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.
Highlights
Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes
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)
We have further investigated the role of the tail region and the C-terminal domain (CTD) (Fig. 1) of Rous sarcoma virus (RSV) IN by site-directed mutagenesis as well as the long-terminal repeat (LTR) DNA sequences on the assembly of the SSC
Summary
Temperature, time, and length of the tail region from the end of the last -strand in the CTD affect assembly of both tetrameric and octameric SSC. As shown previously at 4 °C with all three new C-terminal IN truncation mutants (Fig. 2) (7), the formation of the tetrameric SSC occurred first prior to conversion to the octameric form in a time-dependent manner (data not shown). All of these C-terminal IN truncation mutants had similar concerted integration activity with 3Ј-OH recessed 18R GU3 or blunt-ended 20B GU3 as IN(1–278) (Fig. S3A), and like the other previously defined C-terminal IN truncations (7, 10), they were dimeric in solution (data not shown). There appears to be no major amino acid sequence requirements in the tail region between Gln-271 and Lys-278 for assembly of the octameric SSC in vitro
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