Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide

  1. Erik Lee Snapp
  2. Nicholas McCaul
  3. Matthias Quandte
  4. Zuzana Cabartova
  5. Ilja Bontjer
  6. Carolina Källgren
  7. IngMarie Nilsson
  8. Aafke Land
  9. Gunnar von Heijne
  10. Rogier W Sanders
  11. Ineke Braakman  Is a corresponding author
  1. Janelia Research Campus, United States
  2. Utrecht University, Netherlands
  3. dr heinekamp Benelux B.V., Netherlands
  4. National Institute of Public Health, Czech Republic
  5. Academic Medical Center, Netherlands
  6. Stockholm University, Sweden
  7. Stockholm Unversity, Sweden
  8. Institute of Life Sciences, Netherlands

Abstract

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 that 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.

Article and author information

Author details

  1. Erik Lee Snapp

    Janelia Research Campus, Ashburn, United States
    Competing interests
    Erik Lee Snapp, Has filed a patent application with and licensed technology to Lucigen Corp (U.S. Patent Application 15/152/908). The technology is not related to this manuscript..
  2. Nicholas McCaul

    Cellular Protein Chemistry, Utrecht University, Utrecht, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7888-7815
  3. Matthias Quandte

    dr heinekamp Benelux B.V., Riethoven, Netherlands
    Competing interests
    No competing interests declared.
  4. Zuzana Cabartova

    National Reference Laboratory for Viral Hepatitis, National Institute of Public Health, Šrobárova, Czech Republic
    Competing interests
    No competing interests declared.
  5. Ilja Bontjer

    Department of Medical Microbiology, Academic Medical Center, Amsterdam, Netherlands
    Competing interests
    No competing interests declared.
  6. Carolina Källgren

    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
    Competing interests
    No competing interests declared.
  7. IngMarie Nilsson

    Department of Biochemistry and Biophysics, Stockholm Unversity, Stockholm, Sweden
    Competing interests
    No competing interests declared.
  8. Aafke Land

    Hogeschool Utrecht, Institute of Life Sciences, Utrecht, Netherlands
    Competing interests
    No competing interests declared.
  9. Gunnar von Heijne

    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4490-8569
  10. Rogier W Sanders

    Department of Medical Microbiology, Academic Medical Center, Amsterdam, Netherlands
    Competing interests
    Rogier W Sanders, Is listed as an inventor on patents involving recombinant, soluble native-like Env trimers (EP2975053A1, EP2765138A3, WO/2017/055522A1, WO/2011/108937, WO/2010/041942, WO/2008/103428A2, WO/2003/022869A2). The technology is not related to this manuscript..
  11. Ineke Braakman

    Cellular Protein Chemistry, Utrecht University, Utrecht, Netherlands
    For correspondence
    i.braakman@uu.nl
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1592-4364

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek

  • Nicholas McCaul
  • Matthias Quandte
  • Aafke Land
  • Ineke Braakman

Netherlands AIDS Fund

  • Aafke Land

Seventh Framework Programme (ITN 'Virus Entry')

  • Nicholas McCaul
  • Matthias Quandte
  • Ineke Braakman

National Institutes of Health (NIH AI-51519)

  • Erik Lee Snapp

Swedish Cancer Foundation

  • IngMarie Nilsson
  • Gunnar von Heijne

Knut and Alice Wallenberg Foundation

  • Gunnar von Heijne

European Research Council (ERC-StG-2011-280829-SHEV)

  • Rogier W Sanders

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Reid Gilmore, University of Massachusetts Medical School, United States

Publication history

  1. Received: February 27, 2017
  2. Accepted: July 26, 2017
  3. Accepted Manuscript published: July 28, 2017 (version 1)
  4. Version of Record published: August 23, 2017 (version 2)

Copyright

© 2017, Snapp et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,242
    Page views
  • 497
    Downloads
  • 28
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Erik Lee Snapp
  2. Nicholas McCaul
  3. Matthias Quandte
  4. Zuzana Cabartova
  5. Ilja Bontjer
  6. Carolina Källgren
  7. IngMarie Nilsson
  8. Aafke Land
  9. Gunnar von Heijne
  10. Rogier W Sanders
  11. Ineke Braakman
(2017)
Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide
eLife 6:e26067.
https://doi.org/10.7554/eLife.26067

Further reading

    1. Biochemistry and Chemical Biology
    2. Cancer Biology
    Stefania Monterisi, Johanna Michl ... Pawel Swietach
    Research Article Updated

    Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in ‘metabolic genes’ will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial respiration (NDUFS1) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as ‘essential’ do not influence the growth of human cancers.

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Jinli Geng, Yingjun Tang ... Xiaodong Liu
    Research Article

    Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. responses to stimuli), and also mediate intracellular signaling cascades that normally take longer time to manifest (e.g., regulations of transcription). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging with long-term probe expression in cortical neurons, which has been designed to eliminate the unwanted interactions between conventional GCaMP indicators and endogenous (apo)calmodulin-binding proteins. By expressing in live adult mice at high levels over an extended time frame, GCaMP-X indicators showed less damage and improved performance in two-photon imaging of acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations. Chronic Ca2+ imaging data (³1 month) were acquired from cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined along with the multiple stages (from neonatal to mature) during neural development. Dysregulations of both neuritogenesis and Ca2+ oscillations were observed typically in 2-3 weeks, which were exacerbated by stronger or prolonged expression of GCaMP. In comparison, neurons expressing GCaMP-X exhibited significantly less damage. By varying the timepoints of virus infection or drug induction, GCaMP-X outperformed GCaMP similarly in cultured mature neurons. These data altogether highlight the unique importance of oscillatory Ca2+ to morphology and health of neurons, presumably underlying the differential performance between GCaMP-X and GCaMP. In summary, GCaMP-X provides a viable option for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.