A minimal uORF within the HIV-1 vpu leader allows efficient translation initiation at the downstream env AUG

Abstract

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.

Upstream AUG Codons in the Simian Immunodeficiency Virus SIVmac239 Genome Regulate Rev and Env Protein Translation

“…We assume that these ribosomes can reinitiate scanning on the RevEnv mRNA toward the Env AUG. Similar regulatory scenarios have been described previously for HIV-1 Vpu and Env translation (1,35,38) (Fig. 8C) but also in other viruses and eukaryotes (34,40,44,50).…”

“…8C). Inactivation of this uAUG reduced Env expression, consistent with the idea that this uAUG augments access to the downstream Env ORF (35). Second, alternatively spliced HIV-1 RNAs with a 5= extension (SA4 in Fig.…”

“…In agreement with the scanning mode of translation, there are rarely any uAUGs present in the large variety of mRNAs produced by HIV and SIV. An exception in HIV is the bicistronic mRNA that encodes both Vpu and Env (1,47), and this mRNA also contains a minimal uORF (35). We now describe a major exception for SIV.…”

“…(C) HIV-1 Env mRNA has two uAUG codons: the Vpu AUG and a minimal uORF directly upstream of Vpu. The HIV-1 uORF has a suppressive effect on Vpu translation and thus an indirect stimulatory effect on Env translation (35). Alternative splicing at SA4 will result in an additional uORF for the Env mRNA: the Rev AUG, which has a suppressive effect on Vpu translation (1).…”

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication

“…Since our HIV-2 G-box mutation results in a weaker Kozak context for AUG1, leaky scanning at this initiation site could enhance the use of at least one of the downstream start codons. Several laboratories have shown that in HIV-1 the strength of upstream AUGs can influence initiation at downstream AUGs (Schwartz et al 1992;Anderson et al 2007;Krummheuer et al 2007). Mutation of a weak upstream HIV-1 rev Kozak sequence to a strong initiating sequence resulted in decreased leaky scanning and poor initiation at the downstream vpu AUG ).…”