1 Departments of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3053 (424 CARL), Research Drive, Durham, NC 27710, USA
2 Medicine, Duke University Medical Center, Box 3053 (424 CARL), Research Drive, Durham, NC 27710, USA
3 Microbiology and Tumor Biology Center and Swedish Institute for Infectious Disease Control, Karolinska Institute, Solna 17182, Sweden
Correspondence
Mariano Garcia-Blanco
garci001{at}mc.duke.edu
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ABSTRACT |
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Present address: Columbia University College of Physicians & Surgeons, Department of Medicine, 630 W 168th Street, New York, NY 10032, USA.
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MAIN TEXT |
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Like HIV-1, equine infectious anaemia virus (EIAV) trans-activates transcription via a Tat protein (eTat), TAR RNA (eTAR) and the equine homologue of Cyclin T1 (eCycT1) (Bieniasz et al., 1999; Taube et al., 2000
). In the 25 nucleotide stemloop EIAV TAR, the CUGC loop sequence, the two
U·G base pairs proximal to the loop and the overall secondary structure are required for function (Carvalho & Derse, 1991
). Importantly, eTat binds eTAR only in the presence of eCycT1 (Bieniasz et al., 1999
). Biochemical and 2-D NMR studies have demonstrated conservation of structure between the hTAR and eTAR terminal loops, despite dissimilar sequence (Colvin et al., 1993
; Hoffman et al., 1993
), and the 3-D NMR structure of eTAR reveals an exposed guanosine extending from the loop, similar to that in hTAR (Hoffman & White, 1995
).
In order to analyse functional interactions between hTat and hTAR, we constructed TAR elements chimeric between HIV-1 and EIAV and tested their activity in canine D17 cells, which are permissive for both hTat (via hTAR) and eTat (via eTAR) trans-activation (see Fig. 2 for the predicted structures of the RNA chimeras). We observed that the TAR loops were not interchangeable and that sequential addition of TAR motifs added to function. Our data are most consistent with the existence of at least two interactions between hTat and hTAR in vivo, each of which partially adds to hTat to hTAR binding.
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The plasmid phTAR, containing the chloramphenicol acetyltransferase (CAT) reporter gene driven by the HIV-1 LTR, was modified by replacing the hTAR loop with the eTAR loop (hTAR-e1), or with the eTAR
U·G base pairs and loop (hTAR-e2) (see diagrams in Fig. 2). The size of the distal stem+loop was kept constant by adding or removing
C·G base pairs. Canine D17 cells were transfected, using Lipofectin (GibcoBRL), in duplicate with 250 ng of the indicated TAR chimera, with or without the indicated Tat, as well as 50 ng luciferase DNA transfection control and pBlueScript and/or pRSPA-s DNA to bring total DNA to 1·15 µg. Constant protein amounts of cell extracts were assayed for CAT activity and the CAT activities of un-transfected wells were subtracted. CAT activity and trans-activation were measured as described previously (Suñe & Garcia-Blanco, 1999; Albrecht et al., 2000
). As expected, hTat activated transcription of hTAR but eTat did not. Replacing the hTAR loop with the eTAR loop (hTAR-e1) partially rescued eTat activity while impairing hTat activity (Fig. 1
). Replacing the hTAR loop and distal
C·G base pair with the eTAR loop and
U·G base pairs (hTAR-e2) fully rescued eTat and further impaired hTat function (Fig. 1
). As expected, neither K41A nor R43G (eTat mutant) activated hTAR-e2 (data not shown). We observed that eTat, but not hTat or R43G, activated transcription via eTAR (data not shown), confirming the ability of D17 cells to support both HIV-1 and EIAV Tat-activation and the inability of hTat to activate eTAR.
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Numerous in vitro observations also support this model. The hTat RNA-binding domain binds the hTAR bulge in vitro, but the affinity and specificity of this hTathTAR interaction are enhanced by amino acid residues in the activation domain (Churcher et al., 1993). Tat from both HIV-1 and HIV-2 binds weakly to HIV-2 TAR RNA in a loop-dependent manner in the absence of hCycT1, and the HIV-2 TAR loop sequence specificity of hTat is lost on truncation of the hTat activation domain (Garber et al., 1998b
). The structure of the bovine immunodeficiency virus (BIV) TatTAR complex reveals Tat in close proximity to the loop (Puglisi et al., 1995
). Using circular dichroism and molecular modelling, amino acids 5972 of hTat have been suggested to interact with UGG at positions 3133 in the hTAR loop in vitro (Loret et al., 1992
). Footprinting experiments suggested that hTat bound the bulge and A-35 of the loop simultaneously, consistent with these residues being on the same face of the upper stem in hTAR (Colvin & Garcia-Blanco, 1992
). Furthermore, hTat has been cross-linked to U31 (Wang et al., 1999
) and to U35 (Farrow et al., 1998
) of the hTAR loop and site-specific RNA cleavage has demonstrated that hTat is located in the proximity of both the bulge and loop of hTAR (Huq & Rana, 1997
). Taken together, these data suggest at least three RNAprotein interactions in the hCycT1hTathTAR complex. In addition to hCycT1 binding to the hTAR loop, we propose at least two interactions between hTat and hTAR, rather than the single interaction of hTat with the hTAR bulge (Fig. 2
). While our results do not definitively prove the existence of this RNAprotein interaction(s), we offer this model as the most parsimonious explanation of the available data.
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ACKNOWLEDGEMENTS |
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Received 10 June 2002;
accepted 7 November 2002.