Retroviral integrase (IN) functions within the intasome nucleoprotein
complex to catalyze the insertion of viral DNA into cellular chromatin. The lack of
lentiviral intasome structural information has hampered the development of anti-HIV
drugs and the understanding of viral resistance. Using cryo-electron microscopy, we
now visualize the functional maedi-visna lentivirus intasome at 4.9 Å resolution. The
intasome, which comprises a homo-hexadecamer of IN with a tetramer-of-tetramers
architecture, harbors eight structurally distinct types of IN protomers including two
catalytically competent subunits. The conserved intasomal core, previously observed in
simpler retroviral systems, is formed between two IN tetramers, with a pair of Cterminal
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 structure-guided design of HIV-1
IN inhibitors.