Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

Abstract

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro

“…In recent years, substantial structural and functional information on retroviral intasomes has provided unprecedented detail on the molecular mechanism of retroviral DNA integration ( 20 , 21 , 22 , 23 , 24 , 25 , 26 ). By contrast, there are no structural data on the Ty1 integration process and the interaction between IN and Pol III.…”

Structure and function of retroviral integrase

“…Akin to functionally equivalent transposase-DNA complexes, retroviral intasomes are resistant to challenge with high-salt conditions, and this property was instrumental in their biochemical isolation. The intasomes from several retroviral species were assembled from purified components and visualised by X-ray crystallography or single-particle cryogenic electron microscopy (cryo-EM) 25,[29][30][31][32][33][34][35][36][37][38][39] (Figs. 2A, 3).…”

“…The structures revealed unexpected architectural diversity among intasomes from different retroviral genera. While the intasomes from the prototype foamy virus (PFV, a simiispumavirus) as well as the δ-retroviruses HTLV-1 and simian T-cell lymphotropic virus (STLV) contain four IN subunits (arranged as a dimer-of-dimers) 25,29,37,38 , those from ASLV and mouse mammary tumour virus (MMTV, β-retrovirus) harbour eight IN chains (a tetramer-of-dimers) 30,31,39 . The intasome from maedi-visna virus (MVV, a lentivirus), containing sixteen IN subunits (a tetramer-oftetramers), is the largest characterized to date 32 .…”

Multimodal Functionalities of HIV-1 Integrase

“…Although at the time it was generally believed that tetramers were the basic catalytic units of all retroviral INs, subsequent intasome structures revealed a surprising array of protein oligomers among the different complexes. Akin to PFV, δ-retroviral intasomes harbor an IN tetramer [ 87 , 88 ], while α-and β-retroviral intasomes contain higher-order octameric IN arrangements [ 78 , 89 , 90 ]. Lentiviral intasomes have revealed more diverse structures, which range from IN tetramers to hexadecamers [ 76 , 91 ]; the field consensus is that the higher-order structures likely represent the physiological arrangement of lentiviral IN on vDNA [ 92 , 93 ] ( Figure 3 ).…”