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 inte grase (IN) protein domains on viral DNA specificity and target site selecti on, 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 chi meric enzymes were expressed in Escherichia coil, purified by affinity chro matography and analysed in vitro for IN-specific endonuclease and integrati on activities on various DNA substrates. Of the 21 purified chimeric IN pro teins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integrati on 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 i nfluence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the rea ction conditions that support optimal enzyme activities of CAEV IN. Also, u nder the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibite d endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detaile d characterization of the activities of CAEV IN in vitro as well as the ana lysis of functional chimeric lentiviral IN proteins.