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Stuart F.J. Le Grice, Ph.D.
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Biography
Dr. Stuart Le Grice received his Ph.D. from the Department of Biochemistry, University of Manchester, UK, in 1976, where he studied the mechanisms of R-factor-mediated multidrug resistance in Escherichia coli. After postdoctoral training in the United Kingdom, Germany, and the United States, he was appointed Senior Scientist in the Central Research Units of Hoffmann La Roche, Basel, Switzerland, where he worked from 1984 to 1990 evaluating HIV-1 and HIV-2 enzymes as therapeutic targets. In 1990, he joined the faculty in the Division of Infectious Diseases, Department of Medicine, Case Western Reserve University (CWRU), Cleveland, OH. Initially recruited as an Associate Professor of Medicine, he was awarded tenure in 1992, and in 1995 was promoted to Professor of Medicine, Biochemistry, and Oncology. From 1994 to 1999, he served as Director of the NIH-funded CWRU Center for AIDS Research. Dr. Le Grice joined the National Cancer Institute in 1999 as Chief of the Resistance Mechanisms Laboratory in the HIV Drug Resistance Program, Center for Cancer Research (CCR), and in 2005 was appointed to the Senior Biomedical Research Service. In 2006, he was appointed Head of the Center of Excellence in HIV/AIDS & Cancer Virology, CCR. He is a member of the CCR HIV and Cancer Virology faculty, Chemistry and Biology faculty, and the Steering Committee of the Molecular Targets Discovery Program. In addition to serving on the Editorial Board of the Journal of Biological Chemistry, Dr. Le Grice has been an ad hoc (1990-1999) and permanent Study Section member of NIH AIDS review panels (2000-2004), as well as an ad hoc reviewer for several international funding agencies.Research
Research Focus: Protein/Nucleic Acid Interactions Controlling Retroviral Replication
The primary research objective of the RT Biochemistry Section is dissecting proteins, nucleic acids and their nucleoprotein complexes as they relate to replication of RNA viruses, retroviruses, and LTR-containing retrotransposons. Projects in the laboratory use a combination of biochemical, biophysical, structural, biological, and computational strategies to better understand processes of reverse transcription, RNA export, and genome packaging. Single-molecule spectroscopy has shown HIV-1 reverse transcriptase (RT) to be a highly dynamic enzyme, capable of sliding and changing orientation on its nucleic acid substrate, while at the same time making the novel observation that nonnucleoside RT inhibitors can influence enzyme orientation. We have recently generated high-resolution structures for lentiviral (HIV-1) and gammaretroviral (XMRV) RTs in the presence of a non-polypurine tract (PPT) RNA/DNA hybrid, demonstrating a catalytically competent conformation with respect to nucleic acid in the RNase H active site. These structures provide a platform for both structure/function studies and drug development, where we focus on developing allosteric inhibitors that bind adjacent to the RNase H active site.
Understanding RNA structure and function has taken advantage of a novel chemoenzymatic probing method (SHAPE) that can be used both in vitro and in vivo. Modifications of this technique (ai-SHAPE) allow us to investigate long-range tertiary interactions (i.e., kissing-loop interactions and pseudoknots) that control both genome replication and transport of unspliced RNAs. SHAPE studies are combined with computational methods designed to develop improved algorithms for predicting RNA tertiary structure. This work involves collaborative interactions with several investigators in both the intramural and extramural research communities.
Finally, we are investigating the potential of N-terminal Cu++/Ni++ motifs (ATCUNS) as metallotherapeutics. This tripeptide motif can be appended to the N-terminus of peptides and proteins and induced to release reactive oxygen species, thereby designating them chemical 'nucleases' and 'proteases' capable of irreversibly inactivating their target biomolecule. In addition to their therapeutic potential, ATCUN-derived metallopeptides are also under investigation as through-space cleavage reagents to provide information on RNA tertiary structure.
Research Highlights 2009-2013
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.
JBC online supplemental data related to this article (PDF - 202 K)
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 (Mr = 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.
Chung, N.P.Y., Breun, S.K.J., Bashirova, A., Baumann, J.G., Martin, T.D., Karamchandani, J.M., Rausch, J.W., Le Grice, S.F.J., Wu, L., Carrington, M., and KewalRamani, V.N. (2010) HIV-1 transmission by dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is regulated by determinants in the carbohydrate recognition domain that are absent in liver/lymph node-SIGN (L-SIGN). J. Biol. Chem. 285: 2100-2112.
This multi-disciplinary study identified determinants in DC-SIGN necessary for HIV-1 transmission. While human B cell lines expressing DC-SIGN efficiently capture and transmit HIV-1 to susceptible target cells, cells expressing the related molecule L-SIGN do not. To understand the differences between DC-SIGN and L-SIGN that affect HIV-1 interactions, Raji B cell lines expressing different DC-SIGN/L-SIGN chimeras were developed. Replacing the DC-SIGN carbohydrate recognition domain (CRD) with that of L-SIGN was sufficient to impair virus binding and prevent transmission. Conversely, the ability to bind and transmit HIV-1 was conferred on L-SIGN chimeras containing the DC-SIGN CRD. W258 in the DC-SIGN CRD was found to be essential for HIV-1 transmission. While introducing a K270W mutation at the same position in L-SIGN was insufficient for HIV-1 binding, an L-SIGN mutant molecule with K270W and a C-terminal DC-SIGN CRD subdomain transmitted HIV-1. These data suggest that DC-SIGN structural elements distinct from the oligosaccharide-binding site are required for HIV-1 glycoprotein selectivity.
JBC online supplemental data related to this article (PDF - 469 K)
Legiewicz, M., Zolotukhin, A.S., Pilkington, G.R., Purzycka, K.J., Mitchell, M., Uranishi, H., Bear, J., Pavlakis, G.N., Le Grice, S.F.J., and Felber, B.K. (2010) The RNA transport element of the murine MusD retrotransposon requires long-range intramolecular interactions for function. J. Biol. Chem. 285: 42097-42104.
Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR-retrotransposons (MusD) were reported to contain an RNA export element located upstream of the 3' LTR. Although functionally equivalent, the MusD export element, termed MTE, is distinct from the other retroviral RNA export elements, such as the CTE of simian SRV/MPMV retroviruses and the RTE found in rodent IAP-LTR retrotransposons. Data of Legiewicz et al. show that the MTE comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. Two classes of tertiary interactions, namely kissing loops and a pseudoknot, were identified by a combination of chemoenzymatic footprinting and site-directed mutagenesis. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.
Liu, S., Harada, B.T., Miller, J.T., Le Grice, S.F.J., and Zhuang, X. (2010) Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat. Struct. Mol. Biol. 17: 1453-1460.
Human immunodeficiency virus (HIV) initiates reverse transcription from a cellular tRNALys,3 hybridized to a specific region near the 5' terminus of its (+) viral RNA (vRNA) genome, designated the primer binding site or PBS. This process has been characterized biochemically by a slow initiation phase with specific pauses, followed by a fast elongation phase. However, the mechanisms underlying the slow initiation and the transition to the elongation phase have not been fully elucidated. This publication continues our single-molecule studies by monitoring the dynamics of individual initiation complexes, comprised of the vRNA template, tRNA primer and HIV-1 RT. Dynamic transitions ('flips') between two opposite binding orientations on tRNA:vRNA complexes are exhibited by RT, and the prominent pausing events reflect enzyme binding in an orientation opposite to the polymerase-competent configuration. A stem-loop structure ahead of the PBS is responsible for maintaining the enzyme predominantly in this flipped orientation. Disrupting the stem-loop structure, through site-directed mutagenesis or inclusion of the HIV-1 nucleocapsid protein, triggers enzyme re-orientation and the transition to the elongation phase. These results highlight the important role played by the structural dynamics of the initiation complex in directing transitions between early reverse transcription phases.
Nat. Struct. Mol. Biol. supplemental information related to this article (PDF - 2230 K)
Kenyon, J.C., Tanner, S.J., Legiewicz, M., Phillip, P.S., Rizvi, T.A., Le Grice, S.F.J., and Lever, A.M.L. (2011) SHAPE analysis of the FIV leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization. Nucleic Acids Res. 39: 6692-6704.
Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput chemo-enzymatic footprinting was used to map the secondary structure of the FIV packaging signal RNA. Previous studies indicated four conserved stem-loops, extensive long-range interactions, and a small, palindromic stem-loop within the gag open reading frame that may act as a dimerization initiation site, enabling the virus to package two copies of its genome. The present analysis of wt and mutant RNAs suggests that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form long-range interactions also adopt an alternative, yet similarly conserved conformation, in which the putative dimerization initiation site is occluded, and which may thus favour translational and splicing functions over encapsidation. Dimerization contacts appear to be made between palindromic loop sequences in stem-loop V. As this stem-loop is located within the gag open reading frame, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.
Nucleic Acids Res. supplementary data related to this article
Chung, S., Himmel, D.M., Jiang, J.-K., Wojtak, K., Bauman, J.D., Rausch, J.W., Wilson, J.A., Beutler, J.A., Thomas, C.J., Arnold, E., and Le Grice, S.F.J. (2011) Synthesis, activity, and structural analysis of novel alpha-hydroxytropolone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H. J. Med. Chem. 54: 4462-4473.
We previously demonstrated that the natural product alpha-hydroxytropolone manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibited ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture, most likely reflecting toxicity caused by inhibition of cellular enzymes. Converting the 2-isopropenyl manicol substituent to an epoxide ketone or dihydroxylate permitted synthesis of 14 novel analogs that retained the divalent metal-chelating alpha-hydroxytropolone (2,7-dihydroxy-cyclohepta-2,4,6-trienone) pharmacophore. Screening efforts were augmented by a high-resolution structure of p66/p51 HIV-1 RT containing a nonnucleoside inhibitor (TMC278) in the DNA polymerase domain and manicol in the C-terminal RNase H domain. In this communication, Chung et al. demonstrated that several modified alpha-hydroxytropolones exhibit antiviral activity at noncytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, these studies will aid future structure-based design of improved alpha-hydroxytropolones to complement the nucleoside and nonnucleoside RT inhibitors currently in clinical use.
Chung, S., Miller, J.T., Johnson, B.C., Hughes, S.H., and Le Grice, S.F.J. (2012) Mutagenesis of human immunodeficiency virus reverse transcriptase p51 subunit defines residues contributing to vinylogous urea inhibition of ribonuclease H activity. J. Biol. Chem. 287: 4066-4075.
The vinylogous urea, NSC727447, allosterically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus Type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of the non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied inhibitor-mediated alterations to active site geometry, while mass spectrometry suggested a contribution from alpha-helix I residues Cys280 and Lys281. To further characterize the inhibitor binding site, Chung et al. combined scanning mutagenesis between p51 residues Lys275 and Thr286, (comprising alpha-helix I and portions of the neighboring connecting loops) with a limited vertical scan of Cys280. An important role for Cys280 and Thr286 was suggested by the observation that these reconstituted, selectively mutated p66/p51 heterodimers were significantly resistant to inhibition, as were additional Cys280 mutants. In several instances, alanine substitutions led to significantly increased inhibitor sensitivity. In contrast, mutant enzymes retained equivalent sensitivity to an active site alpha-hydroxytropolone-derived RNase H inhibitor. p66/p51 heterodimers containing short p51 C-terminal truncations also displayed increased sensitivity to vinylogous urea inhibition. Cumulatively, these data suggest a contribution from the p51 thumb subdomain to nucleic acid binding that is compromised following inhibitor binding.
J. Biol. Chem. supplemental data related to this article
Zhou, D., Chung, S., Miller, M., Le Grice, S.F.J., and Wlodawer, A. (2012) Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus. J. Struct. Biol. 177: 638-645.
The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. Zhou et al. report here crystal structures for several variants of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site alpha-hydroxytropolone inhibitor. Enzymatic assays showed that the intact RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H complexed with beta-thujaplicinol clearly showed that coordination by two divalent cations mediated recognition of the inhibitor.
Kessl, J.J., Jena, N., Koh, Y., Taskent-Sezgin, H., Slaughter, A., Feng, L., de Silva, S., Wu, L., Le Grice, S.F.J., Engelman, A., Fuchs, J.R., and Kvaratskhelia, M. (2012) Multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors. J. Biol. Chem. 287: 16801-16811.
HIV-1 integrase (IN) interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate viral DNA into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. 2-(quinolin-3-yl) acetic acid derivatives have been reported to selectively inhibit the IN-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Kessl et al. show here that this class of compounds impairs both IN-LEDGF binding and LEDGF-independent IN catalytic activities with similar IC50 values, defining them as bona fide allosteric inhibitors of IN function. Furthermore, this study shows show that 2-(quinolin-3-yl) acetic acid derivatives block formation of the stable synaptic complex between IN and viral DNA by allosterically stabilizing an inactive multimeric form of IN. 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. Such findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.
Purzycka, K.J., Legiewicz, M., Matsuda, E., Eizentstat, L.D., Lusvarghi, S., Saha, A., Le Grice, S.F.J., and Garfinkel, D.J. (2013) Exploring Ty1 retrotransposon RNA structure within virus-like particles. Nucleic Acids Res. 41: 463-473.
Ty1, a long terminal repeat (LTR) retrotransposon of Saccharomyces, is structurally and functionally related to retroviruses. However, a differentiating aspect between these retroelements is the diversity of the replication strategies used by LTR-retrotransposons. To understand the structural organization of cis-acting elements present on Ty1 genomic RNA from the GAG region that control reverse transcription, Purzcyka et al. applied chemoenzymatic probing (SHAPE) to RNA/tRNA complexes assembled in vitro and to the RNA in virus-like particles (VLPs). By comparing different RNA states, this analysis provides a comprehensive structure of the primer-binding site (pbs), a novel pseudoknot adjacent to the pbs, three regions containing palindromic sequences that may be involved in RNA dimerization or packaging, and candidate protein interaction sites. This study additionally determined the impact of a novel form of transposon control based on Ty1 antisense transcripts that associate with VLPs. The results of this work support the idea that antisense RNAs inhibit retrotransposition by targeting Ty1 protein function rather than annealing with the RNA genome.
Chamanian, M., Purzycka, K.J., Wille, P.T., Ha, J.S., McDonald, D., Gao, Y., Le Grice, S.F.J., and Arts, E.J. (2013) A cis-acting element in retroviral genomic RNA links Gag-Pol ribosomal frameshifting to selective viral RNA encapsidation. Cell Host Microbe 13: 181-192.
During retroviral RNA encapsidation, two full-length genomic RNAs are selectively incorporated into assembling virions. Genome packaging involves a cis-acting packaging element (Y) within the 5’ untranslated region (UTR) of the unspliced genome. However, the mechanism(s) that selects and limits genomic RNAs for packaging remains uncertain. Using a dual complementation system involving bipartite HIV-1 genomic RNA, Chamanian et al. observed that packaging was additionally dependent on a cis-acting RNA element, designated the genomic RNA packaging enhancer (GRPE), found within the Gag p1-p6 domain and overlapping the Gag-Pol ribosomal frameshift signal. Deleting or disrupting the two conserved GRPE stem-loops diminished genome packaging and infectivity >50-fold, while deleting gag sequences between Y and the GRPE had no effect. Downregulating the translation termination factor eRF1 produces defective virus particles containing approximately 20 times more genomic RNA. Thus, only the HIV-1 RNAs employed for Gag-Pol translation may be specifically selected for encapsidation.
Cell Host Microbe supplemental information related to this article (PDF - 2031 K)
Lapkouski, M., Tian, L., Miller, J.T., Le Grice, S.F.J., and Yang, W. (2013) Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat. Struct. Mol. Biol. 20: 230-236.
Although a large number of structures of HIV-1 reverse transcriptase (RT) have been determined, only one contains an RNA/DNA hybrid. In this publication, Lapkouski et al. report three novel structures of HIV-1 RT complexed with a nonnucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, these RNA/DNA structures differ from all prior nucleic acid-RT structures including the polypurine tract (PPT)-containing RNA/DNA hybrid. The enzyme structure also differs from all previous RT-DNA complexes. Thus, the hybrid has ready access to the RNase H active site. These observations collectively indicate that an RT-nucleic acid complex may adopt alternate structural states, namely one competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states.
Huang, Q., Purzycka, K.J., Lusvarghi, S., Li, D., Le Grice, S.F.J., and Boeke, J.D. (2013) Retrotransposon Ty1 RNA contains a 5'-terminal long-range pseudoknot required for efficient reverse transcription. RNA 19: 320-332.
The RNA genome of the Saccharomyces cerevisiae LTR-retrotransposon Ty1 has the potential to fold into a variety of distinct structures, mutation of which has been shown to affect retrotransposition frequency. Huang et al. show in this communication that one potential functional structure is located at the 5' end of the genome and can assume a pseudoknot conformation. Chemoenzymatic probing of wild-type and mutant mini-Ty1 RNAs via SHAPE supports the existence of such a structure, while molecular genetic analyses show that mutations disrupting pseudoknot formation interfere with retrotransposition, indicating that it provides a critical biological function. These defects are enhanced at higher temperatures. When these mutants are combined with compensatory changes, retrotransposition is restored, consistent with pseudoknot architecture. Analyses of mutants suggest a defect in Ty1 reverse transcription. Collectively, data in this study allow modeling of a three-dimensional structure for this novel critical cis-acting signal of the Ty1 genome.
Nowak, E., Potrzebowski, W., Konarev, P.V., Rausch, J.W., Bona, M.K., Svergun, D.I., Bujnicki, J.M., Le Grice, S.F.J., and Nowotny, M. (2013) Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res. 41: 3874-3887.
A key step in proliferation of retroviruses is the converting their RNA genome to double-stranded DNA via a process catalyzed by the multifunctional reverse transcriptase (RT). Dimeric and monomeric RTs have been described, the latter exemplified by enzyme from Moloney murine leukemia virus (Mo-MLV). However, structural information that describes the substrate-binding mechanism for a monomeric RT is lacking. Nowak et al. report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and the single-subunit RT from xenotropic murine leukemia virus-related virus (XMRV), a close relative of Mo-MLV. A comparison with p66/p51 HIV-1 RT shows that substrate binding around the DNA polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length XMRV RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate, highlighting a key difference between monomeric and dimeric RTs.
Nucleic Acids Research online supplementary data related to this article (PDF - 2143 K)
Sztuba-Solinska, J., Teramoto, T., Rausch, J.W., Shapiro, B.A., Padmanabhan, R., and Le Grice, S.F.J. (2013) Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome. Nucleic Acids Res. 41: 5075-5089.
The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. In this study, Sztuba-Solinska et al. performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb2+ ion-induced hydrolysis, and site-directed mutagenesis. This multidisciplinary study highlighted protein-independent 5'-3' terminal interactions involving cis-acting motifs that assume a 'panhandle' structure. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, and internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their predicted interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. Since disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as an autonomous structural/regulatory element. In addition, these studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.
Lusvarghi, S., Sztuba-Solinska, J., Purzycka, K.J., Pauly, G.T., Rausch, J.W., and Le Grice, S.F.J. (2013) The HIV-2 Rev-response element: Determining secondary structure and defining folding intermediates. Nucleic Acids Res., in press.
Interaction between the human immunodeficiency virus (HIV) Rev protein and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced HIV-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, we identified the secondary structures of the HIV-2 RRE and two RNA-folding precursors by combining chemical probing with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, confirm that the RRE2 RNA undergoes a folding transition, and obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. Three-dimensional models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem-loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.
Chung, S., Miller, J.T., Lapkouski, M., Tian, L., Yang, W., and Le Grice, S.F.J. (2013) Examining the role of the HIV-1 reverse transcriptase p51 subunit in positioning and hydrolysis of RNA/DNA hybrids. J. Biol. Chem., in press.
Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has highlighted novel and important contacts between structural elements at the C-terminus of the non-catalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe416-Pro421 was demonstrated to interact with the DNA strand, cross the minor groove of the helix, and finally form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining alpha-helix M', p51 residue Tyr427 forms a hydrogen bond with Asn348, the latter of which, when mutated to Ile, has been implicated in resistance to both nucleoside and nonnucleoside RT inhibitors. Based on this recently reported structural data, Chung et al. analyzed the contribution from p51 C-terminal elements by evaluating selectively mutated p66/p51 heterodimers carrying (i) truncations that encroach on alpha-M, (ii) alterations that interrupt the Asn348:Tyr427 interaction, and (iii) alanine substitutions throughout the region Tyr416-Pro421. Collectively, this strategy supports the notion that the p51 C-terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.
Masaoka, T., Chung, S., Caboni, P., Rausch, J., Wilson, J.A., Taskent-Sezgin, H., Beutler, J.A., Tocco, G., and Le Grice, S. (2013) Exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H. J. Med. Chem., in press.
The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) has been proposed to occupy the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb subdomain of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. In this communication, Masaoka et al. report the synthesis, activity, and antiviral properties of 39 novel thienopyrimidinones bearing substitutions on the thiophene or the C2 position of the pyrimidinone ring. Exploiting a panel of selectively mutated HIV-1 RT mutants allowed identification of four groups of molecules, based on their resistance and sensitivity profiles. Among these, compounds with a 3',4'-dihydroxyphenyl (catechol) substitution displayed activity against both wild-type and drug-resistant RT variants at submicromolar concentrations and, importantly, inhibited HIV-1 replication in cells. Differential scanning fluorimetry indicated that these compounds, in contrast to alpha-hydroxytropolone-derived RNase H inhibitors (such as the natural product manicol), destabilized the RT heterodimer, in some instances lowering the Tm by almost 5oC. Collectively, these data provide an important structural platform for the continued development of thienopyrimidinone-based RNase H inhibitors and highlight the value of genetically engineered HIV-1 RT variants with altered inhibitor sensitivity profiles for secondary screening.
This page was last updated on 5/10/2013.
