A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

Abstract

Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3′ DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.

Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

“…Previous results with RSV IN(1-270) demonstrated that IN with the C-terminal truncation possesses similar properties to those associated with IN(1-286), including robust concerted integration activity using a larger sized (Ͼ1 kb) GU3 substrate (2). We further explored whether IN(1-269) and IN(1-274) displayed different properties than those associated with IN(1-286).…”

“…In summary, these qualitative analyses demonstrated that each independently isolated trapped SC contained the inhibitor. IN(1-286) is dimeric in solution (28), and RSV IN(1-270) appeared to be a dimer upon SEC analysis (2). A stock solution of IN was diluted to 45 M with running buffer in the absence of DNA prior to chromatography.…”

“…Rous sarcoma virus (RSV) IN possesses the canonical three-domain structure consisting of an N-terminal domain (amino acids 1-44), the catalytic core domain (amino acids 50 -214), and the C-terminal domain (amino acids 222-286) (1)(2)(3). Human immunodeficiency virus type 1 (HIV) IN (amino acids 1-288) has similar size analogous domains and linkers between its domains (4,5).…”

Multifunctional facets of retrovirus integrase

“…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] .…”

“…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).…”