Dissecting the Herpesvirus Architecture by Targeted Proteolysis

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Herpesviruses possess a complex structure with an icosahedral capsid, lipid envelope, and a tegument layer composed of numerous proteins.
• Understanding the precise arrangement and interactions of tegument proteins is crucial for deciphering viral assembly and infection mechanisms.
• Current knowledge of tegument architecture relies heavily on indirect evidence from reductionist models.

Purpose of the Study:

• To develop and apply a novel method for probing capsid-tegument interactions in intact herpesvirus particles.
• To precisely map the anchoring interactions of the pUL36 tegument protein with the viral capsid.
• To elucidate the direct and indirect binding of other tegument proteins to the capsid.

Main Methods:

• Engineered tobacco etch virus (TEV) protease sites into the pUL36 protein of pseudorabies virus.
• Utilized fluorescent tags on capsid and tegument components to monitor protein localization.
• Permeabilized purified viral particles and selectively cleaved pUL36 with TEV protease to assess capsid interactions in situ.

Main Results:

• The pUL36 large tegument protein stably anchors to the viral capsid exclusively at its carboxyl terminus.
• Tegument proteins pUL37 and pUS3 are indirectly tethered to the capsid through interactions with pUL36.
• Tegument proteins pUL16, pUL47, pUL48, and pUL49 do not appear to form stable direct or indirect interactions with the capsid.

Conclusions:

• This study provides the first direct evidence for the specific anchoring site of pUL36 on the herpesvirus capsid.
• The findings reveal a hierarchical organization within the tegument layer, with pUL36 acting as a key scaffold.
• The developed methodology offers a powerful tool for dissecting the complex protein interactions within herpesvirus particles.