Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization

Abstract

During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.

Mutations in the U5 Sequences Adjacent to the Primer Binding Site Do Not Affect tRNA Cleavage by Rous Sarcoma Virus RNase H but Do Cause Aberrant Integrations In Vivo

“…Moreover, for the aberrant ends not inserted at the canonical CA, the insertion event does not generate the 5-to 6-bp duplication in the host genome that is characteristic of the normal concerted insertions that involve integrase. Taken together, the data support, but do not prove, a model in which the normal end is inserted by integrase in a reaction similar to the single-end insertions seen in in vitro integrase assays (13,19) and the aberrant end is inserted into the host genome by host enzymes. However, in an RSV-based in vitro integration assay that led to efficient concerted integration events with a substrate with a wild-type sequence at the ends, mutating the concerted CA reduced the concerted integrations and did not appear to produce single-end insertions (1).…”

Fidelity of Target Site Duplication and Sequence Preference during Integration of Xenotropic Murine Leukemia Virus-Related Virus

“…Analyses of various proviruses have revealed that the length of target site duplication, though varying from 4 to 6 bp among the different retroviruses examined, is invariant for each particular retrovirus [12], [23], [39]. The high fidelity of the direct repeat length supports the notion that IN multimers form a stable complex with viral and target DNA and catalyze coordinated processing and integration of the two viral DNA ends [30], [33], [40], [41], [42]. In addition, reaction conditions in vitro and in vivo that promote uncoordinated integration of the two ends often produce deletions and duplications of various lengths in the target DNA [25], [26], [27], [28], [29], [31], [39], [43].…”

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

“…1). The isolated products were ligated and subsequently used to transform MC1061/P3 cells (Invitrogen) (41,42). The donor-target junctions of individually isolated recombinants were sequenced using HIV-1 U3 and U5 sequence-specific primers (46).…”