Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase

Abstract

Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes. The atomic structures of five different retrovirus IN-DNA complexes, termed intasomes, have revealed varying IN subunit compositions ranging from tetramers to octamers, dodecamers, and hexadecamers. Intasomes containing two IN-associated viral DNA ends capable of concerted integration are termed stable synaptic complexes (SSC), and those formed with a viral/target DNA substrate representing the product of strand-transfer reactions are strand-transfer complexes (STC). Here, we investigated the mechanisms associated with the assembly of the Rous sarcoma virus SSC and STC. C-terminal truncations of WT IN (286 residues) indicated a role of the last 18 residues ("tail" region) in assembly of the tetrameric and octameric SSC, physically stabilized by HIV-1 IN strand-transfer inhibitors. Fine mapping through C-terminal truncations and site-directed mutagenesis suggested that at least three residues (Asp-268-Thr-270) past the last β-strand in the C-terminal domain (CTD) are necessary for assembly of the octameric SSC. In contrast, the assembly of the octameric STC was independent of the last 18 residues of IN. Single-site substitutions in the CTD affected the assembly of the SSC, but not necessarily of the STC, suggesting that STC assembly may depend less on specific interactions of the CTD with viral DNA. Additionally, we demonstrate that trans-communication between IN dimer-DNA complexes facilitates the association of native long-terminal repeat (LTR) ends with partially defective LTR ends to produce a hybrid octameric SSC. The differential assembly of the tetrameric and octameric SSC improves our understanding of intasomes.

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

“…The RSV IN "tail" region (defined as residues from Ile269 to Ala286; Supplementary Fig. 3) accelerates the conversion of the precursor tetrameric CSC to the mature octameric form in a time and temperature-dependent manner 15,16 . Supplementary Fig.…”

“…The ability of RSV IN to assemble the catalytically active octameric CSC from its tetrameric CSC precursor affords opportunities to investigate this distinct intasome assembly pathway [14][15][16] . The CTD of the distal IN dimers in the CSC plays a critical structural role together with the proximal IN dimers to stabilize the CSC (Figs.…”

“…Escherichia coli BL21 (DE3)pLysS and purified to near-homogeneity as previously described [14][15][16] . The wt Prague A IN subunit is 286 aa in length, designated 1-286.…”

“…The assembly mechanisms of retroviral intasomes are poorly understood. Our previous studies suggested that a tetrameric RSV intasome is the precursor of the octameric intasome [14][15][16] . We have now determined the cryo-EM structure of the RSV octameric intasome stabilized by the HIV-1 IN strand transfer inhibitor (INSTI) MK-2048.…”

Multifaceted HIV integrase functionalities and therapeutic strategies for their inhibition

“…The IN tail region, which is composed of the amino acids C-terminal from the CTD SH3 fold, varies in length from about 5 residues in the lentivirus equine infectious anemia virus to 55 residues in MMTV. The tail region in ␣-retroviral IN, which is 19 residues, can regulate DNA-dependent IN octamer formation (64,65). Although implicating a role for this region of IN in intasome assembly, tail regions are unresolved in all IN and intasome structures solved to date, limiting the interpretation of how the tail might regulate nucleoprotein complex formation.…”