Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli

Leuthardt, Andreas; Grice, Stuart F. J. Le

doi:10.1016/0378-1119(88)90596-3

Cited by 31 publications

(24 citation statements)

References 12 publications

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“…The availability of large amounts of nearly authentic (560 amino acids long with an apparent molecular weight of * Corresponding author. 66,000) enzymatically active and soluble recombinant HIV type 1 (HIV-1) RT (13,18,19), which was indistinguishable from the virus-purified enzyme (13, 29), enabled us to search for specific inhibitors of the distinct viral RT activities.Illimaquinone (Fig. 1), a secondary metabolite isolated from the Red Sea sponge Smenospongia sp.…”

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confidence: 99%

Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase

Loya

Tal

Kashman

et al. 1990

Antimicrob Agents Chemother

125

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We studied the effect of the natural marine substance mimaquinone on the catalytic activities of reverse transcriptase from human immunodeficiency virus type 1. Illimaquinone inhibited the RNase H activity of the enzyme at concentrations of 5 to 10 ,ug/ml, whereas RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities were considerably less susceptible to this inhibition. Two synthetic derivatives of illimaquinone, in which the 6'-hydroxyl group at the ortho position to one of the carbonyl groups of the quinone ring was modified, proved ineffective in inhibiting the human immunodeficiency virus type 1 reverse transcriptase RNase H function, suggesting involvement of the 6'-hydroxyl group in blocking the enzymatic activity.Reverse transcriptases (RTs) are key enzymes in the life cycle of retroviruses, since they are responsible for the transcription of the viral RNA into the provirus DNA that is subsequently integrated into the host cell DNA (34). RT is a multifunctional enzyme; the same protein molecules exhibit both RNA-dependent DNA polymerase (RDDP) and DNAdependent DNA polymerase (DDDP) activities as well as an inherent RNase H activity (32). Drugs acting at the reverse transcription level block specifically the replication of human immunodeficiency virus (HIV), the human retrovirus that causes acquired immunodeficiency syndrome, by preventing the unique transcription of genomic viral RNA. Several compounds with diverse molecular structures, including various 2',3'-dideoxynucleosides (14, 21, 22) and 3'-azidothymidine (9, 23, 35) that inhibit the viral RT in the form of 5'-triphosphates, foscarnet (28, 33), suramine (2, 7), rifabutin (1), and HPA 23 (26), have been shown to be active against HIV RT in vitro. In contrast to the relatively large volume of research on the inhibition of the DNA polymerase activities associated with various retroviral RTs, there is a paucity of information on the inhibition of the RT-associated RNase H function. RNase H specifically degrades the RNA in the RNA-DNA heteroduplex after the minus-strand DNA has been synthesized. Removal of the RNA strand enables the synthesis of the plus strand of DNA and the formation of the proviral double-stranded DNA, which is subsequently inserted into the host cellular genome (34). The fact that there is a distinction between the DNA polymerase and RNase H activities of retroviral RTs, including that of HIV (11,25,30,31), could be utilized to develop drugs that will specifically inhibit the RNase H function without appreciably affecting the DNA-polymerizing function. Since the physiological significance of RNase H in mammalian cells is far from clear (6), it is possible that even a partial inhibition of this activity would have minimal effects on the metabolism of normal cells but a substantial inhibitory effect on the virus. The availability of large amounts of nearly authentic (560 amino acids long with an apparent molecular weight of * Corresponding author. 66,000) enzymatically active and soluble recombinant HIV type 1 (HI...

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mentioning

confidence: 99%

Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase

Loya

Tal

Kashman

et al. 1990

Antimicrob Agents Chemother

125

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“…The reverse transcriptase isolated from purified HIV-1 has a molecular mass of 95 (14) to 110 (15) kDa, as determined by gel filtration and glycerol gradient centrifugation, respectively. However, the 98-kDa nonprocessed polyprotein expressed in Escherichia coli displayed little or no reverse transcriptase activity (16). The p66/pSi heterodimer expressed in E. coli has an apparent molecular mass of 120-130 kDa and was active (5,6).…”

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Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.

Kang

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Nevertheless one cannot rule out the possibility that the expression of such a trans-frame protein in bacteria is required for proper folding, stability and solubility, leading to an active recombinant protein that does not exist in virons. However, all RTs expressed so far as recombinant proteins are both soluble in bacteria and fully active without any added sequences at their N-terminus, namely RTs of HIV-1, HIV-2, EIAV, MLV, feline immunodeficiency virus and bovine leukaemia virus ( [14][15][16][17][18][19][20][21], and M. Perach and A. Hizi, unpublished work). Therefore the possible involvement of the protease sequence in expressing an enzymically active and soluble RT needs to be investigated further.…”

Section: Discussionmentioning

confidence: 99%

“…, all of which were expressed as recombinant proteins [14][15][16][17][18][19][20][21], have been investigated extensively, including by threedimensional analyses [22][23][24]. Therefore we were interested in expressing and investigating the molecular and catalytic properties of this unique type-B retroviral RT.…”

Section: Several Other Rts Of Both Type C Retroviruses [Such As Murinmentioning

confidence: 99%

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Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization

TAUBE¹,

LOYA²,

Avidan³

et al. 1998

Biochemical Journal

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We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the k1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni# + nitriloacetic acid\agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-

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Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli

Cited by 31 publications

References 12 publications

Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase

Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase

Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.

Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization

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