Monoclonal antibodies against Rous sarcoma virus integrase protein exert differential effects on integrase function in vitro

Abstract

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.

Conformational aspects of HIV‐1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide

“…DNA concentration was one‐eighth that of IN as in the above biological assays. At this ratio the whole amount of IN is supposed to be fixed to the DNA as shown in previous experiments [48]. The whole mixture was filtered on a nitrocellulose membrane.…”

“…Interestingly, similar results have been obtained by Müller et al . (1995) [48] by size exclusion chromatography on a complex formed between IN and a selected Fab fragment that inhibits the binding of IN to DNA. Since anti‐K159 reacts with a distinct epitope on each molecule, it can be deduced that in the conditions selected for filter‐binding assays IN is a homodimer.…”