Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners.
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.
Vera
Jorge
J
Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Parissi
Vincent
V
Bordeaux, F-33000 France; IFR 66 'Pathologies Infectieuses et Cancers', Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.|CNRS UMR 5097, Bordeaux, F-33000 France; Université Victor Segalen Bordeaux 2, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
García
Andrea
A
Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Zúñiga
Roberto
R
Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Andreola
Marie-Line
ML
Bordeaux, F-33000 France; IFR 66 'Pathologies Infectieuses et Cancers', Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.|CNRS UMR 5097, Bordeaux, F-33000 France; Université Victor Segalen Bordeaux 2, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
Caumont-Sarcos
Anne
A
Bordeaux, F-33000 France; IFR 66 'Pathologies Infectieuses et Cancers', Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.|CNRS UMR 5097, Bordeaux, F-33000 France; Université Victor Segalen Bordeaux 2, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
Tarrago-Litvak
Laura
L
Bordeaux, F-33000 France; IFR 66 'Pathologies Infectieuses et Cancers', Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.|CNRS UMR 5097, Bordeaux, F-33000 France; Université Victor Segalen Bordeaux 2, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
Leon
Oscar
O
Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
D000818:Animals; D002868:Chromosomal Proteins, Non-Histone; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C066190:SNF5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D019426:Integrases