1887

Journal of General Virology header logo

Volume 76, Issue 8

Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production Free

Abstract

The retrovirus protease (PR), an aspartic PR, is composed of two identical subunits, each containing a conserved tripeptide sequence present at the active site of the enzyme. Asp-Ser-Gly is found in avian sarcoma leukaemia viruses (ASLV) and Asp-Thr-Gly in mammalian oncoretroviruses. We have mutated the conserved sequence at the active site of ASLV PR by converting the Ser and Gly residues to Thr and Ala, respectively. Replacement of Gly with Ala yielded an ASLV PR devoid of proteolytic activity. The Ser to Thr conversion did not alter the substrate specificity of the enzyme. Both wild-type and mutated PRs correctly cleaved viral precursors expressed in bacterial cells, as well as synthetic peptides homologous to ASLV and human immunodeficiency virus type 1 cleavage sites. Bacterially produced ASLV PR with Thr instead of Ser had increased enzymatic activity, as shown by hydrolysis of synthetic peptides. However, this mutation reduced the production of reverse transcriptase-containing particles and infectious virus following transfection of permissive cells with virus DNA.

  • Received:
  • Accepted:
  • Published Online:
© Journal of General Virology 1995
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-76-8-1917
1995-08-01
2023-05-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/76/8/JV0760081917.html?itemId=/content/journal/jgv/10.1099/0022-1317-76-8-1917&mimeType=html&fmt=ahah

References

  1. Burstein H., Bizub D., Skalka A. M. 1991; Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers. Journal of Virology 65:6165–6172
     [Google Scholar]
  2. Casadaban M. J., Cohen S. N. 1980; Analysis of gene control signals by DNA fusion and cloning in E.coli . Journal of Molecular Biology 138:179–207
     [Google Scholar]
  3. Coffin J. M. 1990; Retroviridae and their replication. In Virology vol 2 pp 1437–1500 Edited by Fields B. N., Knipe D. M. New York: Raven Press;
     [Google Scholar]
  4. Copeland T. D., Oroszlan S. 1988; Genetic locus, primary structure, and chemical synthesis of human immunodeficiency virus protease. Gene Analysis Techniques 5:109–115
     [Google Scholar]
  5. Crawford S., Goff S. P. 1985; A deletion mutation in the 5′ part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. Journal of Virology 53:899–907
     [Google Scholar]
  6. Crowl R., Seamans C., Lomedico P., McAndrew S. 1985; Versatile expression vector for high-level synthesis of cloned gene product in Escherichia coli . Gene 38:31–38
     [Google Scholar]
  7. Debouck C., Gormiak J. G., Strickler J. E., Meek T. D., Metcalf B. W., Rosenberg M. 1987; Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proceedings of the National Academy of Sciences, USA 84:8903–8904
     [Google Scholar]
  8. Demetrios A. S., Welkie N. M. 1984; Expression of exogenous DNA in mammalian cells. In Transcription and Translation pp 1–15 Edited by Hames B. D., Higgins S. J. Boca Raton: IRL Press;
     [Google Scholar]
  9. Dittmar M. J., Moelling K. 1978; Biochemical properties of pl5–associated protease in an avian RNA tumor virus. Journal of Virology 28:106–118
     [Google Scholar]
  10. Goff S., Traktman P., Baltimore D. 1981; Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. Journal of Virology 38:239–248
     [Google Scholar]
  11. Grinde B., Cameron C. E., Leis J., Weber I. T., Wlodaver A., Burstein H., Bizub D., Skalka A. M. 1992; Mutations that alter the activity of the Rous sarcoma virus protease. Journal of Biological Chemistry 267:9481–9490
     [Google Scholar]
  12. Harlow E., Lane D. 1988 Antibodies: A Laboratory Manual pp 53–138 New York: Cold Spring Harbor Laboratory;
     [Google Scholar]
  13. Hughes S., Kosik E. 1984; Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors. Virology 136:89–99
     [Google Scholar]
  14. Katoh I., Yoshinaka Y., Rein A., Shibuya M., Okada T., Oroszlan S. 1985; Murine leukemia virus maturation: protease region is required for conversion from ‘immature’ to ‘mature’ core form and for virus infectivity. Virology 145:280–292
     [Google Scholar]
  15. Kohl N. E., Emini E. A., Schleif W. A., Davis L. J., Heimbach J. C., Dixon R. A. F., Scolnick E. M., Sigal I. S. 1988; Active human immunodeficiency virus protease is required for viral infectivity. Proceedings of the National Academy of Sciences, USA 85:4686–4690
     [Google Scholar]
  16. Kotler M., Katz R. A., Skalka A. M. 1988a; Activity of avian retrovirus protease expressed in Escherichia coli . Journal of Virology 62:2696–2700
     [Google Scholar]
  17. Kotler M., Katz R. A., Danho W., Leis J., Skalka A. M. 1988b; Synthetic peptides as substrates and inhibitors of a retroviral protease. Proceedings of the National Academy of Sciences, USA 85:4185–4189
     [Google Scholar]
  18. Kotler M., Danho W., Katz R. A., Lies J., Skalka A. M. 1989; Avian retrovirus protease and cellular aspartic protease are distinguished by activities on peptide substrates. Journal of Biological Chemistry 264:3428–3435
     [Google Scholar]
  19. Kramer R. A., Schaber M. D., Skalka A. M., Ganguly K., Wong-Staal F., Reddy E. P. 1986; HTLV III gag protein is processed in yeast cells by the virus pol-protease. Science 231:1580–1584
     [Google Scholar]
  20. Krausslich H.-G., Wimmer E. 1988; Viral proteinases. Annual Review of Biochemistry 57:701–754
     [Google Scholar]
  21. Krausslich H.-G. 1991; Human immunodeficiency virus proteinase dimer as a component of the viral polyprotein prevents particle assembly and viral infectivity. Proceedings of the National Academy of Sciences, USA 88:3213–3217
     [Google Scholar]
  22. Le Grice S. F. J., Mills J., Mous J. 1988; Active site mutagenesis of the AIDS virus protease and its alleviation by transcomplementation. EMBO Journal 7:2547–2553
     [Google Scholar]
  23. Leider J. M., Palese P., Smith F. L. 1988; Determination of the mutation rate of a retrovirus. Journal of Virology 62:3084–3091
     [Google Scholar]
  24. Lu A. H., Soong M. M., Wong P. K. Y. 1979; Maturation of Moloney murine leukemia virus. Virology 93:269–274
     [Google Scholar]
  25. Miller R. H. 1987; Proteolytic self-cleavage of hepatitis B virus core proteiu may generate serum antigen. Science 236:722–725
     [Google Scholar]
  26. Moelling K., Scott A., Dittmar K. E. J., Owada M. 1980; Effect of p 16-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors. Journal of Virology 33:680–688
     [Google Scholar]
  27. Morinaga Y., Franceschini T., Inouye S., Inouye M. 1984; Improvement of oligonucleotide-directed mutagenesis using doublestranded plasmid DNA. Bio/Technology 2:636–639
     [Google Scholar]
  28. Moscovici C., Moscovici M., Jimenez H., Lai M. M. C., Hayman M. J., Vogt P. K. 1977; Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell 11:95–103
     [Google Scholar]
  29. Mous J., Heimer E. P., Le Grice S. F. J. 1988; Processing protease and reverse transcriptase from human immunodeficiency virus type-1 polyprotein in Escherichia coli . Journal of Virology 62:1433–1436
     [Google Scholar]
  30. Oroszlan S., Luftig R. B. 1990; Retroviral proteinases. Current Topics in Microbiology 157:153–185
     [Google Scholar]
  31. Pearl L. H., Taylor W. R. 1987; A structural model for the retroviral protease. Nature 329:351–354
     [Google Scholar]
  32. Pepinski R. B. 1983; Localization of lipid-protein and protein-protein interaction within the murine retrovirus gag precursor by a novel peptide-mapping technique. Journal of Biological Chemistry 258:11229–11235
     [Google Scholar]
  33. Resnick-Roguel N., Burstein H., Hamburger J., Panet A., Eldor A., Vlodavsky I., Kotler M. 1989; Cytocidal effect caused by the envelope glycoprotein of a newly isolated avian hemangioma-inducing retrovirus. Journal of Virology 63:4325–1330
     [Google Scholar]
  34. Sedlacek J., Fabry M., Coward J. E., Horeisi M., Strop P., Luftig R. B. 1993; Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus. Virology 192:667–672
     [Google Scholar]
  35. Schwartz D. E., Tizard R., Gilbert W. 1983; Nucleotide sequence of Rous sarcoma virus. Cell 32:853–869
     [Google Scholar]
  36. Toh H., Ono M., Saigo K., Miyata T. 1985; Retroviral proteaselike sequences in the yeast transposon Ty-1. Nature 315:691
     [Google Scholar]
  37. Tomasseli A. G., Howe W. J., Sawyer T. K., Wlodawer A. 1991; The complexities of AIDS: an assessment of the HIV protease as a therapeutic target. International Journal of Biochemistry and Biotechnology 4:7–27
     [Google Scholar]
  38. Tozser J., Blaha I., Copeland T. D., Wondrak E. M., Oroszlan S. 1991; Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag–Pol polyproteins. FEBS Letters 281:77–80
     [Google Scholar]
  39. Tozser J., Weber I. T., Gustchina A., Blaha I., Copeland T. D., Louis J. M., Oroszlan S. 1992; Kinetic and modeling studies of S3-S3′ subsides of HIV proteinases. Biochemistry 31:4793–4800
     [Google Scholar]
  40. Vogt V. M., Wight W., Eisenman R. 1979; In vitro cleavage of avian retrovirus gag proteins by viral protease pi 5. Virology 98:154–167
     [Google Scholar]
  41. Von Der Helm K. 1977; Cleavage of Rous sarcoma viral polypeptide precursor into internal structure proteins in vitro involves viral protein pl5. Proceedings of the National Academy of Sciences, USA 74:911–915
     [Google Scholar]
  42. Wlodawer A., Miller M., Jaskolski M., Sathyanarayana B. K., Baldwin E., Weber I., Selk L. M., Clawson L., Schneider J., Kent S. B. H. 1989; Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 245:616–621
     [Google Scholar]
  43. Yoshinaka Y., Luftig R. B. 1977; Properties of a p70 proteolytic factor of murine leukemia viruses. Cell 12:709–719
     [Google Scholar]
  44. Yoshinaka Y., Luftig R. B. 1981; A comparison of avian and murine retrovirus polyprotein cleavage activities. Virology 111:239–250
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-76-8-1917
Loading
Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production
J. Gen. Virol. 76, 1917 (1995); https://doi.org/10.1099/0022-1317-76-8-1917
/content/journal/jgv/10.1099/0022-1317-76-8-1917
/content/journal/jgv/10.1099/0022-1317-76-8-1917
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error