Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy.

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.

• Authors: Xuekui Yu, Phong Trang, Sanket Shah, Ivo Atanasov, Yong-Hwan Kim, Yong Bai, Z Hong Zhou, Fenyong Liu
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 102 Issue 20 Pg. 7103-8 (May 17 2005) ISSN: 0027-8424 [Print] United States
• PMID: 15883374 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Capsid Proteins
• DNA Primers
• RNA, Catalytic
• Viral Proteins
• Ribonuclease P
• Endopeptidases
• human cytomegalovirus protease UL80

Topics

• Blotting, Northern
• Blotting, Western
• Capsid Proteins (metabolism)
• Cells, Cultured
• Cryoelectron Microscopy
• Cytomegalovirus (genetics, metabolism, physiology)
• DNA Primers
• Endopeptidases (metabolism)
• Gene Expression
• Humans
• RNA, Catalytic (metabolism)
• Ribonuclease P (metabolism)
• Viral Proteins (metabolism)
• Virus Replication (physiology)