Nucleic Acids Research, 1992, Vol. 20, No. 16 4291-4297
© 1992
MOLECULAR BIOLOGY |
Variable inhibition of cell-free translation by HIV–1 transcript leader sequences
Department of Molecular Medicine, Fred Hutchinson Cancer Reserach Center 1124 Columbia Street, Seattle, WA 98104, USA
Received April 20, 1992. Revised July 18, 1992. Accepted July 18, 1992.
The 5' ends of all human immunodeficiency virus type I (HIV–1) transcripts have the potential to coordinately regulate translation of HIV–1 mRNAs. Conflicting observations of the translational impact of these sequences in various systems stimulated these analyses of translation in reticulocyte lysates. We report a sensitive, rapid, quantitative, and inexpensive cell-free translation assay in which translational efficiency is monitored by enzymatic assay of the translation products. Using this assay and conventional radlolabeling assays, we demonstrate that the HIV–1 transcript leader inhibits downstream translation and that the stem-loop structure is required. Under our assay conditions, this Inhibition occurs predominantly in els and is not mediated by the 68 kD, interferon-Induced, double-stranded RNA-activated kinase (p68). However, under other assay conditions the HIV–1 leader may activate p68 and inhibit translation in trans. We show that variation between individual preparations of cell-free extracts can dramatically alter the magnitude of the translational inhibition by the HIV–1 leader. Further, we provide evidence that a heat-labile factor Is required for efficient translation of transcripts containing the HIV–1 leader. These observations provide a foundation for identifying factors required fortranslation of HIV–1 transcripts.
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