A rapid method for quantitating products of the oligodeoxynucleotide processing reaction in vitro has been developed to facilitate enzymatic studies of the retroviral integrases. Unlike earlier procedures, this assay does not depend on polyacrylamide gel electrophoresis but separates products by batch adsorption to PEI-cellulose. A joining assay has also been modified, to facilitate measurement of the two distinct steps in the integration reaction under parallel conditions. Since these methods allow quantitation of numerous samples in a short period of time, they are especially useful for investigation of kinetic parameters and to measure the effects of possible inhibitors of integrase. These assay systems were used to examine the enzymatic activity of wild-type Rous sarcoma virus integrase and selected mutant proteins with substitutions of single conserved amino acids. In contrast to previous studies, reactions were performed under conditions of substrate excess, and rates, rather than yields of product generated after a given period of incubation, were determined. The results showed that substitutions of several highly conserved residues in what is most likely an evolutionarily conserved catalytic domain of the integrases resulted in a 4-to 10-fold decrease in the apparent rate of processing relative to wild type, under optimized standard conditions. Changing an invariant acidic residue reduced the rate by -60-fold. When joining activity was determined, the relative effects of the substitutions tested generally paralleled the results with processing. However, with both wild-type and mutant integrase proteins, the linear phase of the joining reaction was preceded by what appears to be an exponential "burst" phase.After a retrovirus infects a cell, the reverse-transcribed DNA copy of the viral genome is integrated into the host DNA. This integration requires interaction between two retroviral components: the viral enzyme integrase (IN) and specific sequences located at the termini of the linear viral DNA. A number of studies have shown that integration of viral DNA into the host DNA depends on at least two temporally and biochemically distinguishable activities of IN, processing and joining (for review, see refs. 1-3).During processing, a site-specific endonuclease activity of IN removes 2 nt from each of the 3'-OH ends of the viral DNA, creating new CA-3'-OH ends and dinucleotides (4-7). In the joining reaction, the newly processed 3'-OH ends of the viral DNA are coordinately joined to 5'-phosphate ends produced by staggered cleavage of the host DNA by IN (6,[8][9][10].In vitro assays for the processing and joining activities of IN have been described (4, 9, 10). The substrates are short duplex oligodeoxynucleotides homologous to either end of the viral DNA. The processing activity generates products analogous to those produced in vivo: an oligonucleotideThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in acc...

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We have prepared and characterized several monoclonal antibodies (MAbs) against the Rous sarcoma virus integrase protein (IN) with the aim of employing these specific reagents as tools for biochemical and biophysical studies. The interaction of IN with the purified MAbs and their Fab fragment derivatives was demonstrated by Western blot (immunoblot), enzyme-linked immunosorbent assay, and size exclusion chromatography. A series of truncated IN proteins was used to determine regions in the protein important for recognition by the antibodies. The MAbs described here recognize epitopes that lie within the catalytic core region of IN (amino acids 50 to 207) and are likely to be conformational. A detailed functional analysis was carried out by investigating the effects of Fab fragments as well as of intact MAbs on the activities of IN in vitro. These studies revealed differential effects which fall into three categories. (i) One of the antibodies completely neutralized the processing as well as the joining activity and also reduced the DNA binding capacity as determined by a nitrocellulose filter binding assay. On the other hand, this MAb did not abolish the cleavage-ligation reaction on a disintegration substrate and the nonspecific cleavage of DNA by IN. The cleavage pattern generated by the IN-MAb complex on various DNA substrates closely resembled that produced by mutant IN proteins which show a deficiency in multimerization. Preincubation of IN with substrate protected the enzyme from inhibition by this antibody. (ii) Two other antibodies showed a general inhibition of all IN activities tested. (iii) In contrast, a fourth MAb stimulated the in vitro joining activity of IN. Size exclusion chromatography demonstrated that IN-Fab complexes from representatives of the three categories of MAbs exhibit different stoichiometric compositions that suggest possible explanations for their contrasting effects and may provide clues to the relationship between the structure and function of IN.

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