Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.

[1]  M. Braddock,et al.  Blocking of Tat-dependent HIV-1 RNA modification by an inhibitor of RNA polymerase II processivity , 1991, Nature.

[2]  P. Sharp,et al.  Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[3]  R. Gaynor,et al.  tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. , 1991, Genes & development.

[4]  I. Sim.  The Human Immunodeficiency Virus TAT Protein , 1990 .

[5]  P. Luciw,et al.  Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. , 1989, Genes & development.

[6]  A. Blanchard,et al.  HIV‐1 TAR RNA‐binding proteins control TAT activation of translation in Xenopus oocytes , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  D. Crothers,et al.  RNA recognition by Tat-derived peptides: Interaction in the major groove? , 1991, Cell.

[8]  F. Kashanchi,et al.  Direct interaction of human TFIID with the HIV-1 transactivator Tat , 1994, Nature.

[9]  B. Berkhout,et al.  Efficient trans-activation by the HIV-2 Tat protein requires a duplicated TAR RNA structure. , 1990, Nucleic acids research.

[10]  A. Blanchard,et al.  An adenosine at position 27 in the human immunodeficiency virus type 1 trans-activation response element is not critical for transcriptional or translational activation by Tat , 1992, Journal of virology.

[11]  W. Kaczmarski,et al.  Lupus autoantigen Ku protein binds HIV-1 TAR RNA in vitro. , 1993, Biochemical and biophysical research communications.

[12]  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.

[13]  B. Peterlin,et al.  Trans-activation by HIV-1 Tat via a heterologous RNA binding protein , 1990, Cell.

[14]  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.

[15]  M. White,et al.  Imaging of firefly luciferase expresssion in single mammalian cells using high sensitivity charge-coupled device cameras. , 1990 .

[16]  M. Rounseville,et al.  Binding of a host cell nuclear protein to the stem region of human immunodeficiency virus type 1 trans-activation-responsive RNA , 1992, Journal of virology.

[17]  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.

[18]  Eli Gilboa,et al.  Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication , 1990, Cell.

[19]  A. Chambers,et al.  A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction , 1990, Cell.

[20]  M. Mathews,et al.  Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

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

[22]  Michael R. Green,et al.  Activation of transcription by HIV-1 Tat protein tethered to nascent RNA through another protein , 1990, Nature.

[23]  J. Karn,et al.  Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

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

[25]  K. Jeang,et al.  In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor , 1993, Journal of virology.

[26]  K. Jones,et al.  Two distinct nuclear transcription factors recognize loop and bulge residues of the HIV-1 TAR RNA hairpin. , 1991, Genes & Development.

[27]  B. Cullen,et al.  Mechanism of action of regulatory proteins encoded by complex retroviruses , 1992, Microbiological reviews.

[28]  N. Sonenberg,et al.  Structural requirements for trans activation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by tat: importance of base pairing, loop sequence, and bulges in the tat-responsive sequence , 1990, Journal of virology.

[29]  M. Green,et al.  The HIV-1 Tat protein activates transcription from an upstream DNA-binding site: implications for Tat function. , 1991, Genes & development.

[30]  A. Rice,et al.  Specific interaction of the human immunodeficiency virus Tat proteins with a cellular protein kinase. , 1993, Virology.

[31]  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.

[32]  B. Sullenger,et al.  Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation , 1991, Journal of virology.

[33]  B. Cullen,et al.  Genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 Tat function , 1993, Journal of virology.

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

[35]  B. Cullen.  Does HIV-1 Tat induce a change in viral initiation rights? , 1993, Cell.

[36]  B. Berkhout,et al.  Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. , 1991, Science.