Studies of nucleoprotein complexes involved in retroviral DNA integration

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The goal of the project is to understand the detailed molecular mechanism of HIV-1 DNA integration, the structures of the nucleoprotein complexes that mediate DNA integration, the mechanism of action of integrase inhibitors, and how the virus can the virus can evolve resistance to these inhibitors. Integration of a DNA copy of the viral genome into cellular DNA is an essential step for replication of HIV-1 and other retroviruses. Integration is mediated by the virally encoded integrase protein in complex with viral and target DNA; complexes of integrase associated with a pair of viral DNA are collectively called intasomes. The first intasome on the integration reaction pathway is the Stable Synaptic Complex (SSC) intasome that comprises a complex of integrase and the pair of viral DNA ends. Integrase cleaves two nucleotides from each 3' end of the viral DNA (3' end processing) within the SSC to form the Cleaved Stable Synaptic Complex (cCSC) and then integrates these 3' ends into target DNA (DNA strand transfer) to form the Strand Transfer Complex (STC) intasome. The FDA has now approved four drugs that target HIV-1 integrase, Raltegravir, Elvitegravir, Dolutegravir, and Bictegravir, and more are in the pipeline. These drugs are highly effective and provide a new class of drugs for combination antiviral therapy. They specifically target the DNA strand transfer step of integration and are known as Integrase Strand Transfer Inhibitors (INSTIs) and bind to the assembled cSSC intasomes after 3' end processing rather than free integrase protein. High-resolution structural studies of HIV-1 intasomes are therefore required to understand the detailed mechanism of action of inhibitors and mechanisms of escape by mutations that confer resistance. Our current efforts have been made possible by our earlier biochemical studies that overcame the many obstacles to high-resolution structural studies of HIV-1 intasomes. Previous structural studies of retroviral intasomes and their drug interactions with retroviral intasomes were limited to prototype foamy virus (PFV) intasomes. Although these studies elucidated the basic mechanism of inhibition, sequence divergence between PFV and HIV-1 so great that it limits the ability to model drug interactions with HIOV-1 intasomes; fifteen of the most common resistance mutations in HIV-1 integrase are within 10 A of the active site; only six of these are conserved between PFV integrase. Our first HIV-1 intasome structures, determined, in collaboration with Dmitry Lyumkis at the Salk Institute, were of STC intasomes. More recent efforts are focused on cSSC intasomes and their interactions with drugs. Our research over the past year has continued to focus on how HIV-1 integrase can develop resistance to integrase inhibitors that are now a front-treatment for HIV-1. Based on our earlier biochemical work on developing methodologies to assemble HIV-1 intasomes in vitro, we have obtained cryo-EM structures of HIV-1 intasomes in the resolution range of 2.2 to 3 Angstroms with bound drugs, both with wildtype integrase and integrase with mutations that confer drug resistance. Our results reveal the molecular details of how integrase can acquire resistance to one of the leading clinically used drugs, dolutegravir, and provide a tool to develop new drugs with an improved resistance profile. To complement our expertise, we benefit with close collaborations with Dmitry Lyumkis at the Salk Institute, Stephen Hughes (Virology) and Terrance Burke at (Chemistry) at NCI Frederick, and Ron Levy at Temple University (computer simulations, statistical mechanics. Despite improvements, in methodology for in vitro assembly of intasomes, preparation of intasomes for cryo-EM is laborious due to the vast majority of intasomes being in the form of aggregates and the many steps of purification that are required to obtain samples suitable for single particle cryo-EM. The aggregates are stacks formed by domain swapping of intasomes and we find that stacks can be virtually eliminated from the intasome preparations by assembly in the presence of the isolated C-terminal domain of HIV integrase. This greatly simplifies the steps needed to obtain samples suitable for grid preparation, increases the abundance of single particles, reduces orientation bias, and has enabled us to obtain HIV intasome structures at 2 resolution. These structures have revealed residues 271-279 of the C-terminal tail that are partially disordered in previous intasome structures but are essential for integration activity both in vitro and in vivo. Residues in this tail region make numerous inter-domain interactions within the intasome and we propose these interactions contribute to intasome stability.