Structural Syntax of the Vaccinia Virus Entry-Fusion Complex

Area of Science:

• Virology
• Structural Biology
• Computational Biology

Background:

• Poxviruses utilize a unique, uncharacterized mechanism for cell entry.
• This process involves an 11-chain entry-fusion complex (EFC) anchored to the viral envelope.

Purpose of the Study:

• To computationally model the structure of the complete 11-chain entry-fusion complex (holoEFC).
• To elucidate the structural organization and potential assembly pathways of the EFC.
• To investigate the interaction of fusion inhibitor A26 with the EFC.

Main Methods:

• Utilized AlphaFold for computational modeling of the holoEFC.
• Integrated crosslinking mass spectrometry (XLMS) and monolink data for model validation.
• Employed pDockQ, dG_separated, and dSASA for assessing subunit interface authenticity.
• Applied quantitative protein mass spectrometry to determine subunit stoichiometry.

Main Results:

• A computationally confident model of the holoEFC containing all 11 chains was generated.
• Identified novel structural features: an 11-chain transmembrane bundle and a pseudo beta helical connector (PBHC).
• The holoEFC model revealed two distinct subregions with mobility around the PBHC, and a potential assembly pathway for the subregion 2 hexamer.
• Fusion inhibitor A26 was modeled in complex with EFC subcomplex A28:H2:A16:G9, primarily interacting with G9.
• Subunits F9 and J5 were found to be sub-stoichiometric in mature Vaccinia virus preparations.

Conclusions:

• The study provides a high-resolution computational model of the poxvirus EFC, offering insights into its unique cell entry mechanism.
• The identified structural elements, including the PBHC, are critical for understanding EFC function and viral entry.
• The findings lay the groundwork for developing novel antiviral strategies targeting poxvirus entry.