Characterization of chimeric enzymes between caprine arthritis--encephalitis virus, maedi--visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli.
In order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1). The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates. Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction. Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme. The N terminus appears to have no considerable influence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN. Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.
Berger
Nicola
N
Federal Research Centre for Virus Diseases of Animals, Institute for Immunology, Paul-Ehrlich-Strasse 28, D-72076 Tübingen, Germany1.
Heller
Astrid E
AE
Federal Research Centre for Virus Diseases of Animals, Institute for Immunology, Paul-Ehrlich-Strasse 28, D-72076 Tübingen, Germany1.
Störmann
Klaus D
KD
Federal Research Centre for Virus Diseases of Animals, Institute for Immunology, Paul-Ehrlich-Strasse 28, D-72076 Tübingen, Germany1.
Pfaff
Eberhard
E
Federal Research Centre for Virus Diseases of Animals, Institute for Immunology, Paul-Ehrlich-Strasse 28, D-72076 Tübingen, Germany1.
D016088:Arthritis-Encephalitis Virus, Caprine / Q000201:enzymology*; D002678:Chimera / Q000378:metabolism / Q000821:virology*; D004247:DNA / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D016662:Virus Integration; D014790:Visna-maedi virus / Q000201:enzymology*
D011994:Recombinant Proteins; D004247:DNA; D019427:HIV Integrase; D019426:Integrases; D004720:Endonucleases