Herpes simplex virus 1 (HSV-1) is a complex virus that infects mucosal epithelial cells and establishes lifelong latent infections. Its assembly involves a multi-step process, and many of its genes have uncharacterized functions. Previous studies using transmission electron microscopy (TEM) have provided insights, but the extensive sample processing associated with TEM can distort ultrastructure and complicate interpretation. In this study, we utilized an emerging 3D correlative imaging strategy, combining structured illumination microscopy on cryopreserved samples (cryoSIM) and cryo-soft-X-ray tomography (cryoSXT), to investigate the roles of nine HSV-1 genes in virus assembly.
We generated fluorescent HSV-1 mutants, each lacking a different viral protein, and compared their importance in different stages of HSV-1 morphogenesis. The relative importance of five viral proteins in nuclear egress was ranked based on the levels of attenuation observed. Correlative imaging also revealed the roles of five viral proteins in cytoplasmic envelopment. Our findings demonstrate that tomographic 3D correlative imaging is a powerful tool for shedding new light on viral protein functions and virion morphogenesis.
The results of our study provide an extensive and comparative ultrastructural analysis of herpesvirus morphogenesis, revealing new insights into the virion assembly pathway. We observed that the absence of certain viral proteins led to defects in nuclear egress and cytoplasmic envelopment, highlighting the critical roles these proteins play in virus assembly. Our 3D imaging approach captured different assembly stages and determined that envelopment occurs by particle budding rather than wrapping.
Overall, this research contributes to a deeper understanding of HSV-1 assembly and the functions of its viral proteins. The use of advanced imaging techniques allows for a more accurate and detailed analysis, providing valuable insights into the complex processes involved in viral morphogenesis.
