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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.
Highlights
Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits
Rous sarcoma virus (RSV) IN 1–278, which has a C-terminal truncation of 8 amino acids from the full-length 286-residue IN forms a stable octameric complex with 3′ OH recessed viral DNA substrates[15] (Supplementary Figs. 1–3)
The final map had an overall resolution of 3.2 Å (Supplementary Figs. 4, 5) and local resolution reaching ~2.8 Å for the core region of the complex containing the two IN catalytic sites bound with IN strand transfer inhibitor (INSTI) and viral DNA (Supplementary Fig. 5), which was significantly improved from the previously reported RSV strand transfer complex (STC) X-ray crystal structure that was determined at 3.8 Å resolution[5]
Summary
Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. The structures of different retroviral IN-DNA complexes capable of concerted integration show diverse organizations but possess significant mechanistic similarities These structures, generally termed intasomes, contain multimers of IN subunits ranging from 4 for the simiispumavirus prototype foamy virus (PFV)[1,2] and delta-retroviruses human T-cell leukemia virus type 1 (HTLV-1)[3] and simian T-lymphotropic virus type 1 (STLV-1)[4] to 8 for alpha-retrovirus Rous sarcoma virus (RSV)[5] and betaretrovirus mouse mammary tumor virus (MMTV)[6]. Stabilizing interactions that occur between the INSTIs, recessed viral DNA ends and Mg++
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