Biomedical subjects

S F Le Grice

Publications and source records attributed to S F Le Grice.

At least 19 recordsLinked to original sources

PubMed1995

Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase.

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.

Base Sequence↗

PubMed1995

An expanded model of replicating human immunodeficiency virus reverse transcriptase.

Replication complexes containing wild-type and RNase H-deficient p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) were analyzed by DNase I and S1 footprinting. While crystallography and chemical footprinting data demonstrate that 15-18 bases of primer and template occupy the DNA polymerase and RNase H active centers, enzymatic footprinting suggests that a larger portion of substrate is encompassed by the replicating enzyme. Independent of the position of DNA synthesis arrest, template nucleotides +7 to -23 and primer nucleotides -1 to -25 are nuclease resistant. On both DNA strands, position -20 remains accessible to DNase I cleavage, suggestive of an alteration in nucleic acid structure between exiting the RNase H catalytic center and leaving the C-terminal p66 domain. A model of HIV-1 RT containing an extended single-stranded template and duplex region was constructed on the basis of the structure of an RT/DNA complex. Mapping of footprint data onto this model shows consistency between biochemical and structural data, implicating a contribution from domains proximal to the catalytic centers.

Base Sequence↗

PubMed1995

Truncating alpha-helix E' of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer.

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.

Amino Acid Sequence↗

PubMed1995

HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

Base Sequence↗

PubMed1995

Footprint analysis of replicating murine leukemia virus reverse transcriptase.

Replication complexes that contained either murine leukemia virus reverse transcriptase (MLV RT) or a variant reverse transcriptase without a ribonuclease (RNase) H domain (delta RH MLV RT) were visualized by enzymatic footprinting. Wild-type MLV RT protected template nucleotides +6 to -27, and primer nucleotides -1 to -26 of primers that had first been extended by one or four nucleotides. Although it catalyzed DNA synthesis, delta RH MLV RT stably bound template-primer only under conditions of reduced ionic strength and protected the duplex portion only as far as position -15. Despite altered hydrolysis profiles, both enzymes covered primarily the template-primer duplex, contradicting recent predictions based on the structure of rat DNA polymerase beta.

DNA↗

PubMed1994

Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization.

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.

Amino Acid Sequence↗

PubMed1994

Alternative modes of polymerization distinguish the subunits of equine infectious anemia virus reverse transcriptase.

A comparative study of recombinant 51- and 66-kDa subunits comprising equine infectious anemia virus reverse transcriptase (EIAV RT) is reported. Both polypeptides sedimented as stable homodimers (molecular mass, 102 and 132 kDa, respectively) when analyzed by rate sedimentation through glycerol gradients. Consistent with their dimer composition, each preparation displayed considerable levels of both RNA- and DNA-dependent DNA polymerase activity on different homopolymeric template/primer combinations. However, a detailed analysis of the polymerization products indicated qualitative differences. Whereas p66 EIAV RT proceeded essentially unimpaired along both RNA and DNA templates, p51-catalyzed DNA synthesis was interrupted close to or in the immediate vicinity of the primer. A series of "programmed" 2-step polymerization reactions suggests that p51 EIAV RT enters an abortive mode of polymerization. Duplication of this observation with p51 human immunodeficiency virus-1 RT, together with recent observations from murine RT, suggests that lack of a ribonuclease H domain and loss of contact with the nascent product from the polymerase active center have profound consequences on the mode of polymerization.

Base Sequence↗

PubMed1994

Modulation of HIV-1 reverse transcriptase function in "selectively deleted" p66/p51 heterodimers.

A contribution of the 51-kDa subunit of human immunodeficiency virus type-1 reverse transcriptase to activities of the parental heterodimer (p66/p51) was assessed in "selectively deleted" heterodimers whose p51 component contained C-terminal truncations of 13, 19, or 25 residues. Analyses included (i) efficiency of reconstitution into heterodimer, (ii) retention of polymerase and ribonuclease H (RNase H) function, and (iii) interaction with the HIV replication primer, tRNA(Lys,3). Our data suggest that these features of heterodimer reverse transcriptase can be modulated by the extent of the C-terminal p51 deletion. Severely impaired tRNA binding in a selectively deleted heterodimer whose 51-kDa subunit lacks 13 residues, despite retention of enzymatic functions, strengthens arguments for p51 involvement in tRNA binding.

Amino Acid Sequence↗

PubMed1994

Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H.

In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.

Endoribonucleases↗

PubMed1993

Fluorimetric analysis of recombinant p15 HIV-1 ribonuclease H.

We have exploited the sole tryptophan residue (Trp535) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (p15 RNase H) by fluorescence spectroscopy. Incubation of purified p15 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in Trp535 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.5 x 10(6) M-1 for p15 RNase H complexed with poly(rA)/oligo(dT)12-18 and an occluded site size of 4 nucleotides. A cooperativity coefficient (omega) of 910 was also determined which indicated that nearly three logs of the Kapp were due to cooperativity effects. Recombinant p15 RNase H preparations containing mutations at position 478 (Glu478-->Gln478) or 539 (His539 -->Phe539), which are highly conserved between bacterial and retroviral RNases H, were also analyzed. Under the same conditions, these mutants failed to bind the RNA/DNA hybrid, although they were structurally similar to the wild type polypeptide. Fluorescence spectroscopy thus appears to be an alternative and sensitive means of analyzing functional properties of the purified RNase H domain of HIV-1 RT under a variety of conditions.

Amino Acid Sequence↗

PubMed1993

Hydroxyl radical footprint analysis of human immunodeficiency virus reverse transcriptase-template.primer complexes.

Human immunodeficiency virus type 1 reverse transcriptase protects sugar moieties of a model template.primer DNA in a region from positions +3 to -15 from hydroxyl radical attack. A protected region of equivalent size migrates in concert with the translocating enzyme, as shown by hydroxyl radical footprints of replication complexes after primer extension by 4, 10, and 19 nt. The pattern of these footprints suggests that the DNA template.primer is in the A conformation when complexed with reverse transcriptase. Enhanced accessibility of the DNA template strand around position -15 to hydroxyl radicals indicates a conformational change in the template induced by the C-terminal RNase H-containing domain of p66 reverse transcriptase.

Base Sequence↗

PubMed1993

Nuclease footprinting of human immunodeficiency virus reverse transcriptase/tRNA(Lys-3) complexes.

Nuclease footprinting has been used to probe features of binary complexes of type 1 human immunodeficiency virus reverse transcriptase (HIV-1 RT) with both natural and synthetic preparations of its cognate replication primer, tRNA(Lys-3). In addition to heterodimeric RT (p66/p51), ribonucleoprotein complexes containing either the p66 or p51 subunit were analyzed. Footprinting experiments employed both structure- and sequence-specific nucleases. Our results indicate a similar mode of interaction for the three RT preparations tested, suggesting contact with each loop of the tRNA primer (D, anticodon, and T psi C), as well as minor perturbation of the anticodon stem. Although there is little evidence for extensive disruption of the 3'-acceptor stem. RNase A footprinting data with natural and synthetic tRNA suggests that potential base pairing between the T psi C and D loops is disrupted in the presence of RT.

Anticodon↗

PubMed1993

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.

Animals↗

PubMed1992

Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase.

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.

Chromatography, Gel↗

PubMed1992

Domain structure of the human immunodeficiency virus reverse transcriptase.

The spatial arrangement of subunits p51 and p66 of the HIV-1 reverse transcriptase and the position of the RNase H containing domain, p15, have been determined by means of neutron small-angle scattering. The reverse transcriptase (p66/p51) is a flat molecule, which can be approximated by an ellipsoid with the half axes of 5.2 nm, 4.8 nm and 1.4 nm. The two subunits p51 and p66 having a centre-to-centre distance of 3.3 +/- 0.3 nm are attached at their flat sides, slightly shifted sideways. The p15 domain is located at the long axis of the ellipsoidal reverse transcriptase having a distance of 5.0 +/- 0.5 nm to the centre of the p51d domain, which is part of the p66 subunit, and a distance of 5.3 +/- 1.2 nm to the centre of the neighbouring p51s subunit.

Chromatography, Affinity↗

PubMed1991

Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase.

Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits. By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose. Homologous RT subunits rapidly associated to form the respective heterodimers (1-p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed. Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits. In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme. RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66-kDa subunit.

Chimera↗

PubMed1991

Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase.

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.

Base Sequence↗

PubMed1991

Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3).

Using synthetic oligonucleotides, a gene encoding the HIV-1 replication primer, tRNA(Lys,3), was constructed and placed downstream from a bacteriophage T7 promoter. In vitro transcription of this gene yielded a form of tRNA(Lys,3) which lacks the modified bases characteristic of the natural species and the 3' -C-A-dinucleotide. Synthetic tRNA(Lys,3) annealed to a pbs-HIV1 RNA template can prime cDNA synthesis catalysed by recombinant HIV-1 reverse transcriptase. Trans-DDP crosslinking indicates that this synthetic tRNA is still capable of interacting with HIV-1 RT via a 12-nucleotide portion encompassing the anticodon domain. Gel-mobility shift and competition analyses imply that the affinity of synthetic tRNA for RT is reduced. In contrast to earlier observations, synthetic tRNA is readily competed from RT by natural tRNA(Pro). The reduced affinity of synthetic tRNA(Lys,3) for RT is not appreciably affected by mutations in positions within the loop of the anticodon domain. These results would imply that the overall structure of the anticodon domain of tRNA(Lys,3) is an important factor in its recognition by HIV-1 RT. In addition, modified bases within this, although not absolutely required, would appear to make a significant contribution to the enhanced stability of the ribonucleoprotein complex.

Base Sequence↗