16th International AIDS Conference Toronto, Canada - August 13 - 18, 2006

BOTH LINEAR AND DISCONTINUOUS RIBOSOME SCANNING ARE USED FOR TRANSLATION INITIATION FROM BICISTRONIC HIV-1 ENV MRNAS

Int Conf AIDS. 2006 Aug 13-18;16 Abstract No. TuAa0104

Johnson A.¹, Anderson J.¹, Howard J.¹, Krummheuer J.², Hauber I.³, Kammler S.², Hauber J.³, Schaal H.², Purcell D.^1
1University of Melbourne, Microbiology and Immunology, Parkville, Australia, ²Heinrich-Heine-University, Institute for Virology, Dusseldorf, Germany, ³Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany

BACKGROUND: HIV-1 generates 16 alternatively spliced env mRNA’s for the translation of Env. All 16 env mRNA isoforms are bicistronic for both the Vpu and Env open reading frames, but differ in their 5’ untranslated regions (UTR). A subset of env RNA has the extra upstream Rev initiation codon in the 5’ UTR.

METHODS: We mutated the native translation signals in an Env-GFP reporter plasmid and quantified expression of Env-GFP after HeLa cell transfection.

RESULTS: Vpu expression was minimal from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon. However, every env mRNA isoform expressed a similar level of Env. Mutations that removed, altered the strength, or introduced upstream AUGs dramatically altered Vpu expression, but did not alter the consistent Env expression. These data show the different isoforms of env mRNA regulate Vpu production in HIV-1 infected cells. However, translation initiation at the env AUG did not conform to the leaky ribosomal scanning mechanism. The 5’ leader sequences of env mRNA’s contains a minimal upstream open reading frame (uORF) of two codons that overlaps with the Vpu initation codon and is conserved across many strains of HIV-1. Mutating the translational start and stop codons of this uORF resulted in up to five fold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpunitiation sequence abolished Env expression from subgenomic constructs.

CONCLUSIONS: We propose that a minimal upstream uORF before vpu is used in an alternative translation initiation mechanism for Env, such as a ribosome shunting mechanism. This atypical mechanism may be responsible for the maintenance of Env expression even in the presence of upstream AUG’s.

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2006-08-13
TuAa0104

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