The integrity of the stem structure of human immunodeficiency virus type 1 Tat-responsive sequence of RNA is required for interaction with the interferon-induced 68,000-Mr protein kinase.
A number of eucaryotic viruses have devised strategies to minimize the deleterious effects on protein synthesis caused by activation of the interferon-induced, double-stranded-RNA-activated protein kinase, P68. In a recent report, we described the down regulation of the P68 protein kinase in cells infected by human immunodeficiency virus type 1 (HIV-1) (S. Roy, M. G. Katze, N. T. Parkin, I. Edery, A. G. Hovanessian, and N. Sonenberg, Science 247:1216-1219, (1990). We now present evidence that such a decrease in amounts of P68 could be essential for HIV-1 replication because of the presence of the Tat-responsive sequence (TAR sequence) present in the 5' untranslated region of HIV-1 mRNAs, which activates the P68 kinase. We found that poly(A)+ mRNAs prepared from HIV-1-infected cells efficiently activated the protein kinase as did mRNAs from stably transformed cell lines constitutively expressing the TAR region. Furthermore, we found that TAR-containing RNAs complexed with purified P68 protein kinase in vitro by two independent assays and could be cross-linked to P68 kinase present in a HeLa cell extract. Experiments using in vitro-synthesized wild-type and mutant TAR RNAs revealed that both the efficient binding to and the activation of P68 kinase were dependent on the TAR RNA stem structure. The TAR-P68 complex could be competed out by a synthetic RNA that bound to and activated the protein kinase but not by a synthetic RNA that bound with low affinity and did not activate P68. The possible biological consequences of a P68-TAR interaction that may include the switch from latent to active virus replication are discussed.
Department of Biochemistry, McGill University, Montréal, Canada.
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Roy
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Agy
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Hovanessian
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Sonenberg
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Katze
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011494:Protein Kinases / Q000096:biosynthesis / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics* / Q000528:radiation effects; D014466:Ultraviolet Rays; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D007372:Interferons; D011494:Protein Kinases