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. 1993 Dec;2(12):2167–2176. doi: 10.1002/pro.5560021216

Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease H as substrates of the viral protease.

A G Tomasselli 1, J L Sarcich 1, L J Barrett 1, I M Reardon 1, W J Howe 1, D B Evans 1, S K Sharma 1, R L Heinrikson 1
PMCID: PMC2142316  PMID: 7507754

Abstract

A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV-1) to digestion by the HIV-1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440-Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227-14232). The COOH-terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483-Leu484 and Tyr532-Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436-Ala437 and at Phe440-Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.

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Selected References

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  1. Ashorn P., McQuade T. J., Thaisrivongs S., Tomasselli A. G., Tarpley W. G., Moss B. An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7472–7476. doi: 10.1073/pnas.87.19.7472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chattopadhyay D., Evans D. B., Deibel M. R., Jr, Vosters A. F., Eckenrode F. M., Einspahr H. M., Hui J. O., Tomasselli A. G., Zurcher-Neely H. A., Heinrikson R. L. Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease. J Biol Chem. 1992 Jul 15;267(20):14227–14232. [PubMed] [Google Scholar]
  3. Chattopadhyay D., Finzel B. C., Munson S. H., Evans D. B., Sharma S. K., Strakalaitus N. A., Brunner D. P., Eckenrode F. M., Dauter Z., Betzel C. Crystallographic analyses of an active HIV-1 ribonuclease H domain show structural features that distinguish it from the inactive form. Acta Crystallogr D Biol Crystallogr. 1993 Jul 1;49(Pt 4):423–427. doi: 10.1107/S0907444993002409. [DOI] [PubMed] [Google Scholar]
  4. Chou K. C., Zhang C. T., Kézdy F. J. A vector projection approach to predicting HIV protease cleavage sites in proteins. Proteins. 1993 Jun;16(2):195–204. doi: 10.1002/prot.340160206. [DOI] [PubMed] [Google Scholar]
  5. Darke P. L., Nutt R. F., Brady S. F., Garsky V. M., Ciccarone T. M., Leu C. T., Lumma P. K., Freidinger R. M., Veber D. F., Sigal I. S. HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. Biochem Biophys Res Commun. 1988 Oct 14;156(1):297–303. doi: 10.1016/s0006-291x(88)80839-8. [DOI] [PubMed] [Google Scholar]
  6. Evans D. B., Brawn K., Deibel M. R., Jr, Tarpley W. G., Sharma S. K. A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active. J Biol Chem. 1991 Nov 5;266(31):20583–20585. [PubMed] [Google Scholar]
  7. Griffiths J. T., Phylip L. H., Konvalinka J., Strop P., Gustchina A., Wlodawer A., Davenport R. J., Briggs R., Dunn B. M., Kay J. Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites. Biochemistry. 1992 Jun 9;31(22):5193–5200. doi: 10.1021/bi00137a015. [DOI] [PubMed] [Google Scholar]
  8. Hellen C. U., Kräusslich H. G., Wimmer E. Proteolytic processing of polyproteins in the replication of RNA viruses. Biochemistry. 1989 Dec 26;28(26):9881–9890. doi: 10.1021/bi00452a001. [DOI] [PubMed] [Google Scholar]
  9. Henderson L. E., Benveniste R. E., Sowder R., Copeland T. D., Schultz A. M., Oroszlan S. Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne). J Virol. 1988 Aug;62(8):2587–2595. doi: 10.1128/jvi.62.8.2587-2595.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hostomska Z., Matthews D. A., Davies J. F., 2nd, Nodes B. R., Hostomsky Z. Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. J Biol Chem. 1991 Aug 5;266(22):14697–14702. [PubMed] [Google Scholar]
  11. Hui J. O., Tomasselli A. G., Zürcher-Neely H. A., Heinrikson R. L. Ribonuclease A as a substrate of the protease from human immunodeficiency virus-1. J Biol Chem. 1990 Dec 5;265(34):21386–21389. [PubMed] [Google Scholar]
  12. Katayanagi K., Miyagawa M., Matsushima M., Ishikawa M., Kanaya S., Ikehara M., Matsuzaki T., Morikawa K. Three-dimensional structure of ribonuclease H from E. coli. Nature. 1990 Sep 20;347(6290):306–309. doi: 10.1038/347306a0. [DOI] [PubMed] [Google Scholar]
  13. Kay J., Dunn B. M. Viral proteinases: weakness in strength. Biochim Biophys Acta. 1990 Jan 30;1048(1):1–18. doi: 10.1016/0167-4781(90)90015-t. [DOI] [PubMed] [Google Scholar]
  14. Kohl N. E., Emini E. A., Schleif W. A., Davis L. J., Heimbach J. C., Dixon R. A., Scolnick E. M., Sigal I. S. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686–4690. doi: 10.1073/pnas.85.13.4686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Oroszlan S., Luftig R. B. Retroviral proteinases. Curr Top Microbiol Immunol. 1990;157:153–185. doi: 10.1007/978-3-642-75218-6_6. [DOI] [PubMed] [Google Scholar]
  18. Oswald M., von der Helm K. Fibronectin is a non-viral substrate for the HIV proteinase. FEBS Lett. 1991 Nov 4;292(1-2):298–300. doi: 10.1016/0014-5793(91)80888-a. [DOI] [PubMed] [Google Scholar]
  19. Phylip L. H., Richards A. D., Kay J., Kovalinka J., Strop P., Blaha I., Velek J., Kostka V., Ritchie A. J., Broadhurst A. V. Hydrolysis of synthetic chromogenic substrates by HIV-1 and HIV-2 proteinases. Biochem Biophys Res Commun. 1990 Aug 31;171(1):439–444. doi: 10.1016/0006-291x(90)91412-l. [DOI] [PubMed] [Google Scholar]
  20. Poorman R. A., Tomasselli A. G., Heinrikson R. L., Kézdy F. J. A cumulative specificity model for proteases from human immunodeficiency virus types 1 and 2, inferred from statistical analysis of an extended substrate data base. J Biol Chem. 1991 Aug 5;266(22):14554–14561. [PubMed] [Google Scholar]
  21. Rivière Y., Blank V., Kourilsky P., Israël A. Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection. Nature. 1991 Apr 18;350(6319):625–626. doi: 10.1038/350625a0. [DOI] [PubMed] [Google Scholar]
  22. Rosé J. R., Salto R., Craik C. S. Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions. J Biol Chem. 1993 Jun 5;268(16):11939–11945. [PubMed] [Google Scholar]
  23. Tomasselli A. G., Howe W. J., Hui J. O., Sawyer T. K., Reardon I. M., DeCamp D. L., Craik C. S., Heinrikson R. L. Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2. Proteins. 1991;10(1):1–9. doi: 10.1002/prot.340100102. [DOI] [PubMed] [Google Scholar]
  24. Tomasselli A. G., Hui J. O., Adams L., Chosay J., Lowery D., Greenberg B., Yem A., Deibel M. R., Zürcher-Neely H., Heinrikson R. L. Actin, troponin C, Alzheimer amyloid precursor protein and pro-interleukin 1 beta as substrates of the protease from human immunodeficiency virus. J Biol Chem. 1991 Aug 5;266(22):14548–14553. [PubMed] [Google Scholar]
  25. Tomasselli A. G., Hui J. O., Sawyer T. K., Staples D. J., Bannow C., Reardon I. M., Howe W. J., DeCamp D. L., Craik C. S., Heinrikson R. L. Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2. J Biol Chem. 1990 Aug 25;265(24):14675–14683. [PubMed] [Google Scholar]
  26. Tomasselli A. G., Hui J. O., Sawyer T. K., Staples D. J., FitzGerald D. J., Chaudhary V. K., Pastan I., Heinrikson R. L. Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease. J Biol Chem. 1990 Jan 5;265(1):408–413. [PubMed] [Google Scholar]
  27. Tomasselli A. G., Olsen M. K., Hui J. O., Staples D. J., Sawyer T. K., Heinrikson R. L., Tomich C. S. Substrate analogue inhibition and active site titration of purified recombinant HIV-1 protease. Biochemistry. 1990 Jan 9;29(1):264–269. doi: 10.1021/bi00453a036. [DOI] [PubMed] [Google Scholar]
  28. Tomaszek T. A., Jr, Moore M. L., Strickler J. E., Sanchez R. L., Dixon J. S., Metcalf B. W., Hassell A., Dreyer G. B., Brooks I., Debouck C. Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease. Biochemistry. 1992 Oct 27;31(42):10153–10168. doi: 10.1021/bi00157a003. [DOI] [PubMed] [Google Scholar]
  29. Tözsér J., Bláha I., Copeland T. D., Wondrak E. M., Oroszlan S. Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag-Pol polyproteins. FEBS Lett. 1991 Apr 9;281(1-2):77–80. doi: 10.1016/0014-5793(91)80362-7. [DOI] [PubMed] [Google Scholar]
  30. Wlodawer A., Erickson J. W. Structure-based inhibitors of HIV-1 protease. Annu Rev Biochem. 1993;62:543–585. doi: 10.1146/annurev.bi.62.070193.002551. [DOI] [PubMed] [Google Scholar]
  31. Yang W., Hendrickson W. A., Crouch R. J., Satow Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein. Science. 1990 Sep 21;249(4975):1398–1405. doi: 10.1126/science.2169648. [DOI] [PubMed] [Google Scholar]
  32. di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [DOI] [PubMed] [Google Scholar]

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