Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex...

Area of Science:

• Virology
• Cell Biology
• Microscopy

Background:

• Herpes simplex virus-1 (HSV-1) assembly involves numerous viral genes with poorly understood functions.
• Previous studies lacked correlative light microscopy to identify specific viral components during viral morphogenesis.

Purpose of the Study:

• To investigate the function and relative importance of viral proteins in HSV-1 assembly.
• To characterize viral protein roles in nuclear egress and cytoplasmic envelopment using correlative imaging.

Main Methods:

• Utilized correlative cryo-structured illumination microscopy (cryoSIM) and cryo-soft-X-ray tomography (cryoSXT).
• Imaged fluorescent capsid (eYFP-VP26) and envelope (gM-mCherry) proteins in nine fluorescent HSV-1 mutants.
• Compared viral protein importance in nuclear egress and cytoplasmic envelopment stages.

Main Results:

• Ranked the importance of five viral proteins for nuclear egress (pUL34 >pUL21>VP16>pUL16>pUS3).
• Identified roles for five viral proteins in cytoplasmic envelopment, including VP16 in capsid delivery.
• Observed novel features of stalled envelopment and cytoplasmic viral particle clusters in mutants lacking pUL11, pUL51, gK, and gE.

Conclusions:

• Correlative 3D imaging provides new insights into viral protein functions and HSV-1 morphogenesis.
• Established that HSV-1 envelopment occurs via particle budding, not wrapping.
• Highlighted the utility of tomographic 3D correlative imaging as an emerging technology in virology research.