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.

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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.

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.

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  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

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