Integration of a DNA copy of the human immunodeficiency virus (HIV)-1 RNA genome into the human genome is an essential step in the viral replication cycle. This process is catalysed by a viral protein, integrase (IN). The first key reaction in the overall integration process is the cleavage of a dinucleotide from each 3¢-end of the viral DNA (substrate DNA), a reaction termed 3¢-end processing. In the second step, DNA strand transfer, a pair of processed DNA ends of the same viral DNA is inserted into the host cellular DNA (target DNA). The 3¢-end processing reaction requires a conserved nucleotide sequence at the viral DNA ends, but the second reaction does not absolutely require specific sequences within the host DNA. The 3¢-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium ⁄ kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a singleturnover mode and is intrinsically very slow (single-turnover rate constant ¼ 0.004 min )1 ), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.Abbreviations HIV, human immunodeficiency virus; IN, integrase; LTR, long terminal repeat; PIC, preintegration complex; r, anisotropy; RSV, Rous sarcoma virus.

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn 2؉ or Mg 2؉ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg 2؉ than in the presence of Mn 2؉ . Modifications of base pairs 7-9 affected 3-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.Following reverse transcription, a DNA copy of the human immunodeficiency virus, type 1 (HIV-1), 2 RNA is integrated into the genome of infected cells. Integration is a prerequisite for viral replication and is catalyzed by the viral enzyme integrase (IN). IN binds to sequences located at the end of U3 and U5 parts of long terminal repeats (LTRs) of viral DNA and catalyzes the trimming, or 3Ј-end processing, of the terminal dinucleotide from the 3Ј-ends of both strands of the DNA. IN then mediates a strand transfer reaction that inserts the viral DNA into the host DNA. During this reaction, IN must bind simultaneously to viral and target DNA. However, IN interacts with these two DNA molecules in different ways as follows: binding to host DNA does not depend directly on host DNA sequence, whereas interaction with the viral DNA is a sequence-specific process. Nevertheless, the U5 and U3 sequences recognized by IN are not exactly identical.Strand transfer and 3Ј-end processing reactions may be carried out in vitro, using recombinant HIV IN, DNA duplexes mimicking U3 or U5 sequences of LTRs, and divalent metal ions, such as Mg 2ϩ or Mn 2ϩ . However, the Mn 2ϩ -and Mg 2ϩ -dependent activities of IN are not equivalent, with lower specificity reported for Mn 2ϩ -dependent IN (1, 2). Moreover, the inhibition of HIV-1 IN by compounds such as ␤-diketo acids, which interact with the active site of HIV-1 IN, is also metal-de...

Integrase of the human immunodeficiency virus type-1 (HIV-1) recognizes specific sequences located in the U3 and U5 regions at the ends of viral DNA. We synthesized DNA duplexes mimicking the U5 region and containing either 2¢-aminonucleosides or non-nucleoside 1,3-propanediol insertions at the third and terminal positions and studied their interactions with HIV-1 integrase. Both modifications introduced a local structural distortion in the DNA double helix. Replacement of the terminal nucleosides by corresponding 2¢-aminonucleosides had no significant effect on integrase activity. We used an integrase substrate bearing terminal 2¢-aminonucleosides in both strands to synthesize a duplex with cross-linked strands. This duplex was then used to determine whether terminal base pair disruption is an obligatory step of retroviral DNA 3¢-processing. Processing of the cross-linked analog of the integrase substrate yielded a product of the same length as 3¢-processing of the wild-type substrate but the reaction efficiency was lower. Replacement of the third adenosine in the processed strand by a corresponding 2¢-aminonucleoside did not affect integrase activity, whereas, its replacement by 1,3-propanediol completely inhibited 3¢-processing. Both modifications of the complementary thymidine in the nonprocessed strand increased the initial rate of 3¢-processing. The same effect was observed when both nucleosides, at the third position, were replaced by corresponding 2¢-aminonucleosides. This indicates that the local duplex distortion facilitated the cleavage of the phosphodiester bond. Thus, a localized destabilization of the third A-T base pair is necessary for efficient 3¢-processing, whereas 3¢-end-fraying is important but not absolutely required.Keywords: integrase; 2¢-aminonucleoside; interstrand crosslinking; DNA modification.Human immunodeficiency virus type-1 (HIV-1) integrase is the retroviral enzyme that mediates the integration of viral DNA into the host DNA. This process is the key step in retroviral replication. Integrase catalyzes two successive reactions: the first is the cleavage of GT dinucleotides from each 3¢-end of the viral DNA; this reaction is called 3¢-processing. The second reaction, named strand transfer, involves the attack of internucleotide phosphates in the host DNA by the 3¢-hydroxyl groups generated during 3¢-processing; it results in the integration of viral DNA by the transesterification mechanism. Integrase is currently the least studied HIV-1 enzyme, and this fact considerably delays the preparation of efficient integrase inhibitors.Integrase consists of three domains: the N-terminal, catalytic and C-terminal domains. The structures of each domain and of two-domain fragments have been determined by X-ray crystallography or NMR-spectroscopy [1][2][3][4], but the structures of the whole enzyme and of the integrase-viral DNA complex have not yet been determined. The protein-DNA interactions through which integrase mediates the formation of a stable, catalytically active complex with the vir...

Due to their ability to integrate into the host cell's genome, retroviruses represent an optimal basis for the creation of gene therapy vectors. The integration reaction is carried out by a viral enzyme integrase: thus, a detailed research of this enzyme is required. In this work, the catalytic properties of human foamy virus integrase were studied. This virus belongs to the Retroviridae family. The dissociation constant was determined, together with the kinetics of integrase catalytic activity. The data obtained were compared to those for the human immunodeficiency virus integrase and a considerable similarity in the activity of the two enzymes was observed.

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