Structural insights into inhibitor mechanisms on immature HIV-1 Gag lattice revealed by high-resolution in situ single-particle cryo-EM

HIV-1 inhibitors, such as Bevirimat (BVM) and Lenacapavir (LEN), block the production and maturation of infectious virions. However, their mechanisms remain unclear due to the absence of high-resolution structures for BVM complexes and LENs structural data being limited to the mature capsid. Utilizing perforated virus-like particles (VLPs) produced from mammalian cells, we developed an approach to determine in situ cryo-electron microscopy (cryo-EM) structures of HIV-1 with inhibitors. This allowed for the first structural determination of the native immature HIV-1 particle with BVM and LEN bound inside the VLPs at high resolutions. Our findings offer a more accurate model of BVM engaging the Gag lattice and, importantly, demonstrate that LEN not only binds the mature capsid but also targets the immature lattice in a distinct manner. The binding of LEN induces a conformational change in the capsid protein (CA) region and alters the architecture of the Gag lattice, which may affect the maturation process. These insights expand our understanding of the inhibitory mechanisms of BVM and LEN on HIV-1 and provide valuable clues for the design of future inhibitors.