Understanding the structural interactions of HIV assembly, maturation and replication is critical to linking the current structural knowledge of two major enzymatic targets: HIV protease and reverse transcriptase, with their polyprotein precursors, substrates and effectors. Expanding the evolving structural knowledge of the HIV integrase and its interactions with host proteins will provide a more complete picture of the mechanisms of the third major drug target. The HIV Interaction and Viral Evolution (HIVE) Center is characterizing at the atomic level the structural and dynamic relationships between interacting macromolecules in the HIV life cycle. We focus on interactions of the major HIV enzymes with their partners and effectors since they encompass key processes in the viral life cycle and as existing drug targets provide a rich base of structural, biological and evolutionary data that inform our goals. We explore resistance evolution in HIV as an opportune platform upon which to characterize the dynamic relationships between interacting macromolecular structures at the atomic level. Our approach is significant due to the promise of new structural insights into the interdependence of viral mechanisms and the direct potential for new drug design methodologies and therapeutic strategies.
The HIVE Center comprises a group of investigators with expertise in HIV crystallography, virology, molecular biology, biochemistry, synthetic chemistry and computational biology. We study the mechanistic implications of viral macromolecular interactions and dynamics and its broader impacts of the evolution of drug resistance to address several biological questions:
- How do structures of the HIV polyprotein precursors direct assembly, maturation, and replication?
- What novel HIV–Host interactions drive DNA replication and integration?
- How does dynamics impact viral function and fitness and how can it be exploited for therapeutic targeting?
- What are the structural and dynamic consequences of resistance mutations in the HIV life cycle?
Arthur Olson, DirectorArthur J. Olson brings to the Center two decades of research and development in computational docking and virtual screening, and the largest distributed-computing resource currently addressing HIV biology: FightAIDS@Home.
The HIVE Center characterizes assemblies of HIV and host molecules in multiple states and their transitions, by combining structural studies of HIV protein interactions with chemical and evolutionary probes and computational modeling to elucidate macromolecular interactions and mechanisms critical for the viral life cycle. Previous work by Center structural biologists have characterized all of the HIV enzymes, with over 300 unique structure depositions in the PDB, and the work within HIVE will reveal their interaction and maturation from viral polyproteins, and their interactions within the viral lifecycle. HIVE laboratories are approaching this challenge with a variety of experimental methods. The evolution of HIV under the selection pressure of small molecule effectors provides a functional window on the underlying macromolecular interactions. Chemistry gives us the capability to design and refine new atomic level probes to explore mechanism. Computational modeling guides the establishment of structural hypotheses and enables the integration of multi-scale dynamic data into a coherent physical picture.
The HIVE Center recently submitted an application for renewal of the Center. We are proposing to build on the productive collaborative work of the HIVE Center Team over the past four years, focusing work on four overall specific aims with the goal of understanding, at the atomic, biophysical and evolutionary level, the system interdependency of interacting HIV macromolecules and their assemblies which shape the HIV life cycle:
- the structural biology of retroviral polyproteins and their components in retroviral assembly and maturation, including study of structural determinants for integrase pleotropism in viral maturation and assembly, and maturation of HIV and PFV polyproteins;
- interactions of HIV with host factors during reverse transcription and integration, including initiation of reverse transcription, inhibition by APOBEC3 family proteins, and the mechanisms of integration into cellular chromatin and their consequences on the formation and reactivation of latent proviruses;
- evolution of antiviral resistance mutations and their biological and biophysical implications, building on computational analysis of full-length genomes obtained from patient samples using an innovative new sequencing technology;
- develop and characterize small molecule probes to understand the biological function of critical molecules and assemblies in the HIV life cycle, including the innovative SuFEx chemistry approach for discovering highly selective covalent inhibitors and computational approaches for discovering new molecules and characterizing the large mesoscale assemblies that are targets of these molecules.
We are excited to add four new research groups for this proposed work, adding orthogonal experimental techniques for addressing these aims in new ways. Dmitry Lyumkis and Karin Musier-Forsyth, will be new HIVE principal investigators after having been an active part of the current Center through our Collaborative Development Grant program. Lyumkis brings his expertise in cryoelectron microscopy that has revealed the role of higher-order oligomers in HIV integrase. Musier-Forsyth bringings her expertise in the use of SHAPE and SAXS to probe the structure and interactions of RNA throughout the HIV lifecycle.
Two new proposed HIVE investigators are David Millar and Jamie Williamson. 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. 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.