Abstract
Proteolytic processing of the gag/pol precursor by the human immunodeficiency virus type 1 proteinase is essential for the production of infectious viral particles. Although the sites of virus-specific cleavages have been determined, the primary amino acid sequences surrounding these sites are heterogeneous and the determinants that direct the cleavage specificity exhibited by human immunodeficiency virus type 1 proteinase remain largely undefined. We performed mutational analysis of the Tyr/Pro site, which produces the amino terminus of the viral capsid protein, and the Phe/Pro site, which produces the amino terminus of the proteinase. Mutations were made in a clone encoding a frameshift mutation that results in the expression of equimolar amounts of the substrate and proteinase in the form of a truncated gag/pol precursor. After single-amino-acid substitutions were made, their effects on proteolytic processing were examined by in vitro transcription and in vitro translation of the synthetic mRNA; translation products were then processed by exogenously added purified proteinase. Single-amino-acid substitutions yielded both substrates which were processed with wild-type efficiency and substrates on which processing was impaired. At the Tyr/Pro site in gag, processing was severely inhibited by substitutions within the P4, P2, P1, and P2' positions. The Phe/Pro site in pol, however, demonstrated far greater tolerance to amino acid substitution. These data suggest that the primary amino acid sequence around a scissile bond is more critical for cleavage of the Tyr/Pro site than the Phe/Pro site.
Full text
PDF![3938](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/a6a08c4fc90b/jvirol00063-0386.png)
![3939](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/817aff561a2c/jvirol00063-0387.png)
![3940](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/b6b777b68992/jvirol00063-0388.png)
![3941](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/eb72910b1083/jvirol00063-0389.png)
![3942](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/42bfe9a0b1ab/jvirol00063-0390.png)
![3943](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/2c71dd071842/jvirol00063-0391.png)
![3944](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/342801639d22/jvirol00063-0392.png)
![3945](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/ddbd30d6ffbe/jvirol00063-0393.png)
![3946](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/702b21f7c482/jvirol00063-0394.png)
![3947](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fda6/249689/b857c4c55442/jvirol00063-0395.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Billich S., Knoop M. T., Hansen J., Strop P., Sedlacek J., Mertz R., Moelling K. Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease. J Biol Chem. 1988 Dec 5;263(34):17905–17908. [PubMed] [Google Scholar]
- Blundell T., Pearl L. Retroviral proteinases. A second front against AIDS. Nature. 1989 Feb 16;337(6208):596–597. doi: 10.1038/337596a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Dreyer G. B., Metcalf B. W., Tomaszek T. A., Jr, Carr T. J., Chandler A. C., 3rd, Hyland L., Fakhoury S. A., Magaard V. W., Moore M. L., Strickler J. E. Inhibition of human immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9752–9756. doi: 10.1073/pnas.86.24.9752. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Göttlinger H. G., Sodroski J. G., Haseltine W. A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5781–5785. doi: 10.1073/pnas.86.15.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Jacks T., Power M. D., Masiarz F. R., Luciw P. A., Barr P. J., Varmus H. E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988 Jan 21;331(6153):280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
- Katoh I., Yasunaga T., Ikawa Y., Yoshinaka Y. Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor. Nature. 1987 Oct 15;329(6140):654–656. doi: 10.1038/329654a0. [DOI] [PubMed] [Google Scholar]
- Katoh I., Yoshinaka Y., Rein A., Shibuya M., Odaka T., Oroszlan S. Murine leukemia virus maturation: protease region required for conversion from "immature" to "mature" core form and for virus infectivity. Virology. 1985 Sep;145(2):280–292. doi: 10.1016/0042-6822(85)90161-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Kotler M., Katz R. A., Danho W., Leis J., Skalka A. M. Synthetic peptides as substrates and inhibitors of a retroviral protease. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4185–4189. doi: 10.1073/pnas.85.12.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kräusslich H. G., Ingraham R. H., Skoog M. T., Wimmer E., Pallai P. V., Carter C. A. Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. Proc Natl Acad Sci U S A. 1989 Feb;86(3):807–811. doi: 10.1073/pnas.86.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kräusslich H. G., Schneider H., Zybarth G., Carter C. A., Wimmer E. Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli. J Virol. 1988 Nov;62(11):4393–4397. doi: 10.1128/jvi.62.11.4393-4397.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Le Grice S. F., Mills J., Mous J. Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation. EMBO J. 1988 Aug;7(8):2547–2553. doi: 10.1002/j.1460-2075.1988.tb03103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leis J., Baltimore D., Bishop J. M., Coffin J., Fleissner E., Goff S. P., Oroszlan S., Robinson H., Skalka A. M., Temin H. M. Standardized and simplified nomenclature for proteins common to all retroviruses. J Virol. 1988 May;62(5):1808–1809. doi: 10.1128/jvi.62.5.1808-1809.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeb D. D., Hutchison C. A., 3rd, Edgell M. H., Farmerie W. G., Swanstrom R. Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases. J Virol. 1989 Jan;63(1):111–121. doi: 10.1128/jvi.63.1.111-121.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meek T. D., Lambert D. M., Dreyer G. B., Carr T. J., Tomaszek T. A., Jr, Moore M. L., Strickler J. E., Debouck C., Hyland L. J., Matthews T. J. Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. Nature. 1990 Jan 4;343(6253):90–92. doi: 10.1038/343090a0. [DOI] [PubMed] [Google Scholar]
- Mervis R. J., Ahmad N., Lillehoj E. P., Raum M. G., Salazar F. H., Chan H. W., Venkatesan S. The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors. J Virol. 1988 Nov;62(11):3993–4002. doi: 10.1128/jvi.62.11.3993-4002.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller M., Jaskólski M., Rao J. K., Leis J., Wlodawer A. Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature. 1989 Feb 9;337(6207):576–579. doi: 10.1038/337576a0. [DOI] [PubMed] [Google Scholar]
- Mous J., Heimer E. P., Le Grice S. F. Processing protease and reverse transcriptase from human immunodeficiency virus type I polyprotein in Escherichia coli. J Virol. 1988 Apr;62(4):1433–1436. doi: 10.1128/jvi.62.4.1433-1436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Navia M. A., Fitzgerald P. M., McKeever B. M., Leu C. T., Heimbach J. C., Herber W. K., Sigal I. S., Darke P. L., Springer J. P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989 Feb 16;337(6208):615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
- Oroszlan S., Copeland T. D. Primary structure and processing of gag and env gene products of human T-cell leukemia viruses HTLV-ICR and HTLV-IATK. Curr Top Microbiol Immunol. 1985;115:221–233. doi: 10.1007/978-3-642-70113-9_14. [DOI] [PubMed] [Google Scholar]
- Pearl L. H., Taylor W. R. A structural model for the retroviral proteases. Nature. 1987 Sep 24;329(6137):351–354. doi: 10.1038/329351a0. [DOI] [PubMed] [Google Scholar]
- Pearl L. H., Taylor W. R. Sequence specificity of retroviral proteases. Nature. 1987 Aug 6;328(6130):482–482. doi: 10.1038/328482b0. [DOI] [PubMed] [Google Scholar]
- Peng C., Ho B. K., Chang T. W., Chang N. T. Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. J Virol. 1989 Jun;63(6):2550–2556. doi: 10.1128/jvi.63.6.2550-2556.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
- Richards A. D., Roberts R., Dunn B. M., Graves M. C., Kay J. Effective blocking of HIV-1 proteinase activity by characteristic inhibitors of aspartic proteinases. FEBS Lett. 1989 Apr 10;247(1):113–117. doi: 10.1016/0014-5793(89)81251-7. [DOI] [PubMed] [Google Scholar]
- Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
- Seelmeier S., Schmidt H., Turk V., von der Helm K. Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6612–6616. doi: 10.1073/pnas.85.18.6612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tang J., James M. N., Hsu I. N., Jenkins J. A., Blundell T. L. Structural evidence for gene duplication in the evolution of the acid proteases. Nature. 1978 Feb 16;271(5646):618–621. doi: 10.1038/271618a0. [DOI] [PubMed] [Google Scholar]
- Tang J., Wong R. N. Evolution in the structure and function of aspartic proteases. J Cell Biochem. 1987 Jan;33(1):53–63. doi: 10.1002/jcb.240330106. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Weber I. T., Miller M., Jaskólski M., Leis J., Skalka A. M., Wlodawer A. Molecular modeling of the HIV-1 protease and its substrate binding site. Science. 1989 Feb 17;243(4893):928–931. doi: 10.1126/science.2537531. [DOI] [PubMed] [Google Scholar]
- Wlodawer A., Miller M., Jaskólski M., Sathyanarayana B. K., Baldwin E., Weber I. T., Selk L. M., Clawson L., Schneider J., Kent S. B. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. doi: 10.1126/science.2548279. [DOI] [PubMed] [Google Scholar]