Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme

Müller, Bárbara; Restle, Tobias; Kühnel, H; Goody, Roger S.

doi:10.1016/s0021-9258(18)98744-2

Cited by 49 publications

(15 citation statements)

References 24 publications

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“…It is evident that MMTV RT is sensitive to OP in all the three catalytic functions, with the RNase H activity being significantly less sensitive than the DNA polymerase functions (of the activities, RDDP was the most sensitive) (Figure 6C). Although the pattern of inhibition of the RNase H activity was quite similar to that of HIV RTs (and was different from that of EIAV RT) [37,38], the inhibition curves of the DNA polymerase activities of MMTV RT were more similar to those of EIAV RT. We have already speculated, on the basis of an assumption that OP is specific to only one Zn# + ion (with no effect on the activities via other mechanisms), that the inhibition pattern of HIV-1 RT by OP can be explained by assuming that each RT molecule binds at least two Zn# + ions.…”

Section: Involvement Of Cysteine Residues In the Mmtv Rt Catalytic Activitiesmentioning

confidence: 80%

“…The results presented in this paper single out another unique feature of the recombinant RT of MMTV. With the exception of mammalian type C retroviruses, all other retroviral RTs are believed to function as heterodimers [22,[35][36][37]. MLV RT forms monomers in solution, although it has been suggested that two monomers bind DNA co-operatively throughout DNA synthesis [38,40].…”

Section: Discussionmentioning

confidence: 99%

“…Most RTs studied so far have been composed of heterodimers [22,[35][36][37]. The one exception is MLV RT, which functions as a monomer of approx.…”

Section: Subunit Composition Of Enzymically Active Mmtv Rtmentioning

confidence: 99%

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

Taube

Loya

Avidan

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 -1 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, Ni2+ 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-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

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Section: Involvement Of Cysteine Residues In the Mmtv Rt Catalytic Activitiesmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

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

Taube

Loya

Avidan

et al. 1998

Biochemical Journal

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“…The column was washed with 5 column volumes of buffer A until the absorbance returned to baseline. The HIV-2 RT heterodimer was washed using 3 column volumes of 80 mM NaCl in buffer A followed by elution using 3 column volumes of 200 mM NaCl in buffer A (Muller et al, 1991). Fractions (0.8 mL) were collected and analyzed for absor-bance (280 nm) and protein concentration by the Bio-Rad Bradford protein assay (Bradford, 1976).…”

Section: Methodsmentioning

confidence: 99%

“…However, based on studies with HIV-1 RT, the protein recovered from the host expression system is usually heterodimeric with a heterogeneous C-terminus of the smaller subunit (Lowe et al, 1988). Another strategy to obtain purified heterodimer is to coexpress the two subunits on a single plasmid (Muller et al, 1991), and this requires a knowledge of the exact C-terminus of the smaller subunit. Thus, it is desirable to obtain heterodimeric HIV-2 RT in which the smaller subunit is generated by specific processing of the larger subunit with the homologous HIV-2 protease.…”

mentioning

confidence: 99%

Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

Fan¹,

Rank²,

Poppe³

et al. 1996

Biochemistry

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Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2.

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Small Molecule Inhibitors Targeting HIV‐1 Reverse Transcriptase Dimerization

et al. 2008

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The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t(1/2) of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.

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Expression of the heterodimeric form of human immunodeficiency virus type 2 reverse transcriptase in Escherichia coli and characterization of the enzyme

Cited by 49 publications

References 24 publications

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

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

Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

Small Molecule Inhibitors Targeting HIV‐1 Reverse Transcriptase Dimerization

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