Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Abstract

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly. Retroviral integrase (IN)3 catalyzes the insertion of viral DNA into the DNA of the infected host cell. IN is one of three retrovirally encoded enzymes that are essential for retroviral replication and therefore is an important target for drugs to treat HIV/AIDS. One IN active site-directed inhibitor, raltegravir, is approved by the Food and Drug Administration for this purpose, and a second, elvitegravir, is in advanced clinical trials.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

“…We recently employed small and wide angle x-ray scattering (SAXS/WAXS) in combination with chemical cross-linking to elucidate the full-length architecture of the apoIN monomer and dimer of ASV (3). We showed that the ASV IN dimer derives its stability from interactions of the NTD of one monomer with the CTD and core of the second monomer and from CTD-CTD contacts between the monomers; core-core interactions are only observed in tetramers.…”

“…Integration of retroviral DNA into the host cell genome is an essential step in viral replication catalyzed by the viral integrase (IN) 3 protein (for a recent review, see Ref. 1).…”

Retroviral Integrase Structure and DNA Recombination Mechanism

“…While not impossible, we favor the straightforward interpretation that 3′ processing activity is catalyzed by the integrase tetramer in the context of the SSC during viral infection. Of note, results of small angle X-ray scattering (SAXS) have yielded novel "reaching dimer" solution conformations for full-length HIV-1 and ASLV integrase in the absence of DNA (137,138). Although extensive CTD-CTD interactions preclude these structures from functionally engaging DNA, they nevertheless could represent intermediates on the pathway to SSC formation (see the chapter by Anna Marie Skalka).…”

Multifunctional facets of retrovirus integrase

“…There are strong structural similarities in the CCD between HIV, PFV and RSV IN (Figure 2) [47,51,52,57,67,76,77] suggesting that under appropriate conditions the highly soluble RSV IN could also be trapped in the presence of the appropriate oligonucleotide (ODN) substrate and STIs. Assembly of a trapped RSV SC at high IN concentrations (1.5 mg/mL; 45 µmol/L) in solution required the presence of 3' OH recessed viral ODN ranging in sizes (16/18R to 18/20R) and DTG, RAL or MK-2048 [50] (Figure 9).…”

“…PFV IN is soluble (10-15 mg/mL) in 0.2 mol/L NaCl [48,49] while RSV IN is highly soluble (30 mg/mL) in 0.2 mol/L (NH4)2SO4 [50] . PFV IN is monomeric [49] while RSV IN is a dimer in solution [50][51][52] . Recombinant HIV IN has been purified as a monomer, dimer and tetramer [47,53,54] .…”