Biomedical subjects

Stuart F J Le Grice

Publications and source records attributed to Stuart F J Le Grice.

At least 19 recordsLinked to original sources

PubMed2006

Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis.

Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5' to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5' terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G-->A or A-->G substitution at PPT positions -2, -4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions -2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting -4G with an analog containing a 6-NH2 moiety.

Adenine↗

PubMed2006

Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference.

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.

DNA↗

PubMed2006

Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function.

We reported previously that substitutions F61L, F61W, F61Y and F61A in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase affect strand displacement synthesis [T. S. Fisher, T. Darden and V. R. Prasad (2003) J. Mol. Biol., 325, 443-459]. We have now determined the effect of these mutations on HIV replication. All mutant viruses were replication defective. Measuring replication intermediates in infected cells did not reveal a specific block as all mutants displayed reduced DNA synthesis (wild-type>F61L>F61W>F61Y>F61A). Analysis of 2-LTR circle junctions revealed that F61W and F61Y mutants generated increased aberrant circle junctions. Circle junctions corresponding to F61Y included 3'-PPT insertions suggesting ribonuclease H defect. In vitro assays mimicking PPT primer generation indicated that F61L, F61W and F61Y mutant RTs were unaffected, while F61A mutant cleaved both at PPT/U3 junction and at +6 with similar efficiencies. In assays measuring cleavage at the RNA/DNA junction to remove the PPT primer, all mutants were significantly affected with F61Y and F61A being most severely impaired. Our results show that (i) replication block of most mutants is due to more than one biochemical defect; (ii) mutations in polymerase domain can affect the function of a distal domain; and (iii) virological analyses of RT mutations can yield insight into structure-function relationship that is otherwise not obvious.

Amino Acid Substitution↗

PubMed2006

Nucleotide excision repair and template-independent addition by HIV-1 reverse transcriptase in the presence of nucleocapsid protein.

During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.

DNA Primers↗

PubMed2005

A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid.

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.

Carbohydrate Conformation↗

PubMed2005

Examining Ty3 polypurine tract structure and function by nucleoside analog interference.

We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy, and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3 by its cognate reverse transcriptase. Locked nucleic acid analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (-)-DNA template, whereas abasic tetrahydrofuran linkages, which lack the nucleobase but preserve the sugar phosphate backbone, were introduced throughout the (-)-strand DNA template and (+)-strand RNA primer. Collectively, our data suggest that both the 5'- and 3'-portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, whereas the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT that make close contact with the Ty3 reverse transcriptase thumb subdomain and RNase H catalytic center, respectively. Achieving a similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features that are important mediators of Ty3 PPT recognition. Finally, the results from introducing tetrahydrofuran lesions around the scissile PPT/unique 3'-sequence junction indicate that template nucleobase -1 is dispensable for catalysis, whereas a primer nucleobase on either side of the junction is necessary.

Base Sequence↗

PubMed2005

Interaction of the Ty3 reverse transcriptase thumb subdomain with template-primer.

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Alanine↗

PubMed2005

Attenuation of DNA replication by HIV-1 reverse transcriptase near the central termination sequence.

Previous pre-steady-state kinetic studies of equine infectious anemia virus-1 (EIAV) reverse transcriptase (RT) showed two effects of DNA substrates containing the central termination sequence (CTS) on the polymerization reaction: reduction of burst amplitude in single nucleotide addition experiments and accumulation of termination products during processive DNA synthesis [Berdis, A. J., Stetor, S. R., Le Grice, S. F. J., and Barkley, M. D. (2001) Biochemistry 40, 12140-12149]. The present study of HIV RT uses pre-steady-state kinetic techniques to evaluate the molecular mechanisms of the lower burst amplitudes using both random sequence and CTS-containing DNA substrates. The effects of various factors, including primer/template length, binding orientation, and protein concentration, on the burst amplitude were determined using random sequence DNA substrates. The percent active RT increases with total RT concentration, indicating that reversible dissociation of RT dimer is responsible for substoichiometric burst amplitudes with normal substrates. This finding was confirmed by gel mobility shift assays. Like EIAV RT, HIV RT showed lower burst amplitudes on CTS-containing DNA substrates compared to random sequences. The dissociation kinetics of RT-DNA complexes were monitored by enzyme activity and fluorescence. Biphasic kinetics were observed for both random sequence and CTS-containing DNA complexes, revealing two forms of the RT-DNA complex. A mechanism is proposed to account for reduction in burst amplitude of CTS-containing DNA that is consistent with the results of both single nucleotide addition and dissociation experiments. The two forms of the RT-DNA complex may represent partitioning of primer/template between the P- and N-sites on RT for the nucleic acid substrate.

Base Sequence↗

PubMed2005

Investigating HIV-1 polypurine tract geometry via targeted insertion of abasic lesions in the (-)-DNA template and (+)-RNA primer.

A variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (-)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO(4) footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (-)-DNA template between positions -15 and -13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (-11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile -1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis.

Base Sequence↗

PubMed2005

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones.

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

Anti-HIV Agents↗

PubMed2005

Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase.

Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151-->Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151-->Asn and Asp213-->Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition.

Amino Acid Substitution↗

PubMed2004

Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor.

HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.

Amino Acid Sequence↗

PubMed2004

Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes.

In order to determine the contribution of modified bases on the efficiency with which tRNA(Lys,3) is used in vitro as the HIV-1 replication primer, the properties of synthetic derivatives prepared by three independent methods were compared to the natural, i.e. fully modified, tRNA. When prepared directly by in vitro run-off transcription, we show here that the predominant tRNA species is 77 nt, representing a non-templated addition of a single nucleotide. As a consequence, this aberrant tRNA inefficiently primes (-) strand strong stop DNA synthesis from the primer binding site (PBS) on the HIV-1 viral RNA genome to which it must hybridize. In contrast, correctly sized tRNA(Lys,3) can be prepared by (i) total chemical synthesis and ligation of 'half' tRNAs, (ii) transcription of a cassette whose DNA template contained strategically placed 2'-O-Methyl-containing ribonucleotides and (iii) processing from a larger precursor by means of targeted cleavage with Escherichia coli RNase H. When each of these 76 nt tRNAs was supplemented into a (-) strand strong stop DNA synthesis reaction utilizing the HXB2 strain of HIV-1, the amount of product obtained was comparable to that from the fully modified counterpart. Parallel assays monitoring early events in (-) strand strong stop DNA synthesis using either the HXB2 or Mal strain of HIV-1 RNA as the template indicated little difference in the pattern or total product amount when primed with either natural or synthetic tRNA(Lys,3). In addition, nuclease mapping of PBS-bound tRNA suggests inter-molecular base pairing between bases of the tRNA anticodon domain and the U-rich U5-IR loop of the viral 5' leader region is less stable on the HIV-1(HXB2) genome than the HIV-1(Mal) isolate.

DNA Primers↗

PubMed2004

A capillary electrophoretic assay for ribonuclease H activity.

A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type 1 reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program. Since autofluorescent compounds in samples migrated differently from both substrate and product in most cases, the assay was extremely robust for assaying enzymatic inhibition of such samples, in contrast to a simple well-based approach. The assay was broadly applicable to other RNases H, specifically those from human, Escherichia coli, and HIV-2, although product profiles varied for each enzyme.

Base Sequence↗

PubMed2004

Two modes of HIV-1 polypurine tract cleavage are affected by introducing locked nucleic acid analogs into the (-) DNA template.

Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (-) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA.rU tract immediately 5' of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT.rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC.rG and neighboring dT.rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3'-end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (+5/+6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 + 5/+6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.

Base Sequence↗

PubMed2004

Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry.

We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 microM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.

Acetylation↗

PubMed2004

Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3' polypurine tract.

Recent structural analyses indicate that localized regions of abnormal base pairing exist within RNA/DNA hybrids containing the HIV-1 polypurine tract (PPT) and that these distortions may play a role in PPT function. To examine this directly, we have introduced pyrrolo-deoxycytosine (pdC), a fluorescent, environmentally sensitive analog of deoxycytosine (dC), into the DNA strand of PPT-containing hybrids. Steady-state fluorescence analysis of these hybrids reveals that the DNA base 11 nt from the PPT-U3 junction is unpaired even in the absence of reverse transcriptase (RT). Unstable base pairing is also observed within the (rG:dC)6 tract in the downstream portion of the duplex, suggesting that HIV-1 RT may recognize multiple pre-existing distortions during PPT selection. HIV-1 RT hydrolyzes pdC-containing hybrids primarily at the PPT-U3 junction, indicating that the analog does not induce a gross structural deformation of the duplex. However, aberrant cleavage is frequently observed 3 bp from the site of pdC substitution, most likely reflecting a specific interaction between the analog and amino acid residues within the RNase H primer grip. pdC substitution within the template strand of a DNA duplex does not appear to significantly affect RT-catalyzed DNA synthesis. Implications of these findings on the use of pdC to examine nucleic acid structure are discussed.

Adenine↗

PubMed2004

'Binding, bending and bonding': polypurine tract-primed initiation of plus-strand DNA synthesis in human immunodeficiency virus.

During the course of reverse transcription, human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) initiates plus-strand DNA synthesis from two highly conserved, purine-rich RNA segments of the viral genome referred to as the 3' and central polypurine tracts (3' and cPPTs). Processing of these elements occurs in several sequential steps including (1) minus-strand DNA synthesis over the PPT(s), (2) ribonuclease H (RNase H) mediated cleavage at the PPT 3' terminus, (3) plus-strand DNA synthesis from the nascent RNA primer(s), and (4) primer removal. Completing each of these steps precisely and specifically is essential, as failure to do so can result in reduced virus replication and/or impaired integration of viral DNA into the host cell genome. In this review, plus-strand primer processing in HIV-1 is discussed from biochemical, structural, and historical perspectives. A comparative analysis of PPT-processing in different LTR-containing retroelements is also presented.

DNA, Viral↗