Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Abstract

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...

Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

“…To circumvent this limitation, strand transfer activity was inhibited with a nonradiolabeled version of 1. The resulting IC 50 was compared with that for inhibition of the WT complex by 1. The resulting relative IC 50 values indicate that 1 has ϳ2% of the WT affinity toward this combination, similar to the loss of affinity measured for the WT LTR-Q148R complex.…”

“…The resulting IC 50 was compared with that for inhibition of the WT complex by 1. The resulting relative IC 50 values indicate that 1 has ϳ2% of the WT affinity toward this combination, similar to the loss of affinity measured for the WT LTR-Q148R complex. Overall, these results indicate that abasic T 1 and C Ϫ1 sites produce synergistic defects in the assembly and specific strand transfer activity of Q148R, without further reducing already highly attenuated 1 affinity.…”

Relationship between the Oligomeric Status of HIV-1 Integrase on DNA and Enzymatic Activity

“…In particular, the CA sequence preceding the GT dinucleotide cleaved by IN is strictly required. Reaction specificity seems to depend on the catalytic step, because no significant difference in affinity is observed in vitro for different DNA sequences (2,3,5,6). The catalytic mechanism of IN has been extensively studied, but the structural determinants of IN activity remain unclear, and controversy remains concerning the multimeric status of active IN.…”

“…The catalytic core establishes specific contacts with the viral DNA and, together with the C-terminal domain, is involved in DNA binding (1)(2)(3)(4). The 3Ј-processing reaction is highly specific, and the terminal 13 bp of the LTR play a key role for Mg 2ϩ -dependent 3Ј-processing in terms of reaction specificity (4,5). In particular, the CA sequence preceding the GT dinucleotide cleaved by IN is strictly required.…”

Investigation of the Binding Affinity between Styrylquinoline Inhibitors and HIV Integrase Using Calculated Nuclear Quadrupole Coupling Constant (NQCC) Parameters (A Theoretical ab initio Study)

“…[11][12][13][14] The emergence of viral strains resistant against available drugs and the dynamic nature of the HIV-1 genome support a continued effort towards the discovery and characterization of novel targets and anti-viral drugs. Due to its central role in the HIV-1 life cycle, IN represents a promising therapeutic target.…”