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. 2004 Jul 22;183(1):215–224. doi: 10.1016/0042-6822(91)90134-W

Expression and frameshifting but extremely inefficient proteolytic processing of the HIV-1 gag and pol gene products in stably transfected rodent cell lines

Dieter Moosmayer ∗,1, Heide Reil †, Martina Ausmeier †, Jens-Gerd Scharf ∗, Hansjorg Hauser †, Klaus Dieter Jentsch ∗, Gerhard Hunsmann ∗
PMCID: PMC7131782  PMID: 2053281

Abstract

Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.

References

  1. Artelt P., Morelle C., Ausmeier M., Fitzek M., Hauser H. Vectors for efficient expression in mammalian fibroblast, myeloid and lymphoid cells via transfection or infection. Gene. 1988;68:213–219. doi: 10.1016/0378-1119(88)90023-6. [DOI] [PubMed] [Google Scholar]
  2. Barr P.J., Power M.D., Lee-Ng C.T., Gibson H.L., Luclw P.A. Expression of active human immunodeficiency virus reverse transcriptase in Saccharomyces cerevisiae. Biotechnology. 1987;5:486–489. [Google Scholar]
  3. Brierley I., Boursnell M., Birns M., Bilmora B., Block V., Brown T., Inglis S. An efficient ribosomal frameshift signal in the polymerase-encoding region of the corona virus IBV. EMBO J. 1987;6:3779–3785. doi: 10.1002/j.1460-2075.1987.tb02713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colbère-Garapin F., Horodniceau F., Khourilsky P., Garapin A.C. A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Biol. 1981;150:1–13. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  5. Dayton A.I., Terwilliger E.F., Potz J., Kowalski M., Sodroski I.G., Haseltine W.A. Cis-acting sequences responsive to the rev gene product of the human immunodeficiency virus. J. Acquired Immune Defic. Syndr. 1988;1:441–452. [PubMed] [Google Scholar]
  6. Debouck C., Gorniak J., Strickler J.D., Meek T.D., Metcalf B.W., Rosenberg M. Vol. 84. 1987. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor; pp. 8903–8906. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. de Wet J., Wood K.V., Deluca M., Helinski D.R., Subramani S. Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell Biol. 1987;7:725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farmerie W.G., Loeb D.D., Casavant N.C., Hutchison C.A., Edgell M.H., Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in E. coli. Science. 1987;236:305–308. doi: 10.1126/science.2436298. [DOI] [PubMed] [Google Scholar]
  9. Feinberg B.K., Jarrett R.F., Aldovini A., Gallo R.C., Wongstaal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986;46:807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
  10. Felber B.K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G.N. Vol. 86. 1989. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA; pp. 1496–1499. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flexner C., Broyles S.S., Earl P., Chakrabarti S., Moss B. Characterization of human immunodeficiency virus gag/ pol gene products expressed by recombinant vaccinia viruses. Virology. 1988;166:339–349. doi: 10.1016/0042-6822(88)90504-1. [DOI] [PubMed] [Google Scholar]
  12. Gowda S.D., Stein B.S., Steimer K.S., Engleman E.G. Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus. J. Virol. 1989;63:1451–1453. doi: 10.1128/jvi.63.3.1451-1454.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graham F., Van Der Eb.K. A new technique for the assay of infectivity of adenovirus DNA. Virology. 1973;52:456–487. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  14. Hammarskjöld M.-L., Heimer J., Hammarskjöld B., Sangwan I., Albert L., Rekosh D. Regulation of human immunodeficiency virus env expression by the rev gene product. J. Virol. 1989;63:1959–1966. doi: 10.1128/jvi.63.5.1959-1966.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haseltine W.A. Replication and pathogenesis of the AIDS virus. AIDS. 1988;1:217–240. [PubMed] [Google Scholar]
  16. Jacks T., Madhani H.D., Masiarz F.R., Varmus H.E. Signals for ribosomal frameshift in the rous sarcoma virus gag-pol region. Cell. 1988;55:447–458. doi: 10.1016/0092-8674(88)90031-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacks T., Power M.D., Masiarz F.R., Luclw P.A., Barr P.J., Varmus H.E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988;331:280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
  18. Jacks T., Townsley K., Varmus H.E., Majors J. Vol. 84. 1987. Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins; pp. 4298–4302. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katoh I., Yasunaga T., Ikawa Y., Yoshinaka Y. Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor. Nature. 1987;329:654–656. doi: 10.1038/329654a0. [DOI] [PubMed] [Google Scholar]
  20. Kit S., Dubbs D.R., Piekarski L.J., Hsu T.C. Deletion of the thymidine kinase activity from L cells resistant to bromodeoxyuridine. Exp. Cell Res. 1963;31:297–312. doi: 10.1016/0014-4827(63)90007-7. [DOI] [PubMed] [Google Scholar]
  21. 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. Vol. 86. 1988. Active human immunodeficiency virus protease is required forviral infectivity; pp. 4686–4690. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kramer R.A., Schaber M.D., Skalka A.M., Ganguly K., Wongstaal F., Reddy E.D. HTLV III gag protein is processed in yeast cells by the virus poi protease. Science. 1986;231:1580–1584. doi: 10.1126/science.2420008. [DOI] [PubMed] [Google Scholar]
  23. Le Grice S.F.J., Beuck V., Mous J. Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis. Gene. 1987;55:95–103. doi: 10.1016/0378-1119(87)90252-6. [DOI] [PubMed] [Google Scholar]
  24. Le Grice S.F.J., Mills J., Mous J. Active site mutagenesis of the AIDS virus protease and its alleviation by trans-complementation. EMBO J. 1988;8:2547–2553. doi: 10.1002/j.1460-2075.1988.tb03103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leuthardt A., Le Grice S.F.J. Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli. Gene. 1988;68:35–42. doi: 10.1016/0378-1119(88)90596-3. [DOI] [PubMed] [Google Scholar]
  26. Lightfoote M.M., Coligan J.E., Folks T.M., Fauci A.S., Martin M.A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J. Virol. 1986;60:771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Madisen L., Travis B., Hu S.-L., Purchio A.F. Expression of the human immunodeficiency virus gag gene in insect cells. Virology. 1987;158:248–250. doi: 10.1016/0042-6822(87)90262-5. [DOI] [PubMed] [Google Scholar]
  28. Malim M.H., Hauser J., Fenrick R., Cullen B.R. Immunodeficiency virus rev trans-activator modulates expression of the viral regulatory genes. Nature. 1988;335:181–184. doi: 10.1038/335181a0. [DOI] [PubMed] [Google Scholar]
  29. Meek T.D., Dayton B.D., Metcalf B.W., Dreyer G.B., Strickler J.E., Gorniak J.G., Rosenberg M., Moore M.L., Magaard V.W., Debouck C. Vol. 86. 1989. Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease; pp. 1841–1845. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mellor J., Fulton A.M., Dobson M.J., Roberts N.A., Wilson W., Kingsman S.M., Kingsman A.J. A retrovirus-like strategy for expression of a fusion protein encoded by yeast transposon, Tyl. Nature. 1985;313:243–246. doi: 10.1038/313243a0. [DOI] [PubMed] [Google Scholar]
  31. Mervis R.I., Ahmad N., Lillehoi E.P., Raum M.G., Salazar H.R., Chan H.W., Venkatesan S. The gag gene products of human immunodeficiency virus type 1: Alignment within the gag open reading frame, identification of post-translational modifications, and evidence for alternative gag precursors. J. Virol. 1988;62:3993–4002. doi: 10.1128/jvi.62.11.3993-4002.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mous I., Heimer E.P., Le Grice S.F.J. Processing protease and reverse transcriptase from human immunodeficiency virus type I polyprotein in Escherichia coli. J. Virol. 1988;62:1433–1436. doi: 10.1128/jvi.62.4.1433-1436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Muesing M.A., Smith D.H., Cabradilla C.D., Benton C.V., Laskey L.A., Capon D.J. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature. 1985;313:450–458. doi: 10.1038/313450a0. [DOI] [PubMed] [Google Scholar]
  34. 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 I.P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989;337:615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
  35. Overton H., Fuj Y., Price I.R., Jones I.M. The protease and gag gene products of the human immunodeficiency virus: Authetic cleavage and post-translational modification in an insect cell expression system. Virology. 1989;176:107–116. doi: 10.1016/0042-6822(89)90357-7. [DOI] [PubMed] [Google Scholar]
  36. Peterlin B.M., Luclw P.A. Molecular biology of HIV. AIDS. 1988;2:29–40. doi: 10.1097/00002030-198800001-00005. [DOI] [PubMed] [Google Scholar]
  37. 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., Ivanoff L., Petteway S.R., Jr., Pearson M.L., Lautenberger J.A., Papas T.S., Ghrayeb J., Gallo R.C., Wong-Staal F. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985;313:277–283. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  38. Reil H., Hauser H. Test system for detection of HIV-1 frameshifting efficiency in animal cells. Biochim. Biophys. Acta. 1990;1050:288–292. doi: 10.1016/0167-4781(90)90183-3. [DOI] [PubMed] [Google Scholar]
  39. Robey W.G., Safai B., Oroszlan S., Arthur L.0., Gonda M.A., Gallo R.C., Fischinger P.I. Characterization of envelope and core structural gene products of HTLV-III with sera from AIDS patients. Science. 1985;228:593–595. doi: 10.1126/science.2984774. [DOI] [PubMed] [Google Scholar]
  40. Sanchez-Pescador R., Power M.D., Barr P.J., Steimer K.S., Stempien M.M., Brown-Shimer S.L., Gee W.W., Renard A., Randolph A., Levy J.A., Dina D., Luclw P.A. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2) Science. 1985;227:484–492. doi: 10.1126/science.2578227. [DOI] [PubMed] [Google Scholar]
  41. Schneider J., Jurkiewicz E., Wendler I., Jentsch K.D., Bayer H., Desrosiers R.C., Gelderblom H., Hunsmann G. Structural, biochemical and serological comparison of LAV/HTLVIII and STLV-Illmac to primate lentiviruses. In: Gallo R.C., Haseltine W., Klein G., zur Hausen H., editors. Viruses and Human Cancer. A. R. Liss; New York: 1986. pp. 319–332. [Google Scholar]
  42. Schneider J., Kent S. Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. Cell. 1988;54:363–368. doi: 10.1016/0092-8674(88)90199-7. [DOI] [PubMed] [Google Scholar]
  43. Seelmeier S., Schmidt H., Turk V., Von Der Helm K. Vol. 85. 1988. Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A; pp. 6612–6616. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shioda T., Shibuta H. Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV. Virology. 1990;175:139–148. doi: 10.1016/0042-6822(90)90194-v. [DOI] [PubMed] [Google Scholar]
  45. Smith A.J., Cho M.-L., Hammarskjöld M.-L., Rekosh D. Human immunodeficiency virus type 1 Pr55gag and P0 60gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles. J. Virol. 1990;64:2743–2750. doi: 10.1128/jvi.64.6.2743-2750.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sodroski J.G., Goh W.C., Rosen C.A., Dayton A., Terwillinger E., Haseltine W. A second post-transcriptional trans-activator gene required for HLTV-III replication. Nature. 1986;321:412–417. doi: 10.1038/321412a0. [DOI] [PubMed] [Google Scholar]
  47. Toh H., Ono M., Saigo K., Miyata T. Retroviral protease-like sequence in the yeast transposon Ty 1. Nature. 1985;315:691–692. [Google Scholar]
  48. Towbin H., Staehelin T., Gordon J. Vol. 76. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications; pp. 4350–4354. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vara J., Portela A., Ortin I., Jimenez A. Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucl Acids Res. 1986;14:4617–4624. doi: 10.1093/nar/14.11.4617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Veronese F.D., Copeland T.D., Oroszlan S., Gallo R.C., Sarngadharan M.G. Biochemical and immunological analysis of human immunodeficiency virus gag gene products pl7 and p24. J. Virol. 1988;62:795–801. doi: 10.1128/jvi.62.3.795-801.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wain-Hobson S., Sonigo P., Danos 0., Cole, Alizon S. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985;40:9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  52. Wigler M., Silverst S., Lee L.S., Pellicer A., Cheng Y.C., Axel R. Transfer of purified herpes-virus thymidine kinase gene to cultured mouse cells. Cell. 1977;11:223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]
  53. Wilson W., Braddock M., Adams S.E., Rathjen P.D., Kingsman S.M., Kingsman A.J. HIV expression strategies: Ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell. 1988;55:1159–1169. doi: 10.1016/0092-8674(88)90260-7. [DOI] [PubMed] [Google Scholar]
  54. Wirth M., Bode J., Zettlmeissl G., Hauser H. Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure. Gene. 1988;73:419–426. doi: 10.1016/0378-1119(88)90506-9. [DOI] [PubMed] [Google Scholar]

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