THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 5'-PACKAGING SIGNAL STRUCTUREAFFECTS TRANSLATION BUT DOES NOT FUNCTION AS AN INTERNAL RIBOSOME ENTRY SITE STRUCTURE

The role of the RNA secondary structure in the 5' packaging signal reg ion of human immunodeficiency virus type 1 (HIV-1) in initiating trans lation of gag mRNA has been investigated both in vitro and in the pres ence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translat ed products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal s tructure suggested that it might function as an internal ribosome entr y site. However, in both a cell-free system and eukaryotic cells, tran slation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, re sulting in gag protein products with an extra 11 amino acids at the am ino terminus, which, when expressed in T lymphocytes, are confined int racellularly, probably because of the lack of an N-terminal glycine my ristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of mt and rev in trans. Using dicistr onic constructs containing the HIV-1 5' leader cloned between two hete rologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supp ortive of an internal ribosome entry site mechanism. Using mutant prov iruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term repli cation studies, we were unable to detect reverse transcriptase activit y 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 sign al structure causes significant translational inhibition.