The primary research objective of the laboratory is to dissect mechanisms of minus- and plus-strand synthesis in HIV and structurally related lentiviruses, including those of simian, equine, and feline origin. Converting the single-stranded RNA genome of the invading virus into integration competent double-stranded proviral DNA requires the lentiviral enzymes to accommodate A-form duplex RNA (initiation of minus-strand synthesis), non-A/non-B RNA/DNA hybrids (minus-strand synthesis and initiation of plus-strand synthesis), and B-form duplex DNA (plus-strand synthesis). Moreover, the lentiviral enzymes are asymmetrically organized heterodimers whose subunits are derived from the same gene. Understanding the contribution of each subunit to the activities of the parental heterodimer has been possible through a program of 'subunit-selective' mutagenesis developed in my laboratory. A variety of chemical and enzymatic probing techniques have also been applied to reverse transcriptase (RT) variants with impaired DNA polymerase or ribonuclease H (RNase H) function, using model systems closely mimicking the minus- and plus-strand initiation complexes. Biophysical studies in my laboratory include an NMR analysis of the polypurine tract (PPT) primer of plus-strand synthesis and crystallization of HIV-1 RT complexes with small-molecule RNase H inhibitors. More recently, my laboratory has focused on developing strategies to site-specifically introduce unnatural amino acids for key residues of HIV-1 RT in order to gain high-resolution solution information on the protein and nucleic acid components of these complexes.

Research Highlights 2008–2009

Yi-Brunozzi, H.Y., Brinson, R.G., Brabazon, D.M., Lener, D., Le Grice, S.F.J., and Marino, J.P.  (2008)  High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers.  Chem. Biol. 15: 254-262.

A purine-rich region of the (+) RNA genome of retroviruses and long terminal repeat (LTR)-containing retrotransposons, known as the polypurine tract (PPT), is resistant to hydrolysis by the RNase H subdomain of reverse transcriptase (RT), and ultimately serves as a primer for (+) strand DNA synthesis.  The mechanisms underlying PPT resistance and selective processing remain largely unknown.  In this communication, two RNA/DNA hybrids, derived from the PPTs of HIV-1 and the Saccharomyces cerevisiae LTR-retrotransposon Ty3, were probed using high-resolution NMR for pre-existing structural distortions in the absence of RT.  The PPTs were selectively modified through base-pair changes or by incorporation of the non-hydrogen-bonding thymine isostere, 2,4-difluoro-5-methylbenzene (dF), into the DNA strand.  While both wild-type and mutated hybrids adopted global A-form-like helical geometries, structural perturbations in the base-pair and dF-modified hybrids suggested that the PPT hybrids may function as structurally coupled domains.

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Turner, K.B., Brinson, R.G., Yi-Brunozzi, H.Y., Miller, J.T., Rausch, J.W., Le Grice, S.F.J., Marino, J.P. and Fabris, D.  (2008)  Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.  Nucleic Acids Res. 36: 2799-2810.

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA, and swapped-hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT).  Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT.  In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)₄:(dT)₄ tract and the 3' PPT-U3 junction.  Data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin.  These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the RNase H active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

Abbondanzieri, E.A., Bokinsky, G., Rausch, J.W., Zhang, J.X., Le Grice, S.F.J., and Zhuang, X.  (2008)  Dynamic binding orientations direct activity of HIV reverse transcriptase.  Nature 453: 184-189.

HIV catalyzes 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 multifunctional reverse transcriptase (RT) to bind and discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support RNA-directed DNA synthesis, DNA-directed DNA synthesis, and DNA-directed RNA hydrolysis.  However, the mechanism by which substrates regulate the activity of the enzyme remains unclear.  In their recent publication, Abbondanzieri et al. have reported distinct orientational dynamics of the RT observed on different substrates using a single-molecule assay.  The enzyme adopted opposite binding orientations on duplexes containing generic DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively.  On duplexes containing the HIV polypurine tracts, which function as unique primers for plus-strand DNA synthesis, RT binds in both orientations and rapidly switches between the two states.  Switching kinetics were regulated by cognate nucleotides and non-nucleoside RT inhibitors, a major class of anti-HIV drugs.  These results indicate that the enzymatic activities of the RT are determined by its binding orientation on the substrate.

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Nature News and Views feature related to this article:
Arnold, E., and Sarafianos, S.G.  (2008)  Molecular biology: An HIV secret uncovered.  Nature 453: 169-170.

Harvard University Gazette Online feature about this article:
Bradt, S.  (2008)  Research reveals workings of anti-HIV drugs.

CCR Connections feature about this article:
Reverse transcriptase: When function follows direction.  CCR Connections 2 (1): 4.

Efroni, S., Duttagupta, R., Cheng, J., Deghnani, H., Hoeppner, D.J., Dash, C., Bazett-Jones, D.P., Le Grice, S.F.J., McKay, R.D.G., Buetow, K.H., Gingeras, T.R., Misteli, T., and Meshorer, E.  (2008)  Global transcription in pluripotent embryonic stem cells.  Cell Stem Cell 2: 437-447.

The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem (ES) cells are largely unclear.  Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions during differentiation.  Here we show that the ES cell genome is transcriptionally globally hyperactive and undergoes global silencing as cells differentiate.  Normally silent repeat regions are active in ES cells and tissue-specific genes are sporadically expressed at low levels.  Whole genome tiling arrays demonstrate widespread transcription in both coding and noncoding regions in pluripotent ES cells, whereas the transcriptional landscape becomes more discrete as differentiation proceeds.  The transcriptional hyperactivity in ES cells is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery, but not histone-modifying activities.  Interference with several chromatin-remodeling activities in ES cells affects their proliferation and differentiation behavior.  We propose that global transcriptional activity is a hallmark of pluripotent ES cells that contributes to their plasticity and that lineage specification is strongly driven by reduction of the actively transcribed portion of the genome.

Cell Stem Cell Previews feature related to this article:
Turner, B.M.  (2008)  Open chromatin and hypertranscription in embryonic stem cells.  Cell Stem Cell 2: 408-410.

Ehteshami, M., Scarth, B.J., Tchesnokov, E.P., Dash, C., Le Grice, S.F.J., Hallenberger, S., Jochmans, D., and Goette, M.  (2008)  Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against "nucleotide-competing reverse transcriptase inhibitors."  J. Biol. Chem. 283: 29904-29911.

Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1).  Although their structure differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the DNA polymerase active site.  NRTI-associated mutations clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1.  This collaborative effort studied the underlying biochemical mechanism.  Enzymes containing individual mutations M184V and Y115F cause 2- to 3-fold increases in IC50 values, while their combination causes a > 15-fold increase.  K65R can partially counteract these effects.  Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of RT to discriminate against the inhibitor.  Studies with other strategic mutations at residues F61 and A62, as well as the use of chemically modified templates, further illuminated the putative binding site of the inhibitor and ternary complex formation.  An abasic site residue at position n, i.e. opposite the 3' end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect.  Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleosidetriphosphates (dNTPs).  Members of this class of compounds are denoted nucleotide-competing RT inhibitors (NcRTIs).

Legiewicz, M., Badorrek, C.S., Turner, K.B., Fabris, D., Hamm, T.E., Rekosh, D., Hammarskjold, M.-L., and Le Grice, S.F.J.  (2008)  Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element.  Proc. Natl. Acad. Sci. USA 105: 14365-14370.

Nuclear export of certain HIV-1 mRNAs requires an interaction between the retroviral Rev protein and the Rev response element (RRE), a structured element located in the Env region of its RNA genome.  Disrupting this interaction has been an attractive target for drug design and gene therapy, exemplified by RevM10, a transdominant negative protein that, when introduced into host cells, disrupts viral mRNA export and inhibits virus replication.  However, two silent G->A mutations in the RRE (designated RRE61) conferred RevM10 resistance.  This observation prompted Legiewicz et al. to examine RRE evolution at the structural level using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry, a novel footprinting approach that interrogates the base pairing status of all RNA nucleotides in a single reaction.  Structural variations in region III/IV/V of mutant RNAs suggest a stepwise rearrangement of the RRE to RevM10 resistance.  Using high-resolution mass spectrometry, these authors could also demonstrate that the stoichiometry of Rev "loading" onto RRE61 is unaffected by these structural changes, while chemical footprinting highlighted subtle differences between wild-type and mutant Rev and the RRE variants.

Wendeler, M., Lee, H.-F., Bermingham, A., Miller, J.T., Chertov, O., Bona, M.K., Baichoo, N.S., Ehteshami, M., Beutler, J., O'Keefe, B.R., Götte, M., Kvaratskhelia, M., and Le Grice, S.  (2008)  Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity.  ACS Chem. Biol. 3: 635-644.

High-throughput screening of NCI libraries of synthetic and natural compounds, totaling ~230,000, has identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT).  Synergy studies demonstrated that NSC727447 and the active site hydroxytropolone RNase H inhibitor b-thujaplicinol were mutually exclusive in their interaction with the RNase H domain of RT.  Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the p51 thumb subdomain were affected by inhibitor binding.  Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb.  Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity.  The study by Wendeler et al. shows that NSC727447 represents a novel class of allosteric RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.

ACS Chemical Biology online supporting material related to this article (PDF - 336 K)

ACS Chemical Biology feature about Michaela Wendeler:
Introducing our Authors.  Chem. Biol. 3: 593.

ACS Chemical Biology podcast featuring Stuart Le Grice:
October 2008 ACS Chemical Biology Podcast

Liu, S., Abbondanzieri, E.A., Rausch, J.W., Le Grice, S.F.J., and Zhuang, X. (2008)  Slide into action: Dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates.  Science 322: 1092-1097.

HIV reverse transcriptase (RT) catalyzes a series of intricate reactions through which it converts single-stranded viral RNA of the invading virus into integration-competent double-stranded DNA.  This process requires a variety of enzymatic activities encoded within its p66 subunit, including DNA synthesis, RNase H-mediated cleavage of the RNA/DNA replication intermediate, strand transfer, and strand displacement synthesis.  Using single-molecule fluorescence resonance energy transfer (FRET), we have probed interactions between HIV-1 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, the enzyme can spontaneously “flip” into a polymerization orientation.  Sliding kinetics were also 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 and possibly provide insights into a unique mechanism of action of nonnucleoside RT inhibitors.

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An HIV enzyme with a flair for the acrobatic

Science Perspectives feature related to this article:
Sarafianos, S.G., and Arnold, E.  (2008)  Biochemistry: RT slides home… (PDF - 280 K). Science 322: 1059-1060.

Rausch, J.W., Chelico, L., Goodman, M.F., and Le Grice, S.F.J.  (2009)  Dissecting APOBEC3G substrate specificity by nucleoside analog interference.  J. Biol. Chem. 284: 7047-7058.

The APOBEC cytidine deaminase genes clustered on chromosome 22 encode a set of enzymes including APOBEC1 (A1), APOBEC2 (A2), and APOBEC3A-G (A3A-G).  Although each possesses one or more zinc binding motifs conserved among enzymes catalyzing C-->U conversion, the functions and substrate specificities of these gene products vary considerably.  In the publication of Rausch et al., nucleoside analog interference mapping was used to probe APOBEC3G (A3G)-DNA interactions throughout the enzyme-substrate complex, as well as to determine which DNA structural features determine substrate specificity.  This study indicated that multiple components of nucleosides within the consensus sequence are important for substrate recognition by A3G (with base moieties being most critical), whereas deamination interference by analog substitution outside this region is minimal.  Furthermore, exocyclic groups in pyrimidines 1-2 nt 5' of the target cytosine were demonstrated to dictate substrate recognition by A3G, with chemical composition at ring positions 3 and 4 found to be more important than at ring position 5.  Taken together, these results provide insights into how the enzyme selects A3G hotspot motifs for deamination, as well as which approaches might be best suited for forming a stable, catalytically competent cross-linked A3G-DNA complex for future structural studies.

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Wilkinson, T.A., Januszyk, K., Phillips, M.L., Tekeste, S.S., Zhang, M., Miller, J.T., Le Grice, S.F.J., Clubb, R.T., and Chow, S.A.  (2009)  Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase.  J. Biol. Chem. 284: 7931-7939.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) exerts pleiotropic effects in the viral replication cycle.  Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription.  IN and reverse transcriptase (RT) have been demonstrated to interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT.  In this study, Wilkinson et al. used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN-RT interaction.  An IN K258A substitution disrupting reverse transcription in infected cells is located at the putative RT-binding surface, and was shown here to substantially weaken IN CTD-RT interactions.  Two additional IN amino acid substitutions located at the putative RT-binding surface (W243E and V250E) that significantly impair viral replication in tissue culture were also reported.  Results of this investigation strengthen the notion that IN-RT interactions are biologically relevant during HIV-1 replication and also provide insights into this interaction at the molecular level.

Turner, K.B., Yi-Brunozzi, H.Y., Brinson, R.G., Marino, J.P., Fabris, D., and Le Grice, S.F.J.  (2009)  SHAMS – Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.  RNA 15: 1605-1613.

Selective 2'-hydroxyl acylation analyzed by primer extension, or SHAPE, has recently gained popularity as a facile method of examining RNA secondary structure both in vitro and in vivo.  This technique exploits accessibility of the ribose 2'-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations, and is thus not base-specific.  Subsequent primer extension terminates at the site of chemical modification and the terminated products are fractionated by high-resolution gel electrophoresis.  When applying SHAPE to investigate structural features of the wild-type and analog-substituted polypurine tract (PPT)-containing RNA/DNA hybrids, their size (20-25 bp) rendered primer extension impractical.  We therefore reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M_r = 133Da).  Using this approach, data of this communication demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere.  Our "SHAMS" strategy (selective 2'-hydroxyl acylation analyzed by mass spectrometry) will find utility by examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.

Brinson, R.G., Turner, K.B., Yi-Brunozzi, H.Y., Le Grice, S.F.J., Fabris, D., and Marino, J.P.  (2009)  Probing anomalous structural features in polypurine tract-containing RNA-DNA hybrids with neomycin B.  Biochemistry 48: 6988-6997.

During (-)-strand DNA synthesis in retroviruses and long terminal repeat-containing retrotransposons, a purine-rich region of the RNA template, defined as the polypurine tract (PPT), is resistant to RNase H-mediated hydrolysis and subsequently serves as a primer for (+)-strand, DNA-dependent DNA synthesis.  Although HIV-1 and Ty3 PPT sequences share no sequence similarity beyond the fact that both include a contiguous stretch of purine ribonucleotides, it has been suggested that these RNA primers are processed by their cognate reverse transcriptases (RTs) through a common molecular mechanism.  In this communication, Brinson et al. used the aminoglycoside neomycin B (NB) to examine structural features of the Ty3 PPT that contribute to specific recognition and processing by its cognate RT.  Using high-resolution NMR, direct-infusion FTICR mass spectrometry, and isothermal titration calorimetry, they show that NB binds preferentially and selectively adjacent to the Ty3 3' PPT-U3 cleavage junction and in an upstream 5' region where the thumb subdomain of Ty3 RT putatively grips the substrate.  Regions highlighted by NB on the Ty3 PPT are similar to those previously identified on the HIV-1 PPT sequence that are implicated as contact points for substrate binding by its RT.  Collectively, this work supports the notion that common structural features of lentiviral and LTR-retrotransposon PPTs facilitate the interaction with their cognate RT.

Last modified: 28 July 2009