Related Experiment Videos
Moloney murine leukemia virus IN protein from disrupted virions binds and specifically cleaves its target sequence in
L K Ishimoto1, M Halperin, J J Champoux
1University of Washington, School of Medicine, Department of Microbiology, Seattle 98195.
Abstract:
The integration of retroviral DNA plays an essential role in the viral life cycle. Previous studies of the Moloney murine leukemia virus (M-MuLV) have shown that viral integration is mediated by the integrase (IN) protein acting on the 13-bp inverted repeats that flank the linear viral DNA produced during reverse transcription. Prior studies have also shown that when the M-MuLV IN protein is produced in Escherichia coli it retains an ability to specifically associate with the viral inverted repeats (Krogstad and Champoux, 1990). In this study we present evidence that the IN protein present in detergent-disrupted virions is capable of specifically interacting with double-stranded oligonucleotides that correspond to the viral inverted repeats, and that this interaction may change after integration-related processing of the viral att sites. We further present evidence that, in vitro, detergent-disrupted virions are capable of specifically cleaving ds-IR oligonucleotides in an IN-dependent reaction that mimics the trimming step that precedes integration.
Insights
Moloney murine leukemia virus (M-MuLV) integrase (IN) protein in virions binds viral DNA repeats. This IN protein also cleaves these DNA repeats in vitro, mimicking a step before viral integration.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Retroviral DNA integration is a critical step in the viral life cycle.
- Moloney murine leukemia virus (M-MuLV) integration is facilitated by its integrase (IN) protein.
- M-MuLV IN protein specifically binds to viral DNA inverted repeats.
Purpose of the Study:
- To investigate the specific interaction of M-MuLV IN protein within detergent-disrupted virions with viral DNA inverted repeats.
- To explore potential changes in IN-DNA interaction after integration-related processing.
- To examine the in vitro enzymatic activity of M-MuLV IN on viral DNA mimics.
Main Methods:
- Analysis of IN protein binding to double-stranded oligonucleotides representing viral inverted repeats.
- Investigation of IN-DNA interactions using detergent-disrupted virions.
- In vitro cleavage assays using ds-IR oligonucleotides and detergent-disrupted virions.
Main Results:
- M-MuLV IN protein in detergent-disrupted virions specifically interacts with double-stranded oligonucleotides corresponding to viral inverted repeats.
- This IN-DNA interaction may be altered following integration-related processing of viral att sites.
- Detergent-disrupted virions exhibit IN-dependent cleavage of ds-IR oligonucleotides in vitro.
Conclusions:
- The M-MuLV IN protein within virions retains specific DNA-binding capabilities.
- The observed in vitro cleavage activity mimics the processing step preceding viral integration.
- These findings provide insights into the molecular mechanisms of M-MuLV DNA integration.