HIV Interaction and Viral Evolution Center Groups
Olson GroupArthur J. Olson (Director), email@example.com
The Arnold laboratory has been studying HIV-1 RT structure and its implications for function, ligand binding, drug design, and drug resistance since 1987. In a long-term collaboration with Stephen Hughes at NCI Frederick, the Arnold lab has solved the structure of wild-type and mutant HIV-1 RT in many functional states, including complexes with DNA, RNA/DNA, and inhibitors. The Arnold group contributed to the discovery of two anti-AIDS drugs, TMC125/etravirine/Intelence and TMC278/rilpivirine/Edurant, in a multidisciplinary structure-based drug design effort. Key areas of focus within the HIVE Center are structural (cryo-EM with Lyumkis, and crystallographic) and functional studies of the HIV-1 RT initiation complex (HIV-1 RT/vRNA/tRNA), HIV Gag-Pol and Pol polyproteins, prototype foamy virus (PFV) PR-RT and Pol polyprotein, and crystallographic fragment screening of multiple HIV targets. Collaborations within the HIVE Center have included: study of IN-ALLINI interaction and structure-guided fragment-based inhibitor design with Kvaratskhelia, Engelman, Fuchs, Olson, and Levy; structural, biophysical, functional, and expression studies of retroviral polyproteins, including PFV PR-RT, PFV PR-RT complex with DNA, and HIV-1 Gag-Pol and Pol, and PFV Pol, with Marcotrigiano, Torbett, Musier-Forsyth, Hughes, and Lyumkis; hydrogen-deuterium exchange studies to probe RT dynamics and interactions with inhibitors, DNA, and RNA with Griffin, DeStefano, and Musier-Forsyth; chemical labeling using SuFEx and click chemistry of HIV-1 RT and other HIVE targets with Sharpless, Olson, Sample, and Forli; and use of DNA aptamers for structure determination of HIV-1 RT with DeStefano.
The Elder laboratory has been involved in molecular virology over the past 35 years, with emphasis on characterization of molecular mechanisms and life cycle of retroviruses. Within the HIVE Center, the laboratory has expressed, purified and characterized a number of viral proteins employed in successful crystallization efforts, with specific focus on defining the molecular basis of drug resistance involving the viral protease (PR) and the structure and interactions of the Gag and GagPol polyproteins that may impact on drug resistance and hopefully offer additional targets for intervention. Collaborations within the HIVE Center have included: an inhibitor design cycle targeting protease resistance and expression and purification of Gag constructs for crystallographic, NMR, HDX and SAXS studies with Torbett, Finn, Stout, deVera, Cojetin, Griffin, Forli and Olson. In addition, attempts to develop Gag-specific aptamers are in progress with the DeStefano laboratory.
Alan Engelman has >30 years of experience studying retrovirology and >25 years of experience studying HIV DNA integration. Seminal contributions to the integration field include deciphering the mechanisms of IN 3’ processing and strand transfer activities, the roles of LEDGF and CPSF6 in guiding PICs to favored integration sites in the human genome, the initial structure of the IN-LEDGF complex, the fact that ALLINIs inhibit HIV-1 particle maturation, and several intasome structures. Engelman moreover coined the class I and class II terminology to distinguish the different phenotypic effects of IN mutations on HIV-1 replication. Collaborations within the HIVE Center have included: elucidation of the mechanism of action of ALLINIs with Kvaratskhelia; discovery and characterization of new IN inhibitors with Kvaratskhelia, Arnold, Fuchs, Levy and Olson; and structure of integrase and intasomes with Lyumkis.
The Finn laboratory is highly experienced in synthetic chemistry for the development of drug candidates, chemical biology probe molecules, and bioconjugation techniques. He is one of the leading discoverers and developers of click chemistry methods and their applications to the synthesis of inhibitors/high affinity ligands and covalent modification of macromolecules. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) has become one of the most widely used synthetic methods during the last decade. Collaborations within the HIVE Center has included an inhibitor design effort targeting protease resistance with Stout, Elder, Torbett and Olson.
Dr. Griffin is a leading expert in the application of HDX-MS to the study of protein dynamics in the context of mutations, protein-protein interactions, and ligand binding. The Griffin laboratory has built a state-of-the-art platform that includes three high resolution hybrid orbital-trap mass spectrometers each with fully automated liquid handling robots and integrated LC systems. We have also developed automated software for HDX data processing. The HDX-NMR Core addresses central goals of the HIVE Center that include characterization of HIV protein interactions among themselves, with host molecules, or with small molecule probes.
Dr. Kojetin oversees the protein NMR component of the HDX and NMR core for the HIVE center. Dr. Kojetin has extensive experience in protein NMR including solving solution structures, probing protein dynamics, and ligand interaction. The NMR studies compliment all structural and dynamics studies in the HIVE center.
The primary expertise of the Jones lab for many years has been the development of innovative methods for the chemical synthesis of modified nucleosides and nucleic acids. These efforts have included modifications of nucleosides that can be incorporated into strands of DNA and RNA to form cross-linkable tethers that are important for the structure determinations in the HIVE Center.
The Kvaratskhelia laboratory investigates the structure and function of retroviral integrase as a therapeutic target. Some seminal findings include revealing a role of BET proteins in targeting gamma-retroviral integration to transcription start sites, binding of LEDGF/p75 to mononucleosomes containing a specific histone mark (H3K36me3), and a non-catalytic function of integrase in HIV-1 biology as it binds and encapsidates the viral RNA genome during virion morphogenesis. Within the HIVE Center, his collaborative work with Engelman, Arnold, Griffin, Levy, Olson and Fuchs have allowed these investigators to dissect the unexpected mode of action of quinoline-based allosteric integrase inhibitors (ALLINIs) that are currently in clinical trials and to identify entirely new ALLINIs with unique structural scaffolds through fragment-based screening of HIV-1 integrase inhibitors.
The Levy laboratory brings to the HIVE Center more than thirty years of experience and leadership in the development and application of molecular simulation methods to study the structure, folding, and dynamics of proteins and their complexes. Levy works on problems involving the interplay between computational models in structural biology and experiments at different levels of resolution and different time scales. The group uses their multi-scale modeling approaches with experimental restraints provided by HIVE collaborators to elucidate the thermodynamic and kinetic processes by which ALLINIs promote multimerization of HIV integrase, and to build structural models for Gag-Pol polyproteins. Novel high throughput free energy simulations refine the results of protein-ligand docking carried out by HIVE collaborators in order to assist in the design of potent inhibitors of HIV integrase and reverse transcriptase. The Levy group is developing information-theoretic (Potts) statistical inference techniques to identify correlated patterns of resistance mutations on HIV proteins and their partners. In collaboration with other HIVE investigators, Levy is integrating these tools with biophysical and biochemical data and structural models to map the fitness landscapes of HIV proteins, in order to assess how correlated mutations facilitate the development and evolution of drug resistance.
Sharpless GroupBarry Sharpless (PI), firstname.lastname@example.org
The Scripps Research Institute, La Jolla
Barry Sharpless is a highly innovative Nobel Laureate (2001) who developed click chemistry and recently produced an application termed Sulfur (VI) Fluoride Exchange (SuFEx). He has had a long-standing interest in infectious disease and development of chemical reagents to serve as probes and therapeutic agents for HIV. SuFEx compounds are very stable in aqueous solutions near neutral conditions, but become reactive upon encountering a specific protein partner. When the correct SVI--F motif is incorporated into the correct small organic molecule, it may selectively and covalently react with protein targets. Sharpless’s significant contribution to the HIVE Center is to develop novel small-molecule libraries using the SuFEx chemistry for HIV polyprotein and virion screening.
Torbett has 20 years of experience in studies of the biochemical, biophysical, and structural roles of drug resistance mutations in HIV protease and Gag, their macromolecular interplay and fitness. The Torbett group also studies human stem cell restriction to HIV vectors. The Torbett group has shown that HIV protease non-active site (compensatory) mutations stabilize protease and restore enzymatic function and has identified novel inhibitors of nucleocapsid. In collaboration with Okulicz, Levy, and Routh, the Torbett group has utilize deep sequencing to identify novel mutations in protease and Gag resulting from epistatic interaction as HIV evolves resistance to inhibitors. In collaboration with Jamie Williamson and colleagues (CHEETAH Center), as well as the Marcotrigiano and Arnold groups, the Torbett group has been collaborating to define cellular proteins and their role in Gag assembly. Lastly, in collaboration with Olson, Marcotrigiano, Arnold, Sarafianos, and Elder groups, the Torbett group is utilizing SuFEx (Sulfur(VI) Fluoride Exchange) reaction chemistry (with Sharpless) as methodology to develop novel chemical probes for targeting HIV polyproteins.
In Memoriam: C. David Stout
Members of the HIVE Center are saddened by the passing of C. David Stout in 2016. He has been an energetic and insightful member of the HIVE Center, using x-ray crystallography to probe the mechanisms of protease and drug resistance.
Collaborative Development Grant Investigators
Jeffrey DeStefano, University of Maryland, works on understanding the process of reverse transcription including fidelity, recombination, protein-nucleic acid interactions, and design of novel inhibitors. HIVE-related research has focused on developing nucleic acid aptamers that bind with high affinity to retroviral proteins and can aid in structural analysis as well as serve as potential inhibitors or diagnostics. A primer-template mimicking aptamer to HIV RT helped produce (with the Arnold group) the first RT structure with a nucleic acid in the absence of cross-linking agents.
Dmitry Lyumkis, the Salk Institute, brings to the HIVE Center nearly a decade of expertise in the field of single-particle cryo-EM. He has contributed numerous technical advances that broadly aim to improve cryo-EM methods and to resolve increasingly more complex and structurally heterogeneous macromolecular assemblies. He continues to develop tools for pushing the technological capabilities of cryo-EM to gain a deeper understanding into macromolecular structure and function. He also has broad interests in HIV structural biology, and in particular integration. In collaboration with HIVE Center researchers, he solved groundbreaking structures of several intasome complexes, from HIV and related retroviruses.
Karin Musier-Forsyth, Ohio State University, focuses on understanding RNA structure and RNA-protein interactions that are critical for retrovirus replication. Areas of expertise in the lab include RNA structure probing by SHAPE, RNA SAXS analysis, and RNA-protein interactions. An overarching goal is to identify new targets and novel strategies for anti-retroviral therapy. Current research in the lab related to the specific objectives of the HIVE Center includes: structural analysis of the reverse transcription initiation complex (RTIC), assembly and packaging of genomic RNA into HIV-1 by retroviral Gag proteins, and polyprotein structure and function. As a HIVE CDP awardee, exciting collaborations have been established with Arnold (RTIC structure), Lyumkis and Kvaratskhelia (structural analysis of RNA and RNA-IN complexes by Cryo-EM), Griffin (HDX of Gag-RNA complexes), and Levy and Olson (modeling of Gag).
HIVE Center Collaborators
Bridget Carragher, Director, and Clint Potter, Co-Director, National Resource for Automated Molecular Microscopy, NYSBC, runs a national facility for high-resolution cryo-EM and automated electron microscopy, with the goal of developing, testing, and applying technology aimed towards completely automating the processes involved in solving macromolecular structure using cryo-EM. In collaboration with several HIVE members, she has contributed to the advancement of tools for high-resolution cryo-EM studies, and for visualizing microcrystals in the lipidic cubic phase. Her center broadly provides a resource for applying next-gen tools, such as phase plates, for structural biology by cryo-EM.
Stephen H. Hughes, Director HIV Drug Resistance Program, NCI-Frederick, has worked with Arnold to study the structure and function of HIV-1 RT for 25 years. He will participate in the HIV polyprotein precursor studies, providing expression constructs, produce purified foamy virus polyproteins in large amounts for structural studies, study the impact of mutations on proteolytic susceptibility of Pol enzymes, and will test interesting compounds for anti-HIV activity in single-round replication systems. Hughes’ authoritative overview of HIV biology and research will help to guide the Center’s aim of connecting our studies and results throughout the HIV lifecycle.
Jason Okulicz MD, MC, USAF, is on the Infectious Disease Service Staff at Lackland Air Force Base, Texas, will provide clinical data and sera/plasma/cell samples from participates in the U.S. Military HIV Natural History Study. Okulicz will participate within the Center on the antiretroviral-mediated viral co-evolution studies by providing viral samples from individuals for protease and gag sequencing. The sequence information from viral samples will be crucial for modeling the acquisition of resistance mutations.
Alan Rein, Director of the Retroviral Assembly Section, NCI-Frederick, and staff scientist Sid Datta will collaborate on studies of HIV Gag and Gag-Pol polyprotein precursors. Using a defined assembly system that he and his colleagues have developed, Rein and Datta will work with Center investigators to map specific Gag-Gag interactions by HDX and to explore Gag and Gag-Pol interactions in assembly. Their broad expertise in studying retroviral assembly will help to place the Center’s studies of HIV polyproteins in the broader context of HIV assembly and maturation.
Doug Richman MD, Director of UC San Diego Center for AIDS Research, will provide resources from the CFAR, including core services in flow cytometry, molecular biology, translation virology, bioinformatics and genomics, protein expression and proteomics, and as well as clinic investigations. Utilizing the available CFAR Cores will leverage valuable research and clinical services for all Center grant participants.
Robin Wilner, Vice President for Global Community Initiatives, IBM World Community Grid, will continue a long-standing collaboration on the FightAIDS@Home project, which provides a distributed network of over two million internet shared processors for use in docking and drug design.
HIVE Center Administration
Management of the Center builds upon over 25 years of experience in the Structural Biology of HIV program. Principal Investigator Arthur Olson led the TSRI program project for three funding cycles, building a highly collaborative program that effectively combined structure, biology, chemistry and computation into a full drug discovery cycle. The former Center Coordinator Margaret Graber retired in May 2015. Her more than 25 years of experience in administration of NIH and NSF funded program projects, including subawards, continuing with the three previous funding cycles of the Structural Biology of HIV program and organization of national and international meetings, workshops, and conferences were invaluable. The new Center Coordinator as of May 2015, Ms. McCarthy, has 15 years of experience in research administrative assistance with an additional 15 years of administrative assistance experience in various professional business settings.
Co-principal Investigator Eddy Arnold administers the Collaborative Development Program, building on 10 years of experience in administration of program projects. In/Outreach is coordinated by Richard Belew and David Goodsell. Dr. Belew has a long history of development of innovative methods for scientific data mining and communication. He applies this experience to the development of effective online tools for communication and archiving results within the Center and in the wider community, as well as methods for maximizing the effectiveness of face-to-face meeting through use of online preparation and sharing. David Goodsell brings to the Center experience in creation of dynamic visual materials for education and public outreach. He has recently integrated results from HIVE Center members and other Centers into a series of illustrations that capture the current state of knowledge in HIV structure and function.