Abstract Retrovirus integrase (IN) is responsible for integration of viral genome into host DNA. IN multimerizes on viral DNA to produce intasomes. Intasomes produced from lentiviruses including HIV-1 demonstrate an extended architecture compared to other retroviral intasomes. The specific aims of this proposal are to: 1) determine the structure of HIV-2 intasomes by single-particle cryo-electron microscopy (cryo-EM) in the presence of HIV-1 IN strand transfer inhibitors (INSTIs); 2) determine the mechanisms of assembly of extended intasome architectures using HIV-1 and HIV-2 as model systems; and 3) produce and determine the structure of intasomes assembled with untagged native tetrameric HIV-1 and dimeric HIV-2 IN, as fusion tags used to enhance IN solubility may interfere with positioning of IN subunits in intasomes. As proof of concept and to demonstrate expertise, we determined the structure of Rous sarcoma virus octameric (8 IN subunits) cleaved synaptic complex intasome stabilized with INSTI MK-2048 at 3.21Å resolution by cryo-EM. We have obtained preliminary data for HIV-1 and HIV-2 intasomes structures using cryo-EM. The partial intasome structures from other research groups have further determined the mechanisms of HIV-1 INSTIs. However, the distal subunits which are crucial for intasome assembly and catalysis are not resolved in these structures. We will use wt HIV- 1 and HIV-2 IN with native N-terminal residue phenylalanine to produce intasomes for their complete structure determination by cryo-EM. HIV-2 is a significant human pathogen and determining of the structure of HIV-2 intasome with INTSIs will be complementary and confirmatory for HIV-1 studies. These studies will provide a greater understanding of intasome assembly mechanisms and facilitate development of active site and novel allosteric IN inhibitors.