Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state

  1. Miri Shnayder
  2. Aharon Nachshon
  3. Batsheva Rozman
  4. Biana Bernshtein
  5. Michael Lavi
  6. Noam Fein
  7. Emma Poole
  8. Selmir Avdic
  9. Emily Blyth
  10. David Gottlieb
  11. Allison Abendroth
  12. Barry Slobedman
  13. John Sinclair
  14. Noam Stern-Ginossar  Is a corresponding author
  15. Michal Schwartz  Is a corresponding author
  1. Weizmann Institute of Science, Israel
  2. University of Cambridge, United Kingdom
  3. Sydney Cellular Therapies Laboratory, Australia
  4. University of Sydney, Australia

Abstract

Human cytomegalovirus (HCMV) causes a lifelong infection through establishment of latency. Although reactivation from latency can cause life-threatening disease, our molecular understanding of HCMV latency is incomplete. Here we use single cell RNA-seq analysis to characterize latency in monocytes and hematopoietic stem and progenitor cells (HSPCs). In monocytes, we identify host cell surface markers that enable enrichment of latent cells harboring higher viral transcript levels, which can reactivate more efficiently, and are characterized by reduced intrinsic immune response that is important for viral gene expression. Significantly, in latent HSPCs, viral transcripts could be detected only in monocyte progenitors and were also associated with reduced immune-response. Overall, our work indicates that regardless of the developmental stage in which HCMV infects, HCMV drives hematopoietic cells towards a weaker immune-responsive monocyte state and that this anergic-like state is crucial for the virus ability to express its transcripts and to eventually reactivate.

Data availability

Sequencing data have been deposited in GEO under accession code GSE138838

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Miri Shnayder

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  2. Aharon Nachshon

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  3. Batsheva Rozman

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  4. Biana Bernshtein

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  5. Michael Lavi

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  6. Noam Fein

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  7. Emma Poole

    Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3904-6121
  8. Selmir Avdic

    Sydney Cellular Therapies Laboratory, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  9. Emily Blyth

    Sydney Cellular Therapies Laboratory, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  10. David Gottlieb

    Sydney Cellular Therapies Laboratory, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  11. Allison Abendroth

    Discipline of Infectious Diseases and Immunology, Faculty of Medicine and Health, Charles Perkins Centre, University of Sydney, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  12. Barry Slobedman

    Discipline of Infectious Diseases and Immunology, Faculty of Medicine and Health, Charles Perkins Centre, University of Sydney, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9431-6094
  13. John Sinclair

    Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Noam Stern-Ginossar

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    For correspondence
    noam.stern-ginossar@weizmann.ac.il
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3583-5932
  15. Michal Schwartz

    Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
    For correspondence
    michalsc@weizmann.ac.il
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5442-0201

Funding

Infect-ERA (TANKACY)

  • Noam Stern-Ginossar

H2020 European Research Council (starting grant (StG-2014-638142))

  • Noam Stern-Ginossar

Cambridge NIHR BRC Cell Phenotyping Hub

  • John Sinclair

British Medical Research Council (G0701279)

  • John Sinclair

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

Ethics

Human subjects: All fresh peripheral blood samples were obtained after approval of protocols bythe Weizmann Institutional Review Board (IRB application 92-1). Informed written consent was obtained from all volunteers, and all experiments were carried out in accordance with the approved guidelines. The study using HSCT recipient samples was approved by the Human Research Ethics Committee of the University of Sydney and the Western Sydney Local Health District. Informed consent was obtained from all study participants prior to enrolment in accordance with the Declaration of Helsinki.

Copyright

© 2020, Shnayder 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

  • 4,487
    views
  • 650
    downloads
  • 48
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Miri Shnayder
  2. Aharon Nachshon
  3. Batsheva Rozman
  4. Biana Bernshtein
  5. Michael Lavi
  6. Noam Fein
  7. Emma Poole
  8. Selmir Avdic
  9. Emily Blyth
  10. David Gottlieb
  11. Allison Abendroth
  12. Barry Slobedman
  13. John Sinclair
  14. Noam Stern-Ginossar
  15. Michal Schwartz
(2020)
Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state
eLife 9:e52168.
https://doi.org/10.7554/eLife.52168

Share this article

https://doi.org/10.7554/eLife.52168

Further reading

    1. Microbiology and Infectious Disease
    2. Neuroscience
    Aleksandra Prochera, Anoohya N Muppirala ... Meenakshi Rao
    Research Article

    Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions (Prochera and Rao, 2023). To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express the gene Proteolipid protein 1 (PLP1) in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1+ cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1+ glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

    1. Microbiology and Infectious Disease
    Carley N Gray, Manickam Ashokkumar ... Michael Emerman
    Research Article

    The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing toward inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells of people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo, and we detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout, suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Finally, we found that INTS12 more generally limits the efficacy of a variety of LRAs with different mechanisms of action.