Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/ transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development. AIDS | antiretroviral therapy

Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 Å resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.

Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo–electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone that are critical for antagonizing viruses containing the Q148H and G140S mutations. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this class of anti-HIV/AIDS therapeutics.

Retroviral integrase (IN) catalyzes the integration of viral DNA (vDNA) into host target (tDNA), which is an essential step in the lifecycle of all retroviruses1. Prior structural characterization of IN-vDNA complexes, or intasomes, from the spumavirus prototype foamy virus (PFV) revealed a functional IN tetramer2–5, and it is generally believed that intasomes derived from other retroviral genera will employ tetrameric IN6–9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, we determine here an unexpected octameric IN architecture for the β-retrovirus mouse mammary tumor virus (MMTV) intasome. The structure is composed of two core IN dimers, which interact with the vDNA ends and structurally mimic the PFV IN tetramer, and two flanking IN dimers that engage the core structure via their IN C-terminal domains (CTDs). Contrary to the belief that tetrameric IN components are sufficient to catalyze integration, the flanking IN dimers were necessary for MMTV IN activity. The IN octamer solves a conundrum for the β- as well as α-retroviruses by providing critical CTDs to the intasome core that cannot be provided in cis due to evolutionarily restrictive catalytic core domain (CCD)-CTD linker regions. The octameric architecture of the MMTV intasome provides a new paradigm for the structural basis of retroviral DNA integration.