HIV‐1 integrase can process a 3′‐end crosslinked substrate

Abstract

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...

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

“…All these modifications at position 3 destabilized the DNA double helix (supplemental Table S1). This destabilization may stimulate 3Ј-end processing, consistent with previous data demonstrating an increase in 3Ј-processing activity because of destabilization of the 3rd bp (26,35,36).…”

“…Destabilization of the A/T Pair at Position 3 Is Necessary for 3Ј-End Processing-Modification of the thymidine at position 3 in the unprocessed strand, by P insertion or 2Ј-modified nucleosides (U N and U M ), increased 3Ј-end processing rate (Table 3) probably because of local destabilization of the A/T base pair, as suggested previously (36). The processing stimulation following the thymidine replacement likely points to an absence of specific contact between IN and this base.…”

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

“…to adopt a structure intermediate between B-DNA and A-DNA (Figure 4a and c and Figure 5). The highly conserved C14pA15 step in the upper strand is known for its involvement in the viral DNA integration (62,63). The distortions that affect this step could be the foundation upon which the LTR-end of viral DNA is specifically recognized and cleaved by IN.…”

Cytotoxic activity of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide is underlain by DNA interchain cross-linking

“…3A). The retardation of the latter could be caused either by DNAprotein cross-linking or by cross-linking within DNA [17][18][19][20]. Further experiments on purified plasmid and genomic DNA revealed that EDC induced interchain crosslinking within the double-stranded DNA ( fig.…”