Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1990 Jun;64(6):2519–2529. doi: 10.1128/jvi.64.6.2519-2529.1990

Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1.

S Schwartz 1, B K Felber 1, D M Benko 1, E M Fenyö 1, G N Pavlakis 1
PMCID: PMC249427  PMID: 2335812

Abstract

We have used the polymerase chain reaction technique to clone the small multiply spliced mRNA species produced after infection of human cells by a molecular clone of human immunodeficiency virus type 1 (HIV-1). We identified six Rev-expressing mRNAs, which were generated by the use of two splice acceptors located immediately upstream of the rev AUG. The class of small mRNAs included 12 mRNAs expressing Tat, Rev, and Nef. In addition, HIV-1 produced other multiply spliced mRNAs that used alternative splice sites identified by cloning and sequencing. All of these mRNAs were found in the cytoplasm and should be able to produce additional proteins. The coding capacity of the tat, rev, and nef mRNAs was analyzed by transfection of the cloned cDNAs into human cells. The tat mRNAs produced high levels of Tat, but very low levels of Rev and Nef. All the rev mRNAs expressed high levels of both Rev and Nef and were essential for the production of sufficient amounts of Rev. Therefore, HIV-1 uses both alternatively spliced and bicistronic mRNAs for the production of Tat, Rev, and Nef proteins.

Full text

PDF
2519

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmad N., Venkatesan S. Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science. 1988 Sep 16;241(4872):1481–1485. doi: 10.1126/science.3262235. [DOI] [PubMed] [Google Scholar]
  3. Arya S. K., Beaver B., Jagodzinski L., Ensoli B., Kanki P. J., Albert J., Fenyo E. M., Biberfeld G., Zagury J. F., Laure F. New human and simian HIV-related retroviruses possess functional transactivator (tat) gene. Nature. 1987 Aug 6;328(6130):548–550. doi: 10.1038/328548a0. [DOI] [PubMed] [Google Scholar]
  4. Arya S. K., Gallo R. C. Three novel genes of human T-lymphotropic virus type III: immune reactivity of their products with sera from acquired immune deficiency syndrome patients. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2209–2213. doi: 10.1073/pnas.83.7.2209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arya S. K., Guo C., Josephs S. F., Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science. 1985 Jul 5;229(4708):69–73. doi: 10.1126/science.2990040. [DOI] [PubMed] [Google Scholar]
  6. Barré-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., Vézinet-Brun F., Rouzioux C. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):868–871. doi: 10.1126/science.6189183. [DOI] [PubMed] [Google Scholar]
  7. Bilsel P. A., Rowe J. E., Fitch W. M., Nichol S. T. Phosphoprotein and nucleocapsid protein evolution of vesicular stomatitis virus New Jersey. J Virol. 1990 Jun;64(6):2498–2504. doi: 10.1128/jvi.64.6.2498-2504.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  9. Colombini S., Arya S. K., Reitz M. S., Jagodzinski L., Beaver B., Wong-Staal F. Structure of simian immunodeficiency virus regulatory genes. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4813–4817. doi: 10.1073/pnas.86.13.4813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cullen B. R. Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell. 1986 Sep 26;46(7):973–982. doi: 10.1016/0092-8674(86)90696-3. [DOI] [PubMed] [Google Scholar]
  11. Davis J. L., Clements J. E. Characterization of a cDNA clone encoding the visna virus transactivating protein. Proc Natl Acad Sci U S A. 1989 Jan;86(2):414–418. doi: 10.1073/pnas.86.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dayton A. I., Sodroski J. G., Rosen C. A., Goh W. C., Haseltine W. A. The trans-activator gene of the human T cell lymphotropic virus type III is required for replication. Cell. 1986 Mar 28;44(6):941–947. doi: 10.1016/0092-8674(86)90017-6. [DOI] [PubMed] [Google Scholar]
  13. Dayton A. I., Terwilliger E. F., Potz J., Kowalski M., Sodroski J. G., Haseltine W. A. Cis-acting sequences responsive to the rev gene product of the human immunodeficiency virus. J Acquir Immune Defic Syndr. 1988;1(5):441–452. [PubMed] [Google Scholar]
  14. Dorn P. L., Derse D. cis- and trans-acting regulation of gene expression of equine infectious anemia virus. J Virol. 1988 Sep;62(9):3522–3526. doi: 10.1128/jvi.62.9.3522-3526.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emerman M., Guyader M., Montagnier L., Baltimore D., Muesing M. A. The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1. EMBO J. 1987 Dec 1;6(12):3755–3760. doi: 10.1002/j.1460-2075.1987.tb02710.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Emerman M., Vazeux R., Peden K. The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell. 1989 Jun 30;57(7):1155–1165. doi: 10.1016/0092-8674(89)90053-6. [DOI] [PubMed] [Google Scholar]
  17. Feinberg M. B., Jarrett R. F., Aldovini A., Gallo R. C., Wong-Staal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986 Sep 12;46(6):807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
  18. Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Felber B. K., Paskalis H., Kleinman-Ewing C., Wong-Staal F., Pavlakis G. N. The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats. Science. 1985 Aug 16;229(4714):675–679. doi: 10.1126/science.2992082. [DOI] [PubMed] [Google Scholar]
  20. Fisher A. G., Feinberg M. B., Josephs S. F., Harper M. E., Marselle L. M., Reyes G., Gonda M. A., Aldovini A., Debouk C., Gallo R. C. The trans-activator gene of HTLV-III is essential for virus replication. 1986 Mar 27-Apr 2Nature. 320(6060):367–371. doi: 10.1038/320367a0. [DOI] [PubMed] [Google Scholar]
  21. Gallo R. C., Salahuddin S. Z., Popovic M., Shearer G. M., Kaplan M., Haynes B. F., Palker T. J., Redfield R., Oleske J., Safai B. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science. 1984 May 4;224(4648):500–503. doi: 10.1126/science.6200936. [DOI] [PubMed] [Google Scholar]
  22. Gonda M. A., Wong-Staal F., Gallo R. C., Clements J. E., Narayan O., Gilden R. V. Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus. Science. 1985 Jan 11;227(4683):173–177. doi: 10.1126/science.2981428. [DOI] [PubMed] [Google Scholar]
  23. Gourdou I., Mazarin V., Quérat G., Sauze N., Vigne R. The open reading frame S of visna virus genome is a trans-activating gene. Virology. 1989 Jul;171(1):170–178. doi: 10.1016/0042-6822(89)90524-2. [DOI] [PubMed] [Google Scholar]
  24. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  25. Guy B., Kieny M. P., Riviere Y., Le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature. 1987 Nov 19;330(6145):266–269. doi: 10.1038/330266a0. [DOI] [PubMed] [Google Scholar]
  26. Guyader M., Emerman M., Sonigo P., Clavel F., Montagnier L., Alizon M. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature. 1987 Apr 16;326(6114):662–669. doi: 10.1038/326662a0. [DOI] [PubMed] [Google Scholar]
  27. Hadzopoulou-Cladaras M., Felber B. K., Cladaras C., Athanassopoulos A., Tse A., Pavlakis G. N. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J Virol. 1989 Mar;63(3):1265–1274. doi: 10.1128/jvi.63.3.1265-1274.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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 May;63(5):1959–1966. doi: 10.1128/jvi.63.5.1959-1966.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hammes S. R., Dixon E. P., Malim M. H., Cullen B. R., Greene W. C. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9549–9553. doi: 10.1073/pnas.86.23.9549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hauber J., Perkins A., Heimer E. P., Cullen B. R. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6364–6368. doi: 10.1073/pnas.84.18.6364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hess J. L., Clements J. E., Narayan O. cis- and trans-acting transcriptional regulation of visna virus. Science. 1985 Aug 2;229(4712):482–485. doi: 10.1126/science.2990051. [DOI] [PubMed] [Google Scholar]
  32. Kim S., Ikeuchi K., Byrn R., Groopman J., Baltimore D. Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9544–9548. doi: 10.1073/pnas.86.23.9544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  34. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Levy J. A., Hoffman A. D., Kramer S. M., Landis J. A., Shimabukuro J. M., Oshiro L. S. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science. 1984 Aug 24;225(4664):840–842. doi: 10.1126/science.6206563. [DOI] [PubMed] [Google Scholar]
  36. Luciw P. A., Cheng-Mayer C., Levy J. A. Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1434–1438. doi: 10.1073/pnas.84.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Malim M. H., Böhnlein S., Fenrick R., Le S. Y., Maizel J. V., Cullen B. R. Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8222–8226. doi: 10.1073/pnas.86.21.8222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
  39. Mazarin V., Gourdou I., Quérat G., Sauze N., Vigne R. Genetic structure and function of an early transcript of visna virus. J Virol. 1988 Dec;62(12):4813–4818. doi: 10.1128/jvi.62.12.4813-4818.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Meyerhans A., Cheynier R., Albert J., Seth M., Kwok S., Sninsky J., Morfeldt-Månson L., Asjö B., Wain-Hobson S. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell. 1989 Sep 8;58(5):901–910. doi: 10.1016/0092-8674(89)90942-2. [DOI] [PubMed] [Google Scholar]
  41. Muesing M. A., Smith D. H., Cabradilla C. D., Benton C. V., Lasky L. A., Capon D. J. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature. 1985 Feb 7;313(6002):450–458. doi: 10.1038/313450a0. [DOI] [PubMed] [Google Scholar]
  42. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  43. Nagashima K., Yoshida M., Seiki M. A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins. J Virol. 1986 Nov;60(2):394–399. doi: 10.1128/jvi.60.2.394-399.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Niederman T. M., Thielan B. J., Ratner L. Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1128–1132. doi: 10.1073/pnas.86.4.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pavlakis G. N., Felber B. K. Regulation of expression of human immunodeficiency virus. New Biol. 1990 Jan;2(1):20–31. [PubMed] [Google Scholar]
  46. Peterlin B. M., Luciw P. A., Barr P. J., Walker M. D. Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9734–9738. doi: 10.1073/pnas.83.24.9734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ratner L., Fisher A., Jagodzinski L. L., Liou R. S., Mitsuya H., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the virus associated with the acquired immunodeficiency syndrome, HTLV-III/LAV. Haematol Blood Transfus. 1987;31:404–406. doi: 10.1007/978-3-642-72624-8_86. [DOI] [PubMed] [Google Scholar]
  48. Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Rosen C. A., Sodroski J. G., Haseltine W. A. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell. 1985 Jul;41(3):813–823. doi: 10.1016/s0092-8674(85)80062-3. [DOI] [PubMed] [Google Scholar]
  51. Rosen C. A., Terwilliger E., Dayton A., Sodroski J. G., Haseltine W. A. Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2071–2075. doi: 10.1073/pnas.85.7.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sadaie M. R., Rappaport J., Benter T., Josephs S. F., Willis R., Wong-Staal F. Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9224–9228. doi: 10.1073/pnas.85.23.9224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  54. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  55. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schwartz S., Felber B. K., Fenyö E. M., Pavlakis G. N. Rapidly and slowly replicating human immunodeficiency virus type 1 isolates can be distinguished according to target-cell tropism in T-cell and monocyte cell lines. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7200–7203. doi: 10.1073/pnas.86.18.7200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shapiro M. B., Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987 Sep 11;15(17):7155–7174. doi: 10.1093/nar/15.17.7155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sherman L., Gazit A., Yaniv A., Kawakami T., Dahlberg J. E., Tronick S. R. Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat. J Virol. 1988 Jan;62(1):120–126. doi: 10.1128/jvi.62.1.120-126.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sodroski J., Goh W. C., Rosen C., Dayton A., Terwilliger E., Haseltine W. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature. 1986 May 22;321(6068):412–417. doi: 10.1038/321412a0. [DOI] [PubMed] [Google Scholar]
  60. Sodroski J., Patarca R., Rosen C., Wong-Staal F., Haseltine W. Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. Science. 1985 Jul 5;229(4708):74–77. doi: 10.1126/science.2990041. [DOI] [PubMed] [Google Scholar]
  61. Sonigo P., Alizon M., Staskus K., Klatzmann D., Cole S., Danos O., Retzel E., Tiollais P., Haase A., Wain-Hobson S. Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus. Cell. 1985 Aug;42(1):369–382. doi: 10.1016/s0092-8674(85)80132-x. [DOI] [PubMed] [Google Scholar]
  62. Viglianti G. A., Mullins J. I. Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1. J Virol. 1988 Dec;62(12):4523–4532. doi: 10.1128/jvi.62.12.4523-4532.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  64. Wright C. M., Felber B. K., Paskalis H., Pavlakis G. N. Expression and characterization of the trans-activator of HTLV-III/LAV virus. Science. 1986 Nov 21;234(4779):988–992. doi: 10.1126/science.3490693. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES