Vora AC, Grandgenett DP; HIV DRP Symposium Understanding Antiviral Drug Resistance.
Program Abstr HIV DRP Symp Underst Antivir Drug Resist 1st 2000 Chantilly Va. 2000 Dec 3-6; 1: Abstract no. 37.
St. Louis University Health Sciences Center, Institute for Molecular Virology, St. Louis, MO
Integration of the retrovirus linear DNA genome into the chromosome of virus-infected cells is mediated by the viral integrase (IN). The viral genome and IN are present in pre-integration complexes. The blunt-ended DNA termini are recessed by two nucleotides at their 3' OH ends by IN. These complexes are termed intasomes. Intasomes are capable of integrating the recessed DNA termini in a concerted fashion into exogenous target DNA (full-site). We have reconstituted intasome-like complexes with linear 4.5 kbp donors containing the avian U5 and U3 long terminal repeats (LTR) and purified avian myeloblastosis virus IN. The assembly of IN-donor complexes for full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. Assembly of nucleoprotein complexes for full-site integration is readily discernible through their time requirement and low IN concentration dependence when compared to the assembly of other nucleoprotein complexes capable of inserting only one LTR end into circular target DNA (half-site). DNaseI footprint analyses of assembled IN-donor complexes capable of full-site integration shows wt U3 and other LTR ends containing "gain-of-function" attachment (att) site sequences for full-site integration are specifically protected by IN within a defined outer boundary mapping ~20 nucleotides from the termini. The same protected DNaseI footprints are observed with assembly of IN-donor complexes occurring in the presence of target DNA. A donor containing mutations in the att site simultaneously eliminates full-site integration and DNaseI protection by IN. Coupling of either two wt U5 ends or a wt U5 end with a wt U3 end shows binding by IN occurs only at the very terminal nucleotides (<10 bp) on wt U5 for full-site integration. The results suggest assembly of complexes requires a defined number of IN subunits binding asymmetrically to U3 and U5 ends for full-site integration in vitro.ACKNOWLEDGEMENTS: Supported by the National Cancer Institute (CA16312) (DG).
Publication Types:
Keywords:
- Avian myeloblastosis virus
- Base Sequence
- DNA
- DNA Footprinting
- DNA Nucleotidyltransferases
- DNA, Circular
- DNA, Viral
- Genome, Viral
- In Vitro
- Integrases
- Mutation
- Retroviridae
- Terminal Repeat Sequences
- Virion
- genetics
- metabolism
- pharmacokinetics
Other ID:
UI: 102249529
From Meeting Abstracts