Stuart F.J. Le Grice, Ph.D.
Dr. Le Grice is internationally recognized for his pioneering research on retroviral and retrotransposon reverse transcriptase (RT), including development of immobilized metal chelate chromatography for rapid enzyme purification, understanding conformational dynamics by single molecule spectroscopy, and determining the crystal structure of RT-RNA/DNA complexes. These studies are combined with nucleoside analog interference approaches to understand how substrate flexibility controls RT recognition. As a member of the nucleotidyltransferase superfamily of enzymes, his work on HIV RT-associated ribonuclease H extends into developing inhibitors of structurally-related enzymes from alpha-(HSV), beta-(HCMV) and gamma-herpersviruses (KSHV).
More recently, Dr. Le Grice’s research has focused on determining the structure of viral and virus-coded lncRNAs, both in vitro and in vivo. A long-term goal is to exploit this knowledge to screen for small molecule antagonists that interact with cis-acting regulatory RNAs.
The RT Biochemistry Section focuses on nucleoprotein complexes as they relate to replication of RNA and DNA viruses of clinical significance. Projects in the laboratory have used 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 generated high-resolution structures for lentiviral (HIV-1), gammaretroviral (XMRV) and LTR-retrotransposon (Ty3) RTs These structures have provided a platform for structure/function studies and drug development, with emphasis allosteric inhibitors that bind adjacent to the RNase H active site of HIV-1 RT. In an extension of this work, we are investigating whether "RNase H-like" enzymes of a- (type 1 and 2 herpes simplex virus, HSV) b- (human cytomegaolvirus, HCMV) and g-herpesviruses (KSHV) and susceptible to inhibition by chemotypes that chelate divalent metal at the active site. Long-term KSHV studies investigate the feasibility of "kick-and kill" strategies that combine latency activators with inhibitors of key viral enzymes.
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 and SHAPE-MaP) 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. The high sensitivity of these approaches allows us to determine RNA structure in multiple cellular contexts (nuclear, cytoplasmic and virion-associated), evidence by our recent work with a long non-coding RNA (PAN) of Kaposi's sarcoma-associated herpesvirus (KSHV). This work involves collaborative interactions with several investigators in both the intramural and extramural research communities.
Finally, as our understanding of the structural basis through which cis-acting elements control virus replication improves, we have turned our focus to developing small molecule antagonists that recognize structured regulatory RNA, expanding into hepadna- (hepatitis B virus), flavi- (Dengue virus) and filoviruses (Ebola).
Research Highlights 2014-2017
Nowak, E., Miller, J.T., Bona, M.K., Studnicka, J., Szczepanowski, R.H., Jurkowski, J., Le Grice, S.F.J., and Nowotny, M. (2014) Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry. Nat. Struct. Mol. Biol. 21: 389-396.
Retrotransposons represent a class of mobile genetic elements that replicate by converting their single-stranded RNA genome into double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural and biochemical information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)-containing retrotransposons, from which they are evolutionarily derived, are lacking. In this study, Nowak et al. report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR-retrotransposon Ty3 in the presence of its polypurine tract-containing RNA/DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture, whose assembly is substrate dependent. More strikingly, and in contrast to data from other dimeric RTs, this structure, in combination with biochemical analysis via phenotypic mixing, suggests that the RNase H and DNA polymerase activities are contributed by individual subunits of the Ty3 RT homodimer.
Link to Center for Cancer Research (CCR) 'In the Journals' feature related to this article: Novel Structure of Ty3 Reverse Transcriptase, April 2014.
Costi, R., Metifiot, M., Chung, S., Cuzzucoli Crucitti, G., Maddali, K., Pescatori, L., Messore, A., Madia, V.N., Pupo, G., Scipione, L., Tortorella, S., Di Leva, F.S., Cosconati, S., Marinelli, L., Novellino, E., Le Grice, S.F.J., Corona, A., Pommier, Y., Marchand, C., and Di Santo, R. (2014) Basic quinolinonyl diketo acid derivatives as inhibitors of HIV integrase and their activity against RNase H function of reverse transcriptase. J. Med. Chem. 57: 3223-3234.
Based on the similarities of the IN and RNase H catalytic sites and reports on dual activities of a number of compounds against IN and RNase H, Costi et al. tested newly designed quinolonyl DKAs against both HIV-1 enzymes. Among these derivatives, three compounds inhibited HIV-1 IN with IC50 values below 100 nM for strand transfer and showed a two-order of magnitude selectivity over 3' processing. These strand transfer selective inhibitors also inhibited HIV-1 RNase H with low micromolar potencies. Molecular modeling studies based on both the HIV- 1 IN and RNase H catalytic core domains provided new structural insights for the future development of these compounds as dual HIV-1 IN and RNase H inhibitors.
Sztuba-Solinska, J., Shenoy, S.R., Gareiss, P., Krumpe, L., Le Grice, S.F.J., O'Keefe, B.R., and Schneekloth, J., Jr. (2014) Identification of biologically active, HIV TAR RNA-binding small molecules using small molecule microarrays. J. Am. Chem. Soc. 136: 8402-8410 (link is external).
Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to uncover RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. In this communication, Sztuba-Solinska et al. report a strategy to screen a large unbiased library of drug-like small molecules in a microarray format against an RNA target. This strategy identified a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. The candidate thienopyridine binds to and stabilizes the TAR hairpin with a Kd of 2.4 uM. Structure activity relationships demonstrate that this compound achieves activity through hydrophobic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5' UTR of the HIV-1 genome to determine global structural changes in the presence of the molecule. The interaction of this compound could be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5' UTR. Cell-based assays indicated that this novel small molecule inhibits HIV-induced cytopathicity in the micromolar range, while cytotoxicity was not observed at concentrations of 1 mM.
Sherpa, C., Rausch, J.W., Grice, S.F.J., Hammarskjold, M.L., and Rekosh, D. (2015) The HIV-1 Rev Response Element (RRE) exists in two alternative secondary structures which promote different replication activities. Nuc. Acids Res. 43: 4676-4686.
HIV Rev forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, Sherpa et al. determined that the wt NL4-3 RRE comprises a mixture of both structures. To assess functional differences between these RRE ‘conformers’, conformationally locked mutants were created by site-directed mutagenesis. Subgenomic reporters, as well as HIV replication assays, demonstrated that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.
Zhao, H., Lin, Z., Lynn, A.Y., Varnado, B., Beutler, J.A., Murelli, R.P., Le Grice, S.F.J. and Tang, L. (2015) Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism. Nuc. Acids Res. 43: 11003-11016.
Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Zhao et al. show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, these authors observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution.
Crawford, D.W., Blakeley,B.D., Chen,P.-C. Sherpa,C., Le Grice, S.F.J., Laird-Offringa, I.A. and McNaughton, B.R. (2016) An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription. ACS Chemical Biology 11: 2206-2215.
Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. Crawford et al. used yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. The best candidate has truly altered, not simply broadened, RNA-binding selectivity, binding TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). The evolved protein specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR binding reagents reported to date and thus have potential utility as therapeutics and basic research tools. These findings also demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition and modulation of disease-relevant RNA.
Abulwerdi, F.A., Shortridge, M.D., Sztuba-Solinska, J., Wilson, R., Le Grice, S.F.J., Varani, G and Schneekloth, J.S. (2016) Development of Small Molecules with a Highly Selective and Non-Canonical Binding Mode to HIV-1 TAR RNA. J. Med. Chem. 59: 11148-11160
Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has provided an important model system for RNA targeting chemistry. Abulwerdi et al. report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue (15) retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 μM. NMR characterization of a soluble analogue (2) revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by SHAPE indicated specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5′-untranslated region (UTR). These compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design.
Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Ganji et al. probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. To test the effects of long-range attractive forces on flipping efficiency, salt concentration and macromolecular crowding conditions were varied. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when flipping kinetics were analyzed (i.e. the rate and probability of flipping at each condition) this phenomenon was more efficient when RT bound more strongly. Such data is consistent with a view that DNA bound proteins undergo multiple rapid re-binding events (or short hops) that allow the protein to explore other configurations without completely dissociating.
Sztuba-Solinska, J., Diaz, L., Kumar, M.R, Kolb, G., Wiley, M.R., Jozwik, L., Kuhn, J.H., Palacios, G., Radoshitzky, S.R., Le Grice, S.F.J. and Johnson, R.F. (2016). A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8. Nuc. Acids Res. 44: 9831-9846.
Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA were investigated and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. SHAPE analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3΄ stem-loop that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.
Sztuba-Solinska, J., Rausch, J.W., Smith, R., Miller, J.T., Whitby, D and Le Grice, S.F.J. (2017). Kaposi’s sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural fold for nuclear, cytoplasmic and viral proteins. Nuc. Acids Res., In press
Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although numerous nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Sztuba-Solinska et al. used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. This study characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure were identified. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work provides a broad framework for understanding the roles of PAN RNA in KSHV infection.
Selected Key Publications
- Nature. 453: 184-9, 2008. [ Journal Article ]
- Science. 232: 1092-7, 2008. [ Journal Article ]
- Nat. Struct. Mol. Biol. 20: 230-6, 2013. [ Journal Article ]
- Cell. 155: 594-605, 2013. [ Journal Article ]
Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry.Nat. Struct. Mol. Biol.. 21: 389-396, 2014. [ Journal Article ]
Stuart Le Grice received his Ph.D. from the Department of Biochemistry, University of Manchester, UK, in 1976, where he studied RNA polymerase of Escherichia coli. After postdoctoral training in the United Kingdom, Germany, and the United States, he was appointed Senior Scientist at 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. 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, during which time he was designated a CWRU “Million Dollar Professor” in recognition of his NIH funding. Dr. Le Grice joined the National Cancer Institute in 1999 and in 2005 was appointed to the Senior Biomedical Research Service. In 2006, he was appointed Head of the CCR Center of Excellence in HIV/AIDS & Cancer Virology. 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 multiple international funding agencies.
Dr. Le Grice was designated a CCR “Mentor of Merit” in 2007 and 2009, and has been recipient of the NIH Award of Merit (2009) and two NIH Director’s Awards (2012, 2015). In 2015, Dr. Le Grice received the DHHS Career Achievement Award, recognizing his “outstanding administrative and scientific contributions to furthering the national and international mission of the National Institutes of Health.”
|Fardokht Abulwerdi Ph.D.||Postdoctoral Fellow (Visiting)|
|Stefano Ginocchio||Postbaccalaureate Fellow|
|Michelle Kuhlmann Ph.D.||Postdoctoral Fellow (CRTA)|
|Regan Leblanc Ph.D.||Postdoctoral Fellow (CRTA)|
|Jennifer T. Miller Ph.D.||Technical Laboratory Manager|
|Jason W. Rausch, Ph.D.||Staff Scientist|
|Chringma Sherpa Ph.D.||Postdoctoral Fellow (Visiting)|
|Joanna Sztuba-Solinska Ph.D.||Research Fellow|