Stuart F. J. Le Grice

Global transcription in pluripotent embryonic stem cells.

The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem cells (ESCs) are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions. Here we show that the ESC genome is transcriptionally globally hyperactive and undergoes large-scale silencing as cells differentiate. Normally silent repeat regions are active in ESCs, and tissue-specific genes are sporadically expressed at low levels. Whole-genome tiling arrays demonstrate widespread transcription in coding and noncoding regions in ESCs, whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ESCs is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery. We propose that global transcription is a hallmark of pluripotent ESCs, contributing to their plasticity, and that lineage specification is driven by reduction of the transcribed portion of the genome.

Dynamic binding orientations direct activity of HIV reverse transcriptase

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.

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 β-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). β-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 μM, while the IC50 for Escherichia coli and human RNases H was 50 μM and 5.7 μM, respectively. In contrast, the related tropolone analog β-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 μM and 40 μM, 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 β-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 μM, 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 β-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.

Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function

Two single site substitutions (E478 → Q and H539 → F) were introduced into the C‐terminal RNase H domain of HIV‐1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+‐nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

Slide into Action: Dynamic Shuttling of HIV Reverse Transcriptase on Nucleic Acid Substrates

The reverse transcriptase (RT) of human immunodeficiency virus (HIV) catalyzes a series of reactions to convert single-stranded viral RNA into double-stranded DNA for host cell integration. This process requires a variety of enzymatic activities, including DNA polymerization, RNA cleavage, strand transfer, and strand displacement synthesis. We used single-molecule fluorescence resonance energy transfer to probe the interactions between RT and nucleic acid substrates in real time. RT was observed to slide on nucleic acid duplexes, rapidly shuttling between opposite termini of the duplex. Upon reaching the DNA 3′ terminus, RT can spontaneously flip into a polymerization orientation. Sliding kinetics were regulated by cognate nucleotides and anti-HIV drugs, which stabilized and destabilized the polymerization mode, respectively. These long-range translocation activities facilitate multiple stages of the reverse transcription pathway, including normal DNA polymerization and strand displacement synthesis.

[13] Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase

Publisher Summary This chapter discusses the purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Metal chelate chromatography is now finding widespread use in the purification of human immunodeficiency virus (HIV) reverse transcriptase (RT) directly from the high-speed supernatant of bacterial homogenates. Metal chelate affinity chromatography offers a rapid and highly reproducible means of preparing (1) the individual p66 and p51 HIV RT subunits, (2) heterodimer p66/p51, and (3) reconstituted, selectively modified heterodimer directly from bacterial homogenates. The application of a highly selective affinity matrix as the primary purification step has the advantage that bacterial proteases are eliminated at an early stage, avoiding proteolysis of the reconstituted protein. The ease with which HIV RT can be purified by metal chelate affinity chromatography has prompted to develop methodologies for protein mini preparation, where four small columns can be run simultaneously and RT eluted in a batch wise fashion. This procedure has been proven successful in cases where individual domains of the enzyme have been analyzed by insertional mutagenesis.

Multimode, Cooperative Mechanism of Action of Allosteric HIV-1 Integrase Inhibitors

Background: 2-(Quinolin-3-yl)-acetic-acid derivatives target HIV-1 integrase and inhibit viral replication. Results: The compounds are allosteric integrase inhibitors (ALLINIs) that block integrase interactions with viral DNA and its cellular cofactor LEDGF and cooperatively inhibit HIV-1 replication. Conclusion: ALLINIs block multiple steps of HIV-1 integration. Significance: These new properties of ALLINIs will facilitate their further development as potent antiretroviral compounds. The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC50 values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.

Structural basis for the specificity of the initiation of HIV‐1 reverse transcription

Initiation of human immunodeficiency virus type 1 (HIV‐1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV‐1 RNA and tRNA3Lys, but remains poorly understood at the three‐dimensional level. We used chemical probing to gain insight into the three‐dimensional structure of the viral RNA–tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three‐dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA–tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3′ end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure.

Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function

Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3′-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5′-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5′-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3′-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5′-RNA-directed RNase H catalysis.

Restoration of tRNA-primed()-Strand DNA Synthesis to an HIV-1 Reverse Transcriptase Mutant with Extended tRNAs IMPLICATIONS FOR RETROVIRAL REPLICATION

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Δ13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.