Mapping domains of retroviral integrase responsible for viral DNA specificity and target site selection by analysis of chimeras between human immunodeficiency virus type 1 and visna virus integrases

Abstract

Human immunodeficiency virus type 1 (HIV-1) and visna virus integrases were purified from a bacterial expression system and assayed on oligonucleotide substrates derived from each terminus of human immunodeficiency virus type 1 and visna virus linear DNA. Three differences between the proteins were identified, including levels of specific 3-end processing, patterns of strand transfer, and target site preferences. To map domains of integrase (IN) responsible for viral DNA specificity and target site selection, we constructed and purified chimeric proteins in which the N-terminal, central, and C-terminal regions of these lentiviral integrases were exchanged. All six chimeric proteins were active for disintegration, demonstrating that the active site in the central region of each chimera maintained a functional conformation. Analysis of endonucleolytic processing activity indicated that the N terminus of IN does not contribute to viral DNA specificity; this function must reside in the central region or C terminus of IN. In the viral DNA integration assay, chimeric proteins gave novel patterns of strand transfer products which did not match that of either wild-type IN. Thus, target site selection with a viral DNA terminus as nucleophile could not be mapped to regions of IN defined by these boundaries and may involve interactions between regions. In contrast, when target site preferences were monitored with a new assay in which glycerol stimulates IN-mediated cleavage of nonviral DNA, chimeras clearly segregated between the two wild-type patterns. Target site selection for this nonspecific alcoholysis activity mapped to the central region of IN. This report represents the first detailed description of functional chimeras between any two retroviral integrases.

Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases

“…2B) shows that each enzyme performed the DNA joining reaction on each substrate and that the extent of this reaction (i.e., the total amount of longer products) was roughly similar for substrates that matched at the terminal nine positions (compare lanes 1 to 5 with lanes 16 to 20 and lanes 6 to 10 with lanes 11 to 15). As previously noted, the patterns of joined products differed as a function of the target DNA sequence and the source of IN (11). Substrate sequences and quantitation of specific cleavage for multiple replicate reactions are shown in Fig.…”

“…In particular, the substrates derived from the viral U3 termini clearly distinguish between the two enzymes ( Fig. 2A, lanes 1 to 10), a difference we exploited when examining chimeric INs made from the two proteins (11). The differential activity on the two U3 substrates is striking, considering that these sequences differ at only 2 of the final 9 bp at their 3Ј ends, i.e., positions 5 and 6 just internal to the invariant CA dinucleotide ( Fig.…”

“…However, general rules describing which viral DNA bases interact most critically with different integrases have not been defined. Our laboratory has performed comparative studies on the related lentiviral INs of HIV-1 and visna virus to identify regions of the IN protein that interact with different nucleophiles and with features of target DNA during catalysis (10)(11)(12). These studies have permitted a directed and quantitative examination of the roles of subterminal U3 and U5 DNA positions in differential susceptibility to the two enzymes.…”

Retroviral Integrase Structure and DNA Recombination Mechanism

“…Purified integrase proteins display two additional endonucleolytic activities in vitro, disintegration (35) and alcoholysis (36,37). Though it seems unlikely that either of these activities occurs in the context of virus infection, their study has nevertheless yielded valuable information on integrase domain organization and reaction mechanism.…”

Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions

“…(iii) Integrase prefers to integrate into bent regions on target DNA (3,49,(51)(52)(53). The core domain influences the target site preference of the integration reaction (38,50,65). The determinants of integrase's substrate specificity have proven elusive.…”