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Abstract
The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.
Highlights
Despite the success of antiretroviral therapy for HIV infection, the emergence of drug-resistant viral variants and the recognition of long-term drug toxicities leave development of new drug classes a priority [1]
INY15A,F185H formed discrete dimers in solution, retained the ability to bind to the LEDGF integrase binding domain (IBD), and had a reduced level of aggregation in the presence of GSK1264 and GSK002 compared to wild-type IN (S1 Fig and S1 Table)
We favored N-terminal domain (NTD) substitutions for this study because IN constructs containing only the catalytic core domain and the C-terminal domain (CTD) were previously shown to be sufficient for allosteric inhibitor of integrase (ALLINI)-induced aggregation [7,24], suggesting that NTD substitutions would not interfere with understanding ALLINI action
Summary
Despite the success of antiretroviral therapy for HIV infection, the emergence of drug-resistant viral variants and the recognition of long-term drug toxicities leave development of new drug classes a priority [1]. An additional class of IN inhibitors, the allosteric inhibitors of integrase (ALLINIs), act at a second site on HIV IN [3,4,5,6,7,8,9,10,11]. ALLINIs ( referred to as LEDGINs, noncatalytic site integrase inhibitors [NCINIs], or multimodal inhibitors) are highly active against HIV replication in cell culture but have not yet been fully developed for use in patients, motivating close study to inform ongoing inhibitor development. The catalytic core domain (residues 50–212) adopts an RNase H superfamily fold and contains a D,D-35-E motif that binds Mg2+ or Mn2+ ions, which mediate DNA cleaving and joining. The C-terminal domain (CTD; residues 223–268) features an Src homology domain 3 (SH3)-like fold that contributes to DNA binding and is connected to the catalytic core domain by a α-helical linker (residues 213–222)
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