HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes.

The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through -21 and primer nucleotides -1 through -24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe(2+) fails to replace Mg(2+) in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase.

Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry.

Crystallographic studies of the Mn(2+)-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have revealed two bound Mn2+ separated by approximately 4A and surrounded by a cluster of four conserved carboxylates. Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity, implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/ or the doping technique employed. Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT. Three Mn2+ ions bind to HIV-1 RT apo-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results. However, only one Mg2+ ion is bound in the RNase H catalytic center. Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain. The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported.

Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking.

Cys(38) and Cys(280) of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross-linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C(561)) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21. Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies.

Characterization of (+) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome.

Permeabilized preparations of equine infectious anemia virus (EIAV) are shown here to support efficient and accurate synthesis of full-length double-stranded proviral DNA. When (-) and (+) strand products were analyzed by Southern blotting, a discontinuity, mapping approximately to the center of the EIAV genome, could be demonstrated for the (+) strand, predicting a second site for initiation of DNA synthesis and a specific mechanism of (+) strand termination. Precise localization of this (+) strand origin within the integrase (IN) coding region was achieved through its in vitro selection and extension into, and excision from, nascent DNA by purified recombinant p66/p51 EIAV reverse transcriptase (RT), suggesting that the EIAV genome harbors a central polypurine tract (cPPT). In addition, a model system was developed for evaluating whether sequences immediately downstream of the cPPT would terminate (+) strand synthesis in the context of strand displacement. Such a sequence was indeed discovered which functions in a manner analogous to that of the central termination sequence (CTS) of HIV, where A-tract-induced minor groove compression has been suggested to induce localized distortion of the nucleic acid duplex and termination of (+) strand synthesis. This interpretation is reinforced by experiments indicating that read-through of the CTS can be efficiently promoted by substituting 2,6-diaminopurine for adenine, thereby relieving minor groove compression. The nucleotide substitution can also shift the site of termination in strand displacement (+) strand synthesis. Collectively, our data support proposals that lentiviruses may have evolved specialized mechanisms for initiating and terminating (+) strand DNA synthesis at the center of their genomes.

Substituting a conserved residue of the ribonuclease H domain alters substrate hydrolysis by retroviral reverse transcriptase.

Alterations to the highly conserved Asp549 of the retroviral ribonuclease H (RNase H) domain were evaluated in the heterodimeric (p66/p51) reverse transcriptases of human immunodeficiency and equine infectious anemia viruses. In addition to the polymerization-dependent and -independent modes of template hydrolysis, mutants were evaluated via their ability to select and extend the 3' polypurine tract (PPT) primers of these two lentiviruses into (+) strand DNA. Concerted and two-step reactions were designed to evaluate (+) strand priming, the latter of which allows discrimination between selection end extension events. In contrast to enzyme mutated at the highly conserved Glu478, substitution of Asp549 with Asn or Ala reduces, rather than completely eliminates, RNase H activity. When the requirement for RNase H function becomes more stringent, differences in activity are readily evident, most notably in the cleavage events liberating the 5' terminus of the PPT primer. PPT selection thus appears to represent a specialized form of RNase H activity that is more sensitive to minor structural alterations within this domain and may provide a novel therapeutic target.

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Involvement of C-terminal structural elements of equine infectious anemia virus reverse transcriptase in DNA polymerase and ribonuclease H activities.

In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcriptase (EIAV RT), recombinant p66 polypeptides containing a modified ribonuclease H (RNase H) domain were purified and evaluated. Defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of DNA polymerase and RNase H activities, while DNase I footprinting revealed features of replication complexes containing the truncated enzymes. Removal of alpha-helix E' and the conserved beta 5'-alphaE' "His-loop" in p66delta20 RT uncouples the RNase H activities, alters affinity for template-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, DNase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-terminal residues, which includes alpha-helix D', beta-strand 5' and alpha-Helix E', yields an enzyme with distributive DNA polymerase properties closely resembling the purified p51 subunit.

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.