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. 1997 Sep;71(9):6921–6927. doi: 10.1128/jvi.71.9.6921-6927.1997

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

B E Agresta 1, C A Carter 1
PMCID: PMC191975  PMID: 9261419

Abstract

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.

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Selected References

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  1. Aberham C., Weber S., Phares W. Spontaneous mutations in the human immunodeficiency virus type 1 gag gene that affect viral replication in the presence of cyclosporins. J Virol. 1996 Jun;70(6):3536–3544. doi: 10.1128/jvi.70.6.3536-3544.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker E. K., Colley N. J., Zuker C. S. The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J. 1994 Oct 17;13(20):4886–4895. doi: 10.1002/j.1460-2075.1994.tb06816.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Billich A., Hammerschmid F., Peichl P., Wenger R., Zenke G., Quesniaux V., Rosenwirth B. Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus (HIV) type 1: interference with HIV protein-cyclophilin A interactions. J Virol. 1995 Apr;69(4):2451–2461. doi: 10.1128/jvi.69.4.2451-2461.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braaten D., Aberham C., Franke E. K., Yin L., Phares W., Luban J. Cyclosporine A-resistant human immunodeficiency virus type 1 mutants demonstrate that Gag encodes the functional target of cyclophilin A. J Virol. 1996 Aug;70(8):5170–5176. doi: 10.1128/jvi.70.8.5170-5176.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braaten D., Franke E. K., Luban J. Cyclophilin A is required for an early step in the life cycle of human immunodeficiency virus type 1 before the initiation of reverse transcription. J Virol. 1996 Jun;70(6):3551–3560. doi: 10.1128/jvi.70.6.3551-3560.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dorfman T., Göttlinger H. G. The human immunodeficiency virus type 1 capsid p2 domain confers sensitivity to the cyclophilin-binding drug SDZ NIM 811. J Virol. 1996 Sep;70(9):5751–5757. doi: 10.1128/jvi.70.9.5751-5757.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ehrlich L. S., Agresta B. E., Carter C. A. Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro. J Virol. 1992 Aug;66(8):4874–4883. doi: 10.1128/jvi.66.8.4874-4883.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ehrlich L. S., Agresta B. E., Gelfand C. A., Jentoft J., Carter C. A. Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure. Virology. 1994 Nov 1;204(2):515–525. doi: 10.1006/viro.1994.1565. [DOI] [PubMed] [Google Scholar]
  9. Ehrlich L. S., Krausslich H. G., Wimmer E., Carter C. A. Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24). AIDS Res Hum Retroviruses. 1990 Oct;6(10):1169–1175. doi: 10.1089/aid.1990.6.1169. [DOI] [PubMed] [Google Scholar]
  10. Ferreira P. A., Nakayama T. A., Pak W. L., Travis G. H. Cyclophilin-related protein RanBP2 acts as chaperone for red/green opsin. Nature. 1996 Oct 17;383(6601):637–640. doi: 10.1038/383637a0. [DOI] [PubMed] [Google Scholar]
  11. Fischer G., Wittmann-Liebold B., Lang K., Kiefhaber T., Schmid F. X. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature. 1989 Feb 2;337(6206):476–478. doi: 10.1038/337476a0. [DOI] [PubMed] [Google Scholar]
  12. Franke E. K., Yuan H. E., Luban J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature. 1994 Nov 24;372(6504):359–362. doi: 10.1038/372359a0. [DOI] [PubMed] [Google Scholar]
  13. Freskgård P. O., Bergenhem N., Jonsson B. H., Svensson M., Carlsson U. Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase. Science. 1992 Oct 16;258(5081):466–468. doi: 10.1126/science.1357751. [DOI] [PubMed] [Google Scholar]
  14. Fruman D. A., Burakoff S. J., Bierer B. E. Immunophilins in protein folding and immunosuppression. FASEB J. 1994 Apr 1;8(6):391–400. doi: 10.1096/fasebj.8.6.7513288. [DOI] [PubMed] [Google Scholar]
  15. Gamble T. R., Vajdos F. F., Yoo S., Worthylake D. K., Houseweart M., Sundquist W. I., Hill C. P. Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell. 1996 Dec 27;87(7):1285–1294. doi: 10.1016/s0092-8674(00)81823-1. [DOI] [PubMed] [Google Scholar]
  16. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  17. Gitti R. K., Lee B. M., Walker J., Summers M. F., Yoo S., Sundquist W. I. Structure of the amino-terminal core domain of the HIV-1 capsid protein. Science. 1996 Jul 12;273(5272):231–235. doi: 10.1126/science.273.5272.231. [DOI] [PubMed] [Google Scholar]
  18. Hammerschmid F., Billich A., Wasserbauer E., Rosenwirth B. Interactions of HIV-1 proteins with human T-cell cyclophilin A. Ann N Y Acad Sci. 1996 May 15;782:456–461. doi: 10.1111/j.1749-6632.1996.tb40583.x. [DOI] [PubMed] [Google Scholar]
  19. Handschumacher R. E., Harding M. W., Rice J., Drugge R. J., Speicher D. W. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science. 1984 Nov 2;226(4674):544–547. doi: 10.1126/science.6238408. [DOI] [PubMed] [Google Scholar]
  20. Harrison R. K., Stein R. L. Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion. Biochemistry. 1990 Feb 20;29(7):1684–1689. doi: 10.1021/bi00459a003. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Luban J., Bossolt K. L., Franke E. K., Kalpana G. V., Goff S. P. Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell. 1993 Jun 18;73(6):1067–1078. doi: 10.1016/0092-8674(93)90637-6. [DOI] [PubMed] [Google Scholar]
  23. Matouschek A., Rospert S., Schmid K., Glick B. S., Schatz G. Cyclophilin catalyzes protein folding in yeast mitochondria. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6319–6323. doi: 10.1073/pnas.92.14.6319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Momany C., Kovari L. C., Prongay A. J., Keller W., Gitti R. K., Lee B. M., Gorbalenya A. E., Tong L., McClure J., Ehrlich L. S. Crystal structure of dimeric HIV-1 capsid protein. Nat Struct Biol. 1996 Sep;3(9):763–770. doi: 10.1038/nsb0996-763. [DOI] [PubMed] [Google Scholar]
  25. Ott D. E., Coren L. V., Johnson D. G., Sowder R. C., 2nd, Arthur L. O., Henderson L. E. Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain. AIDS Res Hum Retroviruses. 1995 Sep;11(9):1003–1006. doi: 10.1089/aid.1995.11.1003. [DOI] [PubMed] [Google Scholar]
  26. Rassow J., Mohrs K., Koidl S., Barthelmess I. B., Pfanner N., Tropschug M. Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60. Mol Cell Biol. 1995 May;15(5):2654–2662. doi: 10.1128/mcb.15.5.2654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rosé S., Hensley P., O'Shannessy D. J., Culp J., Debouck C., Chaiken I. Characterization of HIV-1 p24 self-association using analytical affinity chromatography. Proteins. 1992 Apr;13(2):112–119. doi: 10.1002/prot.340130204. [DOI] [PubMed] [Google Scholar]
  28. Schönbrunner E. R., Mayer S., Tropschug M., Fischer G., Takahashi N., Schmid F. X. Catalysis of protein folding by cyclophilins from different species. J Biol Chem. 1991 Feb 25;266(6):3630–3635. [PubMed] [Google Scholar]
  29. Stamnes M. A., Rutherford S. L., Zuker C. S. Cyclophilins: a new family of proteins involved in intracellular folding. Trends Cell Biol. 1992 Sep;2(9):272–276. doi: 10.1016/0962-8924(92)90200-7. [DOI] [PubMed] [Google Scholar]
  30. Steinkasserer A., Harrison R., Billich A., Hammerschmid F., Werner G., Wolff B., Peichl P., Palfi G., Schnitzel W., Mlynar E. Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus type 1 (HIV-1): interference with early and late events in HIV-1 replication. J Virol. 1995 Feb;69(2):814–824. doi: 10.1128/jvi.69.2.814-824.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steinmann B., Bruckner P., Superti-Furga A. Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cis-trans-isomerase. J Biol Chem. 1991 Jan 15;266(2):1299–1303. [PubMed] [Google Scholar]
  32. Thali M., Bukovsky A., Kondo E., Rosenwirth B., Walsh C. T., Sodroski J., Göttlinger H. G. Functional association of cyclophilin A with HIV-1 virions. Nature. 1994 Nov 24;372(6504):363–365. doi: 10.1038/372363a0. [DOI] [PubMed] [Google Scholar]
  33. Walsh C. T., Zydowsky L. D., McKeon F. D. Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction. J Biol Chem. 1992 Jul 5;267(19):13115–13118. [PubMed] [Google Scholar]

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