Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N) that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

Highlights d Intramolecular quality control via two post-targeting roles of gp120 signal peptide d Assembly of N and C termini of HIV-1 gp120 triggers signalpeptide cleavage d Redox-active signal-peptide cysteine sustains conformational plasticity

Exposure of neuroblastoma cells to 100% CO(2) does not alter methylation of two prognostic relevant index genes. It seems therefore unlikely that effects on methylation levels within CO(2) pneumoperitoneum lead to epigenetic changes in neuroblastoma.

SummaryThe membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum generally are assumed to be removed during translation, which is a prerequisite for proper folding. Cleavage of the HIV-1 gp120 signal peptide is late however and regulated by gp120 folding. While attached, a conserved cysteine in the signal peptide that is important for viral fitness, sustains disulfide isomerization during gp120 folding. Assembly of the N-terminal β-sandwich with a single C-terminal β-strand sets off signal-peptide cleavage, which releases gp120 from the membrane and from this disulfide-attacking cysteine and stabilizes the native gp120 fold. This is the first example of post-translational regulation of signal-peptide cleavage for intramolecular quality control of folding, with the signal peptide acting as a membrane-embedded propeptide with redox-active cysteine. Considering the ∼15% secretory proteins in our genome, and the frequency of N-C contacts in protein structures, this regulatory role of the signal peptide is bound to be a more common mechanism in secretory protein biosynthesis.