Modeling the Late Steps in HIV-1 Retroviral Integrase-catalyzed DNA Integration

Jonathan Leis,Elena Brin,Jizu Yi,Anna Marie Skalka

doi:10.1074/jbc.m006929200

Abstract

Model oligodeoxyribonucleotide substrates representing viral DNA integration intermediates with a gap and a two-nucleotide 5' overhang were used to examine late steps in human immunodeficiency virus, type 1 (HIV-1) retroviral integrase (IN)-catalyzed DNA integration in vitro. HIV-1 or avian myeloblastosis virus reverse transcriptase (RT) were capable of quantitatively filling in the gap to create a nicked substrate but did not remove the 5' overhang. HIV-1 IN also failed to remove the 5' overhang with the gapped substrate. However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction. The efficiency of this closure reaction was very low. Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions. Addition of Flap endonuclease-1, a nuclease known to remove 5' overhangs, abolished the closure reaction catalyzed by IN. A series of base pair inversions, introduced into the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small decrease in the HIV-1 IN-dependent strand closure reaction. These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA integration. Finally, a combination of HIV-1 RT, Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these model substrates. These results support the hypothesis that cellular enzyme(s) may catalyze the late steps of retroviral DNA integration.

Highlights

Insertion of viral DNA into the host genome is an obligatory step in the retroviral life cycle and requires a concerted mechanism that brings the two LTR1 ends of a single donor into a complex with IN and the host DNA
Model Substrates—In the present study, we have employed a series of model HIV-1 and ASV oligodeoxyribonucleotide substrates to examine the action of viral and/or cellular enzymes in catalyzing the repair and strand closure reactions associated with integration of viral DNA
We have examined several HIV-1 substrate variants in which base pair inversions were introduced at LTR positions 3–7, adjacent to and/or including the conserved CA dinucleotide

Summary

EXPERIMENTAL PROCEDURES

Reagents—[␣-32P]ddATP (3,000 Ci/mmol), [␣-32P]dCTP(3,000Ci/ mmol), and [␥-33P]ATP (2,500 Ci/mmol) were purchased from Amersham Pharmacia Biotech. Plasmid Constructions and Preparations—Plasmid pHHIV2, which was used in this study as a template to amplify donor DNA, is a variation of pBCSKϩ in which a wild type HIV-1 donor DNA polymerase chain reaction product was inserted into pBCSKϩ catalyzed by IN, resulting in the loss of 2 base pairs from the LTR ends. This plasmid was propagated in E. coli MC1061/P3 under the conditions described above. The reaction was terminated by addition of 16 ␮l of 95% formamide, 10 mM EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanole, and reaction products were analyzed by electrophoresis using 20% denaturing polyacrylamide gels as described above. Sequencing was performed using the Thermo-Sequenase kit (U.S Biochemical Corp.)

RESULTS

Base pair duplications in acceptor DNA

DISCUSSION

TTCCAatttaga acgcgcattgACCTT TTCCAgctacagggcgcgt gggcgaaagACCTT