Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

  1. Ke Peng
  2. Walter Muranyi
  3. Bärbel Glass
  4. Vibor Laketa
  5. Stephen R Yant
  6. Luong Tsai
  7. Tomas Cihlar
  8. Barbara Müller
  9. Hans-Georg Kräusslich  Is a corresponding author
  1. Heidelberg University, Germany
  2. German Center for Infection Research, Germany
  3. Gilead Sciences Inc., United States
  4. University Heidelberg, Germany

Abstract

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. Here, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral capsid (CA) protein remains associated with cytoplasmic RTC/PIC, but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.

Article and author information

Author details

  1. Ke Peng

    Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Walter Muranyi

    Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Bärbel Glass

    Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Vibor Laketa

    Partner site Heidelberg, German Center for Infection Research, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Stephen R Yant

    Gilead Sciences Inc., Foster City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Luong Tsai

    Gilead Sciences Inc., Foster City, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Tomas Cihlar

    Gilead Sciences Inc., Foster City, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Barbara Müller

    Department of Infectious Diseases, Virology, University Heidelberg, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Hans-Georg Kräusslich

    Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany
    For correspondence
    hans-georg.kraeusslich@med.uni-heidelberg.de
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Wesley I Sundquist, University of Utah, United States

Version history

  1. Received: July 22, 2014
  2. Accepted: December 15, 2014
  3. Accepted Manuscript published: December 17, 2014 (version 1)
  4. Version of Record published: January 15, 2015 (version 2)

Copyright

© 2014, Peng 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

  • 3,662
    Page views
  • 611
    Downloads
  • 132
    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. Ke Peng
  2. Walter Muranyi
  3. Bärbel Glass
  4. Vibor Laketa
  5. Stephen R Yant
  6. Luong Tsai
  7. Tomas Cihlar
  8. Barbara Müller
  9. Hans-Georg Kräusslich
(2014)
Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid
eLife 3:e04114.
https://doi.org/10.7554/eLife.04114

Share this article

https://doi.org/10.7554/eLife.04114

Further reading

    1. Microbiology and Infectious Disease
    Chiara Andolina, Wouter Graumans ... Teun Bousema
    Research Article

    It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34–501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171–2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Veronica Teresa Ober, George Boniface Githure ... Michael Boshart
    Research Article

    Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.