Previous Article | Next Article ![]()
J. Virol., Mar 1996, 1424-1432, Vol 70, No. 3
AL Wolfe, PJ Felock, JC Hastings, CU Blau and DJ Hazuda
The integration of a DNA copy of the viral genome into the genome of the
host cell is an essential step in the replication of all retroviruses.
Integration requires two discrete biochemical reactions; specific
processing of each viral long terminal repeat terminus or donor substrate,
and a DNA strand transfer step wherein the processed donor substrate is
joined to a nonspecific target DNA. Both reactions are catalyzed by a
virally encoded enzyme, integrase. A microtiter assay for the strand
transfer activity of human immunodeficiency virus type 1 integrase which
uses an immobilized oligonucleotide as the donor substrate was previously
published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini,
Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of
modifications to the method which facilitate study of both the nature and
the dynamics of the interaction between integrase and the donor DNA. The
enzyme which binds to the immobilized donor is shown to be sufficient to
catalyze strand transfer with target DNA substrates added subsequent to
assembly; in the absence of the target substrate, the complex was retained
on the donor in an enzymatically competent state. Assembly required high
concentrations of divalent cation, with optimal activity achieved at 25 mM
MnCl2. In contrast, preassembled complexes catalyzed strand transfer
equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically
distinct functions for the divalent cation in assembly and catalysis,
respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to
promote the multimerization of integrase in the absence of a DNA substrate
and alleviate the requirement for high concentrations of divalent cation
during assembly. The superphysiological requirement for MnCl2 may,
therefore, reflect an insufficiency for functional self- assembly in vitro.
Subunits were observed to exchange during the assembly reaction, suggesting
that multimerization can occur either before or coincident with but not
after donor binding. These studies both validate and illustrate the utility
of this novel methodology and suggest that the approach may be generally
useful in characterizing other details of this biochemical reaction.
Copyright © 1996, American Society for Microbiology
The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
This article has been cited by other articles:
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»