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Updated: Aug 16, 2026
Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions
Published on: May 28, 2017
Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for
Jorge Vera1, Vincent Parissi2,3, Andrea García1
1Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Abstract:
Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in Escherichia coli led the authors to explore the use of Saccharomyces cerevisiae for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel-NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from E. coli and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the in vitro assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, S. cerevisiae was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that S. cerevisiae could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.
Insights
Saccharomyces cerevisiae enables efficient expression of Moloney murine leukemia virus integrase (M-MuLV IN). Yeast-expressed IN is more active and aids in studying its interaction with host factors.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Moloney murine leukemia virus (M-MuLV) integrase (IN) is crucial for viral DNA integration.
- Recombinant M-MuLV IN expressed in E. coli exhibits limited solubility.
- Saccharomyces cerevisiae offers an alternative expression system for M-MuLV IN.
Purpose of the Study:
- To express and purify active M-MuLV IN in yeast.
- To characterize the enzymatic activity and host interactions of M-MuLV IN.
- To validate yeast as a model for studying IN function and host interactions.
Main Methods:
- Expression and purification of M-MuLV IN in Saccharomyces cerevisiae.
- Enzymatic assays to determine IN activity and nuclease contamination.
- Site-directed mutagenesis of Asp184 to Alanine (D184A).
- Analysis of IN's lethal effect in yeast strains with specific gene deficiencies (RAD52, ySNF5).
Main Results:
- Yeast expression yielded highly active, nuclease-free M-MuLV IN.
- Mutation of Asp184 to Alanine (D184A) completely inactivated the enzyme.
- Active M-MuLV IN expression induced lethality in RAD52-deficient yeast, linked to cellular damage.
- The lethal effect was abolished in yeast lacking the ySNF5 transcription factor.
Conclusions:
- Saccharomyces cerevisiae is a suitable system for producing active M-MuLV IN.
- Asp184 is essential for M-MuLV IN catalytic activity.
- M-MuLV IN interacts with yeast cellular components, potentially involving ySNF5, leading to toxicity.
- Yeast serves as a valuable model for investigating M-MuLV IN-host interactions.
