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Investigating HCMV entry into host cells by STEM tomography.

Mohamed E A Abdellatif1, Christian Sinzger2, Paul Walther1

  • 1Central Facility for Electron Microscopy, Ulm University, 89081 Ulm, Germany.

Journal of Structural Biology
|October 24, 2018
PubMed
Summary

This study visualizes human cytomegalovirus (HCMV) entry into cells using advanced 3D imaging, revealing novel intermediate steps and viral particle alterations during cell entry. The findings offer new insights into HCMV infection mechanisms.

Keywords:
ActinCytomegalovirus entryDynamin-2High-pressure freezingSTEM tomographyThree-dimensional

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Area of Science:

  • Virology
  • Cell Biology
  • Microscopy

Background:

  • Human cytomegalovirus (HCMV) entry is a complex process lacking detailed ultrastructural characterization.
  • Previous studies relied on biochemical and inhibitor assays, missing key morphological details of HCMV cell entry.

Purpose of the Study:

  • To provide a clear morphological characterization of human cytomegalovirus (HCMV) entry intermediates at the ultrastructural level.
  • To develop and apply a novel methodological approach for 3D imaging of HCMV entry.

Main Methods:

  • Developed a method involving rapid freezing and cryosubstitution for optimal ultrastructure preservation.
  • Utilized Scanning Transmission Electron Microscopy (STEM) tomography for 3D data acquisition.
  • Optimized cell growth and infection conditions for biologically active HCMV particles.

Main Results:

  • Observed and characterized extracellular and intracellular intermediates of HCMV entry.
  • Identified morphologically altered "Pinocchio particles" associated with cell membranes.
  • Characterized intracellular fusion events between viral envelopes and cellular vesicular membranes.
  • Visualized fusion-like intermediates at the plasma membrane upon inhibiting actin polymerization with Latrunculin-A.
  • Found that Dyngo-4a (dynamin-2 inhibitor) does not impede HCMV internalization.

Conclusions:

  • The study provides unprecedented ultrastructural insights into the dynamic process of HCMV cell entry.
  • Novel intermediate structures and viral particle alterations during entry have been identified.
  • The findings clarify the roles of actin polymerization and dynamin-2 in HCMV internalization.