinase inhibitors and increasing attention is being paid to the physiological significance of these natural inhibitors in pathological processes. The reason for this growing interest is that uncontrolled proteolysis can lead to irreversible damage e.g. in chronic inflammation or tumor metastasis. This review focusses on the possible role of the cystatins, natural and specific inhibitors of the cysteine proteinases, in pathological processes. Key words: Cystatins / Cysteine proteinases / Inflammation / Inhibitors / Pathological processes. Cysteine ProteinasesCysteine Proteinases, synonymous with thiol proteinases, are small proteins with molecular masses varying from 23 to 24 kDa and with two catalytic residues, Cys25 and His159, which are involved in the hydrolytic reaction. Cysteine proteinases catalyze the hydrolysis of various polypeptide substrates and are most active under reducing and mildly acidic conditions (pH 5 -6.5). They have been detected in and isolated from a large number of biological sources including plants, bacteria, animals and humans. Bacterial cysteine proteinases are produced by Clostridium histolyticum, named clostripain, by hemolytic group A streptococci and by the pathogenic anaerobic Porphyromonas gingivalis, a bacterium associated with periodontitis. Bacterial cysteine proteinases probably play a role in the penetration of normal tissues by the bacteria and in the food digestion. Viral cysteine proteinases from picornaviruses, e.g. the poliovirus and rhinovirus type I, are involved in proteolytic cleavage of precursor proteins for virus replication and for the production of new virus particles. A virus-coded cysteine proteinase is involved in this process (Kay and Dunn, 1990; Körant et a/., 1985;. HIV-I also needs proteolytic processing by cysteine proteinases to regulate the expression of viral proteins (Guy ef a/., 1991). Protozoal cysteine proteinases are produced by a number of protozoan parasites to facilitate host invasion, metabolize host proteins, to degrade the host immune molecules or to use them for intracellular replication. Cysteine proteinases are produced by e.g. Trypanosoma cruzi, the causative agent of American trypanosomiasis or by Leishmania species, causing one of the six major parasitic diseases. Furthermore, human malarial parasites produce a broad scala of proteinases including cysteine proteinase which are involved in erythrocyte invasion and rupture (McKerrow, 1993). Mammalian cysteine proteinases are present in the lysosomes of cells. Lysosomes contain different cysteine proteinases, the most important being the cathepsins B, H, L and S Kirschke, 1981, Barrett et a/., 1988). These cathepsins have common ancestors and are related to papain. Cathepsin B has been detected in every tissue or cel...

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Expression and Processing of the AIDS Virus Reverse Transcriptase in Escherichia coli

Cited by 201 publications

References 24 publications

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

Simian immunodeficiency virus reverse transcriptase

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