SPECTRAL-ANALYSIS AND TRYPTIC SUSCEPTIBILITY AS PROBES OF HIV-1 CAPSID PROTEIN-STRUCTURE

The structures of HIV-1 capsid protein (CA, p24) isolated from mature virions and CA protein autoprocessed from a recombinant Gag-Pol precur sor expressed in Escherichia coil were compared using circular dichroi c (CD) spectral analysis. The spectra obtained for the intact recombin ant and viral proteins were indistinguishable, indicating that the bac kbone configurations directed by the primary amino acid sequences of t he proteins were similar or identical. The structure predictions deriv ed from CD were, in general, inconsistent with a model proposing the e ight-stranded beta barrel motif found in several RNA viruses. However, aspects of the model were supported by experiments that identified su rface-exposed regions. Biochemical analysis indicated that the recombi nant CA protein formed nonrandom higher-ordered structures in vitro. U nder physiological conditions, the protein assembled into oligomers co ntaining subunits in two packing arrangements. In one arrangement, the central region near Arg100 was exposed and susceptible to tryptic dig estion at low enzyme concentrations (enzyme:substrate ratios = 1:5000 to 1:100). Also, in this arrangement, the proteins were susceptible to crosslinking by the bifunctional agent DTSSP. Proteins in the other a rrangement were resistant to proteolysis at low enzyme concentrations. The central region of these resistant molecules was inaccessible to m onospecific antibodies that recognized antigenic sites between residue s 94 and 107 and these proteins were not crosslinked by DTSSP or EGS. Following incubation with trypsin, both the resistant molecules and th e fragments derived from the susceptible proteins in the oligomer migr ated as smaller complexes, suggesting that the regions digested by try psin stabilized the oligomer unit. The results indicate that the centr al region of the HIV-1 CA protein plays a role in formation of higher- ordered structures. Moreover, the relative stability of the N- and C-t erminal partial digestion fragments arising from cleavage at Arg100/Gl y101 suggests that this exposed central region separates two structura l domains of the protein. (C) 1994 Academic Press, Inc.