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. We have continued to focus our efforts on study the mechanisms by HIV-1 can evolve resistance to INSTIs . The most frequently encountered resistance mutations to INSTIs are Q148H/K/R and are commonly found in combination with G140A/S. Many other resistance mutations occur around the active site. There are also differences between resistance mutations are easily selected in cell culture and those that commonly arise in the clinical setting. We began by analyzing the structures of dolutegravir-bound intasomes with the single amino acid mutations E138K, G140A, and Q148K. The differences in the structures are quite subtle. In the E138K_DTG structure, a nitrogen of the K138 sidechain approaches within approximately 3Å of the backbone phosphate of the 5’ end of the vial DNA, participating in a salt bridge with the phosphate. In the G140A_DTG structure, the introduction of a methyl group affects the sidechain orientation of the nearby residue Q148, inducing a preference for an altered rotameric configuration. The presence of density for the alternative sidechain configuration of Q148 can be clearly seen in the cryo-EM map, which is resolved to ~2 Å in this region. If the position of the sidechain of Q148 was not altered, the methyl group of A140 would approach the amide moiety of Q148 to within 2.5 Å, which would destabilize the rotamer that is preferred in the WT intasome bound to DTG. The most dramatic changes were observed in the structure of Q148K_DTG, in which the sidechain of K148 impacts multiple nearby residues. We have also determined the structures of the double mutants E138K/G140A, E138K/Q148K_DTG, and the triple mutant E138K/G140A/Q148K_DTG. The structures suggest that the G140A mutation potentiates resistance through its effects on Q148K. This work is published in https://doi.org/10.1126/sciadv.adg5953. A significant technical problem with. structural studies of HIV-1 intasomes is formation of intasome stacks in vitro. These stacks of heterogeneous length result from swapping of flanking C-terminal domains (CTDs) between individual intasomes. The repeating unit within the stacks is an octameric intasome containing a pair of viral DNA ends. The stacks are not biologically relevant as there are only two viral DNA synthesized after infection of cells. However, the majority of the intasomes assembled in vitro form stacks, which presents difficulties with cryo-EM data collection and analysis. We tested whether supplying a large excess of isolated C-terminal in the intasome assembly reaction would break the stacks. With this strategy we can isolate mono disperse intasomes at high yield from a one-step filtration step. This will greatly facilitate our ongoing structural studies of drug resistance mutations which have required very time-consuming purification steps prior to making cryo-EM grids. Intasomes assembled with an excess of isolated CTD were well dispersed on EM grids with minimal stacks and aggregates and more uniform ice. This resulted in a CIC map at 2Å, which is the highest resolution intasome structure we have obtained. The structure revealed the function of the C-terminal tail of HIV-1 integrase. The C-terminal tail of HIV-1 integrase (residues 270-288) is essential for HIV-1 DNA integration both in vitro and in vitro; shortening to residue 281 results in only a modest reduction in DNA integration, but any further shorting completely abolishes DNA integration. This C-terminal tail has not been well-resolved in any previous.structures of HIV-integrase or intasomes. We were able to build an atomic model of the CTD tail of distal IN protomers up to residue 281 with the high-resolution cryo-EM map; the last 7 residues of the C-terminal tail were disordered. At the current resolution, we could see that the C-terminal tail engages in a number of other integrase protomers within the intasome, thus contributing to the stability of the intasome structure. We tested the functional role of the interacting residues by making mutations in integrase and testing for integration activity in vitro. Mutating most of these residues reduced integration efficiency in vitro. To further test the importance of these interactions we collaborated with Alan Engelman to test the effects of these mutations in vivo with HIV virus. The results parallel those observed in vitro. Over the last year we have focused on understanding the mechanism of drug resistance and how novel integrase inhibitors are able to exhibit to exit significantly improved resistance profiles compared with currently approved FDA drugs. Although integrase stand transfer inhibitors are now front-line drugs for the treatment of HIV, the development of drug resistance remains a problem and there is a need to develop drugs with improved resistance profiles. We have continued our studies on the mechanisms by which integrase can evolve drug resistance by mutation. Our chemist collaborators led by Dr. Terrence Burke at NCI Frederick have developed novel integrase strand transfer inhibitors (INSTIs) that have significantly improved resistance profiles compared with those that are currently used clinically. These compounds are naphthyridine-based with a combination of 4-amino and 5-hydroxymethyl groups. We have obtained high-resolution structural data on how these drugs interact with HIV intasomes by cryo-EM. A key feature of these structures is π-π stacking interactions between the naphthyridine core and the terminal adenine nucleobase of the viral DNA. Molecular dynamics simulations together with quantum mechanical and molecular modeling calculations elucidate the roles of intramolecular bonding, stacking geometry, resonance effects, and charge distribution that drug binding within the active site of the intasome. The data mechanistically explain how key interactions contribute to improved antiviral potency against drug-resistant integrase mutants.