HIV-1 TAT “activates” presynthesized RNA in the nucleus

Replication of the human immunodeficiency virus (HIV-1) depends upon the viral TAT protein. TAT stimulates gene expression via a target response sequence (TAR) located within the HIV-1 LTR. As TAR is located in the transcribed region it could act as a signal in either the DNA, the RNA, or both. To test whether TAT acts on transcription and/or posttranscriptionally, we produced TAT in yeast and monitored its activity after microinjection into the nucleus or cytoplasm of Xenopus oocytes. The TAT protein stimulated TAR-dependent expression, but this activation was not inhibited by transcriptional inhibitors. Furthermore, TAR-containing RNA, produced in vitro, was "activated" by TAT after coinjection into oocytes. This activation only occurred, however, when the RNA was injected into the nucleus and not into the cytoplasm. Our data indicate, therefore, that in the Xenopus system TAT acts on presynthesized RNA and that the nucleus is involved in this action.

[1]  J. Sodroski,et al.  The trans-activator gene of the human T cell lymphotropic virus type III is required for replication , 1986, Cell.

[2]  R. Gentz,et al.  Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[3]  W. Richardson,et al.  The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen , 1988, The Journal of cell biology.

[4]  B. Howard,et al.  Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells , 1982, Molecular and cellular biology.

[5]  J. Sodroski,et al.  Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III , 1986, Journal of virology.

[6]  A. Kingsman,et al.  The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins , 1987, Cell.

[7]  K. Jeang,et al.  Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type I tat proteins occurs in the absence of de novo protein synthesis. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Gaynor,et al.  Functional domains required for tat‐induced transcriptional activation of the HIV‐1 long terminal repeat. , 1988, The EMBO journal.

[9]  M. Rosbash,et al.  Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Luciw,et al.  Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product , 1987, Nature.

[11]  B. Cullen,et al.  Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat , 1988, Journal of virology.

[12]  G. Fink,et al.  Transformation of yeast. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Wickens,et al.  The use of Xenopus oocytes for the expression of cloned genes. , 1983, Methods in enzymology.

[14]  D. Melton,et al.  Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. , 1984, Nucleic acids research.

[15]  J. Sodroski,et al.  Post-transcriptional regulation accounts for the trans-activation of the human T-lymphotropic virus type III , 1986, Nature.

[16]  L. C. Moore,et al.  Nuclear envelope permeability , 1975, Nature.

[17]  P. Luciw,et al.  Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[19]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[20]  D. Capon,et al.  A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator , 1988, Molecular and cellular biology.

[21]  Michael B. Mathews,et al.  Transcriptional but not translational regulation of HIV-1 by the tat gene product , 1988, Nature.

[22]  N. Sonenberg,et al.  Mutational analysis of the 5′ non‐coding region of human immunodeficiency virus type 1: effects of secondary structure on translation. , 1988, The EMBO journal.

[23]  Maurice Green,et al.  Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein , 1988, Cell.

[24]  K. Nagai,et al.  Generation of β-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli , 1984, Nature.

[25]  P. Chambon,et al.  Animal DNA-dependent RNA polymerases. 11. Mechanism of the inhibition of RNA polymerases B by amatoxins. , 1974, Biochimica et biophysica acta.

[26]  J. Sodroski,et al.  The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat , 1985, Cell.

[27]  H. Cherwinski,et al.  Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. , 1983, Analytical biochemistry.

[28]  S. Ruben,et al.  Structural and functional characterization of human immunodeficiency virus tat protein , 1989, Journal of virology.

[29]  W. Richardson,et al.  Sequence requirements for nuclear location of simian virus 40 large-T antigen , 1984, Nature.

[30]  G. Pavlakis,et al.  Expression and characterization of the trans-activator of HTLV-III/LAV virus. , 1986, Science.

[31]  A. Kingsman,et al.  The expression of hybrid HIV:Ty virus-like particles in yeast , 1987, Nature.

[32]  B. Moss,et al.  Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein. , 1988, Virology.

[33]  Bryan R. Cullen,et al.  Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism , 1986, Cell.

[34]  C. Feldherr,et al.  Movement of a karyophilic protein through the nuclear pores of oocytes , 1984, The Journal of cell biology.

[35]  D. Capon,et al.  Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein , 1987, Cell.

[36]  R A Laskey,et al.  Protein import into the cell nucleus. , 1986, Annual review of cell biology.

[37]  P. Chambon,et al.  Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein‐DNA complexes. , 1988, The EMBO journal.

[38]  H. M. Sobell The stereochemistry of actinomycin binding to DNA and its implications in molecular biology. , 1973, Progress in nucleic acid research and molecular biology.

[39]  R. Roeder,et al.  Physical analysis of transcription preinitiation complex assembly on a class II gene promoter. , 1988, Science.

[40]  A. Kingsman,et al.  Polyvalent recombinant antigens: a new vaccine strategy. , 1988, Vaccine.

[41]  Eric C. Holland,et al.  HIV-1 tat trans-activation requires the loop sequence within tar , 1988, Nature.

[42]  D. Bredt,et al.  Tat protein from human immunodeficiency virus forms a metal-linked dimer. , 1988, Science.

[43]  A. Kingsman,et al.  Ty: A retroelement moving forward , 1988, Cell.

[44]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[46]  R. Mortimer,et al.  Chromosome Mapping in Saccharomyces: Centromere-Linked Genes. , 1960, Genetics.

[47]  M. Braddock,et al.  Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair. , 1988, Nucleic acids research.

[48]  Michael E. Greenberg,et al.  Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene , 1984, Nature.

[49]  N. Sonenberg,et al.  Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: A novel translational control mechanism , 1989, Cell.

[50]  M. Feinberg,et al.  HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA , 1986, Cell.

[51]  A. C. Chinault,et al.  Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III. , 1979, Gene.

[52]  D. Holmes,et al.  A rapid boiling method for the preparation of bacterial plasmids. , 1981, Analytical biochemistry.

[53]  B. Cullen,et al.  Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[54]  S. Arya,et al.  Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). , 1985, Science.

[55]  M. Malim,et al.  Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast , 1985, Nature.

[56]  Mark L. Pearson,et al.  Complete nucleotide sequence of the AIDS virus, HTLV-III , 1985, Nature.