B-HIVE Center Team Members
Stefan G. Sarafianos has more than 20 years of experience in retroviral structural biology, biochemistry, and virology. His lab has contributed to the development of potent antivirals that act by novel mechanisms of action by characterizing their mechanisms of inhibition and resistance. His group solved the elusive crystal structure of the first native (uncrosslinked) hexameric HIV capsid protein, which serves as a model system for the identification and characterization of novel compounds targeting HIV capsid. He has also recently developed an imaging/microscopy assay (MICDDRP) to visualize viral RNA, DNA, and protein. Collaborations in the HIVE Center include structural and biophysical studies on APOBEC3G complexes with Michael Malim and Arnold, Griffin, Lyumkis, Musier-Forsyth, and Olson; kinetic characterization of HIV RT initiation complexes with Arnold, Millar, Musier-Forsyth, and Lyumkis; reactive docking of novel compounds as probes targeting HIV capsid with Olson and Sharpless; MICDDRP microscopy assays to visualize various aspects of the HIV life cycle with Engelman and Torbett.
Bruce E. Torbett has 15 years of experience in the studies of the biochemical and structural roles of drug resistance mutations in HIV protease and Gag, their macromolecular interplay, and contributions in altering virological fitness. The Torbett group has utilized small molecule protease inhibitors as chemical probes to structurally and mechanistically define the macromolecular interplay during the acquisition of protease inhibitor resistance.
The Group’s expertise will facilitate active and extensive participation among the HIVE Center investigators and projects. The use of the novel protease/Gag assays, developed by the Torbett group, will be integral for undertaking low- and high-resolution studies of protease and Gag polyprotein interaction, small molecule screening, and resistance studies with HIVE Center Investigators. The use of the novel protease/Gag assays, and proteins generated from various protease/Gag constructs will be integral for undertaking low- and high-resolution studies of protease and Gag interaction, necessary to glean insights into the topology of the Gag-Pol polyprotein and its macromolecular partners. Moreover, the Torbett group’s expertise in the identification of chemical probes and the generation of drug resistant viral mutants for interrogating protease and Gag macromolecular interaction is a strong asset for the Center. In this regard, the Gag assay will afford small molecule screening as proposed by Center Investigators.
Eddy Arnold 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.
Terrence Burke utilizes bioorganic and medicinal chemistry to prepare new biologically-active molecules, with an emphasis on peptides and peptide mimetics. His recent work has dealt with the development of inhibitors directed against phosphor-dependent protein-protein interactions, HIV-1 integrase and protein-tyrosine phosphatases. He is also engaged in developing antibody-drug conjugates.
Robert Craigie focuses on mechanistic aspects of retroviral DNA integration. Recent efforts have focused on biochemical and structural studies of HIV-1 intasomes and the mechanism that prevents integrase using the viral DNA as a target for integration.
Rob Dick uses and develops Cryo-Electron Microscopy (cryo-EM) and Cryo-Electron Tomography (cryo-ET) and Subtomogram Averaging (STA) tools to determine the structures of viruses and viral proteins. By understanding the mechanistic details of viral assembly and release from a cell, he aims to support antiviral drug design and discovery.
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.
M.G. Finn combines synthetic chemistry, molecular biology, cell biology, immunology, and materials science with a highly collaborative approach to attack some of the most important problems of today.
Stefano Forli focuses on the design and application of computational methods to structural biology and drug design, performing rational design of molecules capable of modulating biological processes. He combines different techniques, like molecular dynamics, homology modeling and docking to analyze macromolecule structures to identify druggable sites. He then screen large libraries of compounds performing high throughput virtual screening (HTVS) or designing focused virtual libraries based on specific synthetic pathways in order to identify small organic molecules that can act as biological probes and novel therapeutic agents. As a central part of this work, he develops and distributes the AutoDock suite, a series of programs for performing dockings of small organic molecules and peptides against biologically relevant molecules, such as proteins and nucleic acids.
Ashwanth Francis studies how viruses reach their replication centers within the nucleus of living cells. His laboratory uses a combination of quantitative live-cell imaging, single virus tracking and correlative light and cryo-EM (CLEM) techniques to gain mechanistic and structural insights into virus-host interactions, during the many distinct stages of virus replication.
Eric Freed focuses on HIV-1 Gag trafficking, Env incorporation, virus assembly, budding, release, maturation, and drug resistance. He has a special interest in the complex relationship between viral proteins and cellular factors and pathways, believing that characterizing fundamental aspects of the retrovirus replication cycle will suggest novel targets for the development of antiretroviral therapies. Recent work in the Freed lab has also focused on the ability of Env mutations to broadly rescue defects in virus replication, including those conferred by antiretrovirals.
David Goodsell creates materials for outreach to the research and educational communities. For two decades, he has been centrally involved in outreach at the RCSB Protein Data Bank, including the popular “Molecule of the Month” column. In the B-HIVE, he is creating outreach materials for educators, students, and the general public.
Pat Griffin has a broad background in drug discovery and development, as well as the study of protein structure with approaches to modulating protein function via synthetic small molecules that spans the last 20+ years. His research is focused on structure-function and chemical biology studies of nuclear receptors and GPCRs. His laboratory has built an automated platform to profile protein:protein and protein:ligand interactions by hydrogen/deuterium exchange (HDX) mass spectrometry. In the HIVE Center, he serves as a HDX Core Director, providing HDX support to all the Center members, as well as guidance on assay development and using the Fast Track mechanism to transfer assays into the MLPCN network.
Wei-Shau Hu studies how retroviruses transfer genetic information to the next generation, including the transport and trafficking of the viral RNA, packaging of the viral RNA genome, virus assembly, reverse transcription, and recombination. She uses molecular biology and biochemical approaches in combination with state-of-the-art microscopy techniques to study these topics.
Mary Kearney conducts research on the emergence of HIV drug resistance, the persistence of HIV during antiretroviral treatment (ART), and the sources of rebound viremia after stopping ART. Her studies have demonstrated that a diverse population of HIV-infected cells persist during ART, that some infected cells proliferate despite ART, and that residual viremia during ART can result from viral expression from these cells. Dr. Kearney heads the Translational Research Section, which aims to understand the genetics, evolution, and persistence of HIV and other RNA viruses and to design new approaches toward targeting and killing infected cells. Currently, she also serves as a member of the NIH Women Scientists Advisors (WSA) Executive Committee and a member of the CCR WSA Committee; these groups promote career development and address issues affecting women scientists.
Karen Kirby focuses on uncovering fundamental mechanisms of how viruses replicate, how they can be targeted by novel antiviral therapeutics, and how they develop resistance to antivirals. The overarching goal is understand how viruses work and improve antiviral therapies for infected patients. Much of this has focused on HIV and Hepatitis B virus (HBV), as well as Influenza virus and picornaviruses such as Enterovirus and Coxsackievirus, which affect children. Her lab utilizes a variety of tools, including structural biology, biochemical and biophysical methods, and virology.
The goal of Douglas Kojetin’s research is to understand how activation and repression of nuclear receptor transcription is regulated on the structural and molecular level, including the influence of small molecule ligands—natural/endogenous ligands, synthetic ligands, and FDA-approved drugs used clinically. He use biomolecular NMR spectroscopy as a main structural technique, but also apply a variety of structural, computational, biophysical, and functional approaches including X-ray crystallography, molecular dynamics simulations, biophysical and biochemical assays, and cellular assays to connect our molecular and structural findings on nuclear receptors to cellular functions.
Mamuka Kvaratskhelia 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.
Ron Levy 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.
Dmitry Lyumkis 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.
Frank Maldarelli heads the Clinical Retrovirology Section, which develops and implements clinical protocols to elucidate mechanisms underlying the emergence of HIV drug resistance in vivo, the dynamics of infection under treatment, and the role of resistance mutations in the efficacy and failure of subsequent treatments. Representing the clinical research arm of the HIV Dynamics and Replication Program (HIV DRP), Dr. Maldarelli is an Attending Physician in the NIH NIAID/CCMD HIV Clinic and has established extensive collaborations between the HIV DRP in Frederick and both the NCI HIV and AIDS Malignancy Branch and the NIAID HIV clinical research program in Bethesda.
Michael Malim is the Head of the Department of Infectious Diseases at King’s College London. He has >25 years of research experience investigating the molecular pathogenesis of HIV. He has described the anti-viral properties of the human restriction factors APOBEC3G (A3G), a cytidine deaminase that edits viral DNA, and MX2, a dynamin-like GTPase. He will actively participate in the study of the A3G/RT interactions and on the mechanism of RT inhibition by APOBEC3 proteins.
Gregory Melikian has more than 30 years of experience in viral entry and fusion steps of infection. His laboratory has studied HIV-1 entry intermediates and pathways, using a number of functional and live cell imaging techniques. More recently, his laboratory has focused on visualization of single HIV-1 uncoating, docking at the nuclear pore complex, nuclear import and intranuclear trafficking steps of infection.
David Millar has pioneered the application of single-molecule fluorescence methods in HIV-1. He will develop new single-molecule methods to monitor Gag polyprotein assembly, both in a defined in vitro system and in cells and will contribute to real-time visualization of intasome assembly and single-molecule analyses of integrase-vRNA interactions and the RT initiation complex.
Karin Musier-Forsyth 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 B-HIVE Center includes: assembly and packaging of genomic RNA (gRNA) into HIV-1 by retroviral Gag proteins; role of transcriptional start-site heterogeneity on HIV-1 gRNA structure, packaging, and translation; and Gag polyprotein structural dynamics and function in HIV-1 assembly.
Arthur Olson brings to the Center decades of research and development in computational docking and virtual screening, and the largest distributed-computing resource currently addressing HIV biology: FightAIDS@Home. The Olson laboratory focuses on developing and applying computational methods to understand the nature of HIV structure and mechanism and to improve drug design methodology within the context of the evolution of viral drug resistance. Recently the lab is bringing systems biology and structural biology together by integrating proteomics and other bioinformatics with data from structural information at multiple scales. For this task, the lab is developing the autoPACK/cellPACK software, automated tools to construct complex cellular environments at molecular and atomic detail, such as whole virion models of HIV at the molecular level. Collaborations within the HIVE Center have included: an inhibitor design cycle targeting protease resistance with Elder, Finn, Torbett and Stout; development of improved methods for free energy prediction with Levy, and modeling of high-order integrase assemblies with Kvaratskhelia and Engelman.
Vinay Pathak has developed innovative live-cell microscopy methods to show that, in contrast to most HIV-1 replication models, intact viral cores are transported into the nucleus, complete reverse transcription in the nucleus, and disassemble (uncoat) near their integration sites just before integration. He has significantly contributed to our understanding of how HIV-1 replicates in the presence of potent host restriction APOBEC3 proteins and antiviral drugs. Additionally, he played a key role in discovering the origin of a newly identified retrovirus, XMRV, and in quelling a potential public health crisis by refuting the controversial claims associating this virus with chronic fatigue syndrome and prostate cancer.
Andrew Routh combines molecular and cellular virology, next-generation sequencing and computational biology to study well-controlled and highly characterized model systems such as Flock House virus, as well as human pathogens including chikungunya virus, zika virus, coronaviruses and HIV. He studies systems ranging from controlled cell culture, through animal models, into clinical specimens. This multi-strata approach is aimed at gaining a molecule’s-eye view of the mechanisms of RNA replication and recombination in order to understand virus evolution on a population scale.
Greg Voth performs theoretical and computer simulation studies of biomolecular, condensed phase, quantum mechanical, and materials systems. One of his goals is to develop new theory to describe such problems across multiple, connected length and time scales. Another related goal is to develop and apply new computational methods, tied to our multiscale theory, that can explain and predict complex phenomena occurring in these systems. These methods are developed, for example, to probe protein-protein self-assembly, membrane-protein interactions, biomolecular and liquid state charge transport, complex liquids, self-assembly, and energy conversion materials.
Jamie Williamson has extensive experience with biophysical studies of RNA folding and RNP assembly, using NMR, X-ray crystallography, single molecule fluorescence, electron microscopy, and mass spectrometry (MS) to study the process of ribosome assembly. A key feature of this approach is the use of quantitative MS to study the protein composition of intermediates, and the use of stable isotope pulse labeling to study the dynamics of intermediates. This broadly based approach will now be brought to bear on the process of HIV assembly.
Research in the Wysocki group is categorized into four broad areas:(1) development and implementation of surface-induced dissociation onto commercial time-of-flight, Orbitrap and FT-ICR instruments, (2) development and application of native mass spectrometry-guided structural biology approaches, (3) multi-omics approaches to biomarker discovery, disease diagnosis and prognosis using proteomics and metabolomics methods coupled with genomics and transcriptomics, and (4) determination of peptide and other fragment ion structures by IR action spectroscopy.
CDP Awardees Year 1
Sophie Harvey, Research Scientist at The Ohio State University
Department of Chemistry and Biochemistry
Proposal Title: “Characterization of CA inhibitors using native mass spectrometry”
B-HIVE sponsors: Stefan Sarafianos and Bruce Torbett
Junior investigator in Vicki Wysocki’s group (mass spectrometry) who is transitioning to work in the HIV field and currently studies large biological structures by native mass spec.
Luiza Mendonça, Assistant Professor at University of Minnesota
Department of Biochemistry, Molecular Biology and Biophysics
Proposal Title: “Molecular Architecture of the HIV-1 Virological Synapse”
B-HIVE sponsor: Eric Freed
Junior investigator who studies HIV and SARS-Cov-2 processes at the structural level using CryoEM/ET.
John Briggs, PhD, Director, Max Planck Institute of Biochemistry, Germany, will provide CryoET structural information on capsids within virions, for use in all-atom and coarse grain simulations for Projects 1 and 3.
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.
Barry Sharpless, PhD, and John Cappiello, PhD, Professor and Staff Scientist, respectively, Scripps Research Institute. They will provide novel SuFEX molecules for targeting capsid and Gag and Gag-Pol polyproteins. Their broad expertise in devising novel SuFEX compounds will complement efforts that will be provided by Core 1.
Davey Smith MD, Director of UC San Diego Center for AIDS Research, will provide resources from the CFAR, including core services in translation virology, bioinformatics and genomics, and protein expression, and as well as clinic investigations. Utilizing the available CFAR Cores will leverage valuable research and clinical services for all Center grant participants.
Collaborative Development Program
Carol A. Carter, Ph. D.
Department of Microbiology and Immunology, Stony Brook University
John Coffin, Ph. D.
American Cancer Society Professor of Molecular Biology
Douglas D. Richman, M. D.
Director of the AIDS Research Institute, Veteran’s Administration
Department of Pathology, UC San Diego School of Medicine