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Stuart F.J. Le Grice, Ph.D.

HIV DRP Retroviral Replication Laboratory Head, RT Biochemistry Section Senior Investigator Building 535 Room 312 Frederick, MD 21702-1201 Phone:   301-846-5256 Fax:   301-846-6013 E-Mail:   slegrice@ncifcrf.gov Link: Other Homepage

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 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

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.

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: 799-2810.

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.

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.

This page was last updated on 7/11/2008.