Related Experiment Video
Updated: Aug 18, 2026
Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells
Published on: September 7, 2018
Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron
Xuekui Yu1, Phong Trang, Sanket Shah
1Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA 94720, USA.
Abstract:
Human CMV (HCMV) is the leading viral cause of birth defects and causes one of the most common opportunistic infections among transplant recipients and AIDS patients. Cleavage of internal scaffolding proteins by the viral protease (Pr) occurs during HCMV capsid assembly. To gain insight into the mechanism of HCMV capsid maturation and the roles of the Pr in viral replication, an RNase P ribozyme was engineered to target the Pr mRNA and down-regulate its expression by >99%, generating premature Pr-minus capsids. Furthermore, scaffolding protein processing and DNA encapsidation were inhibited by 99%, and viral growth was reduced by 10,000-fold. 3D structural comparison of the Pr-minus and wild-type B capsids by electron cryomicroscopy, at an unprecedented 12.5-angstroms resolution, unexpectedly revealed that the structures are identical in their overall shape and organization. However, the Pr-minus capsid contains tenuous connections between the scaffold and the capsid shell, whereas the wild-type B capsid has extra densities in its core that may represent the viral Pr. Our findings indicate that cleavage of the scaffolding protein is not associated with the morphological changes that occur during capsid maturation. Instead, the protease appears to be required for DNA encapsidation and the subsequent maturation steps leading to infectious progeny. These results therefore provide key insights into an essential step of HCMV infection using an RNase P ribozyme-based inhibition strategy.
Insights
Human Cytomegalovirus (HCMV) protease is essential for viral replication, not capsid maturation. Inhibiting the protease prevents DNA packaging and progeny formation, revealing its role in later stages of HCMV infection.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Human Cytomegalovirus (HCMV) is a significant cause of birth defects and opportunistic infections.
- HCMV capsid assembly involves cleavage of scaffolding proteins by the viral protease (Pr).
Purpose of the Study:
- To investigate the role of the HCMV protease in capsid maturation and viral replication.
- To understand the mechanism of HCMV capsid assembly and maturation.
Main Methods:
- Engineered an RNase P ribozyme to specifically target and down-regulate HCMV protease mRNA expression (>99% reduction).
- Utilized electron cryomicroscopy for 3D structural comparison of Pr-minus and wild-type HCMV capsids at 12.5 angstroms resolution.
Main Results:
- Pr-minus capsids showed inhibited scaffolding protein processing and DNA encapsidation (99% reduction), leading to a 10,000-fold decrease in viral growth.
- Structural analysis revealed identical overall shapes for Pr-minus and wild-type capsids, but Pr-minus capsids lacked core densities and had tenuous scaffold-shell connections.
- Cleavage of scaffolding protein was not linked to morphological changes during capsid maturation.
Conclusions:
- HCMV protease is crucial for DNA encapsidation and subsequent maturation steps, not for initial capsid morphological changes.
- The viral protease plays a vital role in the later stages of HCMV replication.
- RNase P ribozyme-mediated inhibition provides a powerful tool for studying HCMV essential genes.
