Cryogenic Electron Tomography Redefines Herpesvirus Capsid Assembly Intermediates Inside the Cell Nucleus

Area of Science:

• Structural virology
• Molecular biology
• Cellular imaging

Background:

• Herpesviruses form icosahedral nucleocapsids containing double-stranded DNA (dsDNA) within the host cell nucleus.
• The precise roles of different nucleocapsid structures and the capsid vertex-specific component (CVSC) in herpesvirus replication are not fully understood.
• Previous studies on purified capsids have led to controversial interpretations of their function.

Purpose of the Study:

• To determine the atomic structures of herpesvirus capsids and their components, including the CVSC, within infected cells.
• To investigate the structural heterogeneity of capsids and the role of the CVSC in dsDNA packaging and retention.
• To refine the model of herpesvirus capsid assembly and maturation in situ.

Main Methods:

• Integration of cryogenic focused ion beam milling with electron tomography.
• Subtomogram averaging to derive atomic models of viral capsid proteins.
• Focused classification of pentonal vertex densities to analyze CVSC occupancy.

Main Results:

• Atomic models were generated for all protein components of different herpesvirus capsid types within infected cell nuclei.
• Differences in CVSC occupancy were observed between genome-filled and empty capsids, indicating distinct assembly stages.
• Structural heterogeneity and dynamic CVSC roles in dsDNA packaging and retention were revealed in situ.

Conclusions:

• The findings redefine the herpesvirus capsid maturation model based on in situ structural data.
• This study provides crucial insights into herpesvirus replication mechanisms and the function of the CVSC.
• The research highlights the power of in situ electron imaging for studying virus assembly within host cells.