Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.
Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.
Agresta
B E
BE
Department of Molecular Genetics and Microbiology, S.U.N.Y. Stony Brook 11794, USA.
Carter
C A
CA
D000591:Amino Acid Isomerases / Q000502:physiology*; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology*; D018833:Chaperonins / Q000502:physiology*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D019696:Peptidylprolyl Isomerase; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D018833:Chaperonins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase