Stuart F.J. Le Grice, Ph.D.
Basic Research Laboratory
Building 535, Room 312
Frederick, MD 21702-1201
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 2015-2018
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. 45: 6805-6821.
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.
Sherpa C, Rausch JW and Le Grice SFJ: Structural characterization of maternally expressed gene 3 RNA reveals conserved motifs and potential sites of interaction with polycomb repressive complex 2. Nuc. Acids Res., in press, 2018.
Long non-coding RNAs (lncRNAs) have emerged as key players in gene regulation. However, our incomplete understanding of the structure of lncRNAs has hindered molecular characterization of their function. Maternally expressed gene 3 (Meg3) lncRNA is a tumor suppressor that is downregulated in various types of cancer. Mechanistic studies have reported a role for Meg3 in epigenetic regulation by interacting with chromatin-modifying complexes such as the polycomb repressive complex 2 (PRC2), guiding them to genomic sites via DNA-RNA triplex formation. Resolving the structure of Meg3 RNA and characterizing its interactions with cellular binding partners will deepen our understanding of tumorigenesis and provide a framework for RNA-based anti-cancer therapies. In this publication, Sherpa et al. characterized the architectural landscape of Meg3 RNA and its interactions with PRC2 from a functional standpoint.
Okamoto K, Rausch JW, Wakashin H, Yulong F, Chung J-Y, Dummer PD, Shin M, Chandra P, Suzuki K, Shrivastav S, Rosenberg AZ, Hewitt, SM, Ray P, Noiri E, Le Grice SFJ, Hoek M, Han Z, Winkler CA, Kopp JB. APOL1 risk allele RNA contributes to renal toxicity injury by activating protein kinase R. Communications Biology, 2018, in press.
APOL1 risk alleles associate with chronic kidney disease in African Americans, but the mechanisms remain to be fully understood. We show that APOL1 risk alleles activate protein kinase R (PKR) in cultured cells and transgenic mice. This effect is preserved when a premature stop codon is introduced to APOL1 risk alleles, suggesting that APOL1 RNA but not protein is required for the effect. Podocyte expression of APOL1 risk allele RNA, but not protein, in transgenic mice induces glomerular injury and proteinuria. Structural analysis of the APOL1 RNA shows that the risk variants possess secondary structure serving as a scaffold for tandem PKR binding and activation. These findings provide a mechanism by which APOL1 variants damage podocytes and suggest novel therapeutic strategies.
Miller, J.T., Zhao, H., Masaoka, T., Varnado, T., Cornejo Castro, E., Marshall, V.A., Kouhestani, K., Lynn, A.Y., Aron, K.E., Xia, A., Beutler, J.A., Hirsch, D.R., Tang, L., Whitby, D., Murelli, R.P. and Le Grice, S.F.J. Sensitivity of the C-terminal nuclease domain of Kaposi’s sarcoma herpesvirus to two classes of active site ligands. Antimicrobial Agents and Chemotherapy 62: 2018, in press.
Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the Herpesviridae family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, wild-type pORF29C and variants with substitutions at the proposed active-site residues were expressed, purified and characterized. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.
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.”
Sztuba-Solinska J, Rausch JW, Smith R, Miller JT, Whitby D, Le Grice SFJ. Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins. Nucleic Acids Res. 2017;45(11):6805-6821.
Shapiro BA, Le Grice SF. Advances in RNA structure determination. Methods. 2016;103:1-3.
Lapkouski M, Tian L, Miller JT, Le Grice SFJ, Yang W. Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat Struct Mol Biol. 2013;20(2):230-236.
Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang JX, Le Grice SF, Zhuang X. Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature. 2008;453(7192):184-9.
Liu S, Abbondanzieri EA, Rausch JW, Le Grice SF, Zhuang X. Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science. 2008;322(5904):1092-7.
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This page was last updated on February 4th, 2019