Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA

Sarih, Leila; Araya, Alejandro; Litvak, Simón

doi:10.1016/0014-5793(88)80642-2

Cited by 13 publications

(3 citation statements)

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“…In the case of human immunodeficiency virus (HIV), tRNALYs,3 is most probably the primer as deduced from the retroviral genome nucleotide sequence [11]. Previous work has shown that avian reverse transcriptase is involved in the selection of tRNATrP and its annealing to the avian retrovirus PBS [12,13]. More recently we have shown that a retrovirus-encoded small molecular weight nucleic acid binding protein can stimulate the in vitro tRNA-PBS annealing [14].…”

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confidence: 99%

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA^Lys

Bordier¹,

Tarragó-Litvak²,

Sallafranque-Andreola³

et al. 1990

Nucl Acids Res

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Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.

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Section: Introductionmentioning

confidence: 99%

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA^Lys

Bordier¹,

Tarragó-Litvak²,

Sallafranque-Andreola³

et al. 1990

Nucl Acids Res

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“…Retroviral reverse transcriptases initiate DNA synthesis in vivo from the 3'-hydroxyl of a host cell tRNA annealed to its complementary region, or primer binding site, around 650 residues from the 5'-end of the viral genome (Panet et al, 1975). Previous work by Litvak and co-workers (Litvak & Araya, 1982;Sarih et al, 1988) andHaseltine et al (1977) demonstrated specific binding of primer tRNATrp by avian myeloblastosis virus (AMV) reverse transcriptase (RT); the AMV RT holoenzyme also is able to promote positioning of tRNATrp at the primer binding site of the retroviral RNA genome (Litvak & Araya, 1982;Sarih et al, 1988). A re-fThis study was supported in part by awards from the NIH Intramural AIDS Targeted Antiviral Program (S.…”

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Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding

Sobol¹,

Suhadolnik²,

Kumar³

et al. 1991

Biochemistry

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Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component. Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template. The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300. This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

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“…Avian myeloblastosis virus (AMV) RT appears capable of catalyzing the hybridizing of tRNA to the AMV primer-binding site (4). Synthesis using purified AMV RT and added tRNA as a primer has been demonstrated in vitro on a viral genome stripped of endogenous primer and other viral proteins (5). For murine leukemia virus, however, it has been suggested that RT alone is not sufficient for synthesis initiation (6).…”

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Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay.

Kohlstaedt

Steitz

1992

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

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Although that any discrimination that occurs upon binding the tRNA in vivo must be largely due to modified bases in the tRNA (7). Our results suggest that the specificity of tRNA usage by RT does not result solely from sequence-specific recognition of the tRNA by RT but is also a consequence of base-pairing to a specific primer-binding site and of recognition of viral sequences 3' to the primer-binding site. METHODSProtein Purification. HIV RT was purified from an E. coli clone containing the RT gene in an overexpressing vector prepared by D'Aquila and Summers (8). All purification steps were done at 40C. Cells were lysed by a French press in 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) (pH 6.0)/2 mM EDTA/0.02% (wt/vol) 1-hexyl (3-D-glucopyranoside (,BHG) (Calbiochem)/10% (vol/vol) glycerol. The lysate was centrifuged, and the pellet was extracted with 50 mM Mes, pH 6.0/50 mM KCl/50 mM potassium phosphate/0.02% ,3HG/10%o glycerol (HSE buffer) to yield an extract containing the RT activity. Polyethyleneimine was added to 0.05% (vol/vol) to remove nucleic acid, and the RT activity was precipitated with 60% saturated (NH4)2SO4. The ammonium sulfate precipitate was redissolved in HSE buffer and applied to a hydroxyapatite column (Bio-Rad) equilibrated in the same buffer. The column was eluted with a linear gradient from 50 to 250 mM potassium phosphate. Fractions containing RT were pooled, precipitated with (NH4)2SO4, redissolved in 50 mM 1,3-bis[tris(hydroxymethyl)methylamino]-propane (Bis-Tris propane), pH 7.0/100 mM (NH4)2SO4/ 0.02% ,BHG/10% glycerol and applied to a heparin column (Pharmacia) equilibrated in the same buffer. The heparin column was eluted with a linear gradient from 0.1 to 1.0 M (NH4)2SO4. Pure protein was stored at 4°C in 50 mM Bis-Tris propane, pH 7.0/100 mM (NH4)2SO4/0.02% (wt/vol) 3HG/ 10%o glycerol/0.02% (wt/vol) sodium azide. Activity is stable under these conditions for at least 1 yr. Yield was -100 mg from 200 g (wet weight) of cells.When purified RT was passed over a Sephadex G200 column, it eluted as a dimer. SDS/PAGE showed that the purified protein was an equimolar mixture of peptides with molecular masses of -66 and 51 kDa. The concentration of 9652The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA

Cited by 13 publications

References 22 publications

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA^Lys

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA^Lys

Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding

Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay.

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Characterization of the cDNA synthesized by avian retrovirus reverse transcriptase using 35 S avian myeloblastosis virus RNA and an exogenous bovine primer tRNA

Cited by 13 publications

References 22 publications

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNALys

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNALys

Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding

Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay.

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Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA^Lys

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA^Lys