       Document 0640
 DOCN  M9640640
 TI    The human immunodeficiency virus type 1 5' packaging signal structure
       affects translation but does not function as an internal ribosome entry
       site structure.
 DT    9604
 AU    Miele G; Mouland A; Harrison GP; Cohen E; Lever AM; University of
       Cambridge Department of Medicine, Addenbrooke's; Hospital, United
       Kingdom.
 SO    J Virol. 1996 Feb;70(2):944-51. Unique Identifier : AIDSLINE
       MED/96135205
 AB    The role of the RNA secondary structure in the 5' packaging signal
       region of human immunodeficiency virus type 1 (HIV-1) in initiating
       translation of gag mRNA has been investigated both in vitro and in the
       presence of cellular cofactors in vivo. Heat denaturation of the
       structure and mutagenic deletion both lead to an increase in levels of
       translated products, indicating that the structure is a significant
       inhibitor of translation. The proximity of the gag AUG to the packaging
       signal structure suggested that it might function as an internal
       ribosome entry site. However, in both a cell-free system and eukaryotic
       cells, translation will initiate at a novel upstream initiation codon
       introduced within the 5' noncoding region. This codon is utilized
       exclusively, resulting in gag protein products with an extra 11 amino
       acids at the amino terminus, which, when expressed in T lymphocytes, are
       confined intracellularly, probably because of the lack of an N-terminal
       glycine myristoylation signal. Deletion of the secondary structure
       abolishes gag production even in the presence of tat and rev in trans.
       Using dicistronic constructs containing the HIV-1 5' leader cloned
       between two heterologous open reading frames, we were unable to detect
       any significant expression of the second open reading frame that would
       have been supportive of an internal ribosome entry site mechanism. Using
       mutant proviruses either lacking the entire packaging signal structure
       region or containing the introduced upstream initiation codon in
       long-term replication studies, we were unable to detect reverse
       transcriptase activity in culture supernatants. The 5' packaging signal
       structure of HIV-1 does not serve as an internal ribosome entry site.
       The translation of gag is consistent with ribosomal scanning. However,
       the packaging signal structure causes significant translational
       inhibition.
 DE    Amino Acid Sequence  Animal  Base Sequence  Cell Line  Codon, Initiator
       Fusion Proteins, gag-pol/*GENETICS  Hela Cells  Human
       HIV-1/*GENETICS/PHYSIOLOGY  Molecular Sequence Data  *Nucleic Acid
       Conformation  Peptide Chain Initiation/*PHYSIOLOGY  Reticulocytes
       Ribosomes/*METABOLISM  RNA, Messenger/CHEMISTRY/*GENETICS  RNA,
       Viral/CHEMISTRY/*GENETICS  Structure-Activity Relationship  Support,
       Non-U.S. Gov't  Transcription, Genetic  Virus Assembly  JOURNAL ARTICLE

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

