Processing of Viral DNA Ends Channels the HIV-1 Integration Reaction to Concerted Integration*[boxs]

Abstract

Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3 end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are "half-site" products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the halfsite reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.Integration of retroviral DNA into the host chromosomal DNA, an essential step in the retroviral replication cycle, involves two chemical steps (1). The newly synthesized bluntended viral DNA first undergoes 3Ј end processing. In this reaction, two nucleotides are removed from each 3Ј end. Next, the exposed hydroxyl groups attack a pair of phosphodiester bonds on opposite strands of the target DNA to complete the strand transfer step. For human immunodeficiency virus (HIV)-1, 1 the sites of attack are separated by 5 bp, resulting in a five-base duplication of target DNA sequence upon repair of the resulting integration intermediate. In the presence of a divalent metal ion, the viral integrase protein alone is able to carry out both 3Ј processing and DNA strand transfer with simple DNA substrates that mimic the viral DNA ends (2-4). However, under most reaction conditions the products of DNA strand transfer result from the integration of a single viral DNA end into one strand of the target DNA, a reaction that has been termed "half-site" as opposed to concerted integration. Were this to occur in the cell, the viral DNA would fail to integrate, and the viral replication cycle would be aborted. Following reverse transcription, the viral DNA remains associated with integrase and other viral and cellular proteins as part of the preintegration complex (PIC) (5-13). PICs isolated from infected cells efficiently integrate their DNA into a target DNA in vitro. In contras...

Integrase, LEDGF/p75 and HIV replication

“…2. This essential aspect of the in vivo reaction has typically been difficult to recapitulate in vitro with the purified enzyme, although recent adjustments to assay parameters have boosted efficiency [155,156]. The effect of p75 differed sharply between different recombinant lentiviral IN proteins: its addition enhanced full-site integration for EIAV IN while HIV IN was instead directed virtually exclusively to catalyze uncoupled (half-site) integration in which only one viral DNA end is joined to one strand of target DNA [100].…”

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein

“…Complementation studies with two defective forms of HIV-1 IN revealed that subunits can multimerize to become active (38,39). Recent studies indicate that the IN of HIV-1 is a tetramer in its synaptic complex and that multimerization of the C-terminal domain plays an important role in concerted integration (7,40). Our results of gel filtration indicated that full-length Tf1 IN formed a stable dimer.…”

Multifunctional facets of retrovirus integrase

“…The HIV IN monomer purified in the presence of EDTA is converted into a tetramer in the presence of Zn 2+ without the formation of a dimer intermediate [53,54] . Studies have demonstrated that a tetramer of IN is associated with HIV, PFV and RSV SC capable of concerted integration [50,[55][56][57] . The oligomeric state of retrovirus IN play key roles in virus maturation upon release of the virus particle, enzymatic activities for 3' OH processing and concerted integration of viral DNA and possibly for reverse transcription in the cytoplasmic PIC.…”