       Document 0453
 DOCN  M9610453
 TI    Structural requirements for the binding of tRNA Lys3 to reverse
       transcriptase of the human immunodeficiency virus type 1.
 DT    9601
 AU    Oude Essink BB; Das AT; Berkhout B; Department of Virology, Academic
       Medical Center, University of; Amsterdam, The Netherlands.
 SO    J Biol Chem. 1995 Oct 6;270(40):23867-74. Unique Identifier : AIDSLINE
       MED/96007545
 AB    Reverse transcription of the human immunodeficiency virus type 1 (HIV-1)
       RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of
       this tRNA primer during virion assembly is thought to be mediated by
       specific interactions with the reverse transcriptase (RT) protein.
       Portions of the tRNA molecule that are required for interaction with the
       RT protein remain poorly defined. We have used an RNA gel mobility shift
       assay to measure the in vitro binding of purified RT to mutant forms of
       tRNA Lys3. The anticodon loop could be mutated without eliminating RT
       recognition. However, mutations in the T psi C stem were found to
       partially interfere with RT binding, and D arm mutants were completely
       inactive in RT binding. Interestingly, binding of the RT protein to tRNA
       Lys3 facilitates the subsequent annealing of template strand to the
       3'-terminus of the tRNA molecule. Consistent with this finding, we
       demonstrate that mutant HIV-1 virions lacking the RT protein do contain
       a viral RNA genome without an associated tRNA Lys3 primer. We also found
       that a preformed primer tRNA-template complex is efficiently recognized
       by RT protein in vitro. Extension of the template molecule over the T
       psi C loop did result in complete inhibition of RT binding, suggesting
       the presence of additional recognition elements in the T psi C loop.
       These results, combined with a comparative sequence analysis of tRNA
       species present in HIV-1 virions and RNA motifs selected in vitro for
       high affinity RT binding, suggest that RT recognizes the central domain
       of the tRNA tertiary structure, which is formed by interaction of the D
       and T psi C loops.
 DE    Base Sequence  Binding Sites  DNA Primers/GENETICS  Human
       HIV-1/*ENZYMOLOGY  In Vitro  Molecular Sequence Data  Molecular
       Structure  Mutagenesis, Site-Directed  Nucleic Acid Conformation
       RNA-Directed DNA Polymerase/*METABOLISM  RNA, Transfer,
       Lys/*CHEMISTRY/GENETICS/*METABOLISM  RNA,
       Viral/CHEMISTRY/GENETICS/METABOLISM  Support, Non-U.S. Gov't  JOURNAL
       ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

