The HIV-1 Vpu and Env proteins are translated from 16 alternatively spliced bicistronic mRNA isoforms. Translation of HIV-1 mRNAs generally follows the ribosome scanning mechanism. However, by using subgenomic env expression vectors, we found that translation of glycoprotein from polycistronic mRNAs was inconsistent with leaky scanning. Instead a conserved minimal upstream open reading frame (uORF) consisting only of a start and stop codon that overlaps with the vpu start site, appears to augment access to the env start codon downstream. Mutating the translational start and stop codons of this uORF resulted in up to fivefold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpu initiation sequence abolished Env expression from subgenomic constructs and replication of HIV-1, whereas an identical increase in the strength of the minimal uORF initiation site did not alter Env expression.
Human immunodeficiency virus type 1 (HIV-1) generates 16 alternatively spliced isoforms of env mRNA that contain the same overlapping open reading frames for Vpu and Env proteins but differ in their 5 untranslated regions (UTR). A subset of env mRNAs carry the extra upstream Rev initiation codon in the 5 UTR. We explored the effect of the alternative UTR on the translation of Vpu and Env proteins from authentic env mRNAs expressed from cDNA constructs. Vpu expression from the subset of env mRNA isoforms with exons containing an upstream Rev AUG codon was minimal. However, every env mRNA isoform expressed similar levels of Env protein. Mutations that removed, altered the strength of, or introduced upstream AUG codons dramatically altered Vpu expression but had little impact on the consistent expression of Env. These data show that the different isoforms of env mRNA are not redundant but instead regulate Vpu production in HIV-1-infected cells. Furthermore, while the initiation of Vpu translation conforms to the leaky ribosome-scanning model, the consistent Env synthesis infers a novel, discontinuous ribosome-scanning mechanism to translate Env.Like most cellular pre-mRNAs, the human immunodeficiency virus type 1 (HIV-1) 9.2-kb genome-length RNA transcribed from the provirus acquires a 5Ј m 7 GpppN cap and a 3Ј poly(A) tail. Multiple splice donor (SD) and splice acceptor (SA) sites in the genome-length HIV-1 mRNA support alternative splicing to a pool of 4-kb and 2-kb mRNAs that differ in their 5Ј untranslated regions (UTR) (46, 52). These mRNAs contain partly overlapping open reading frames (ORFs) and collectively encode the nine HIV-1 proteins and polyproteins. In the early phase, multiple splicing produces more than 23 different 2-kb HIV-1 mRNAs that are constitutively exported to the cytoplasm for the translation of Rev, Tat, Nef, and Vpr regulatory and accessory proteins (46,52,54). Later, after sufficient levels of Rev accumulate in the nucleus, Rev multimers bind to the highly structured RNA of the Rev-responsive element (RRE) that underlies the Env reading frame. This binding promotes the engagement of proteins required for the nuclear export (22, 42) and cytoplasmic accumulation of unspliced genome-length RNA and 23 different spliced 4-kb mRNAs containing the RRE (reviewed in references 15 and 26). The viral Gag and Gag-Pol structural polyproteins are translated from the 9.2-kb RNA, with a ribosome frameshift producing Gag-Pol on 5% of occasions during translational scanning (24, 36). The structural envelope (Env) glycoprotein and accessory and regulatory Vpu, Vif, and Vpr proteins and amino acids 1 through 72 of Tat (Tat 72 ) are translated from the 4-kb spliced mRNAs (53, 54).The high level of diversity among the spliced HIV-1 mRNAs results in part from the incorporation of the upstream noncoding exons 2 and 3 (46, 52). These exons may be included separately or together or excluded from the 5Ј UTR of alternative 2-kb nef, tat, and rev and 4-kb env and tat mRNAs. Furthermore, the use of alternative SA sit...
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