New 3D imaging approach reveals intricate steps of virus assembly

Left: A 2D CryoSXT projection of an HSV-1 evelopment event. Right: A 3D rendering of the budding assembly stage captured by CLXT. Image credit: Dr Kamal Nahas (CC BY 4.0)

This as a fundamental study that comprehensively examines the roles of nine structural proteins in herpes simplex virus 1 (HSV-1) viral assembly. They say the thoroughly executed research yields compelling data that explain previously unknown functions of HSV-1 structural proteins. 

HSV-1 is a large virus that infects the mucous membranes of the mouth and genitals, causing life-long latent infections. The virus is composed of three layers – a capsid that contains the viral DNA, a protein layer called the tegument and an outer envelope that is studded with viral glycoproteins (proteins with a sugar attached). During replication, newly copied viral genomes are packaged up into this three-layer structure in a process called viral assembly. While some drugs can block the virus’ DNA replication and alleviate symptoms, there is no permanent cure. A deeper understanding of the assembly process could inform the design of novel treatments or cures that inhibit virus formation.. But until now, pinpointing the role of different HSV-1 components in the viral assembly process has proved challenging.

Viral assembly involves a multi-step process, starting in the cell’s nucleus with assembly of capsids, packaging of the DNA to form ‘nucleocapsids’, and transport of these nucleocapsids out of the nucleus via a process of primary envelopment and de-envelopment to travel across the nuclear envelope. This is followed by a secondary envelopment in the cellular area surrounding the nucleus, called the cytoplasm (cytoplasmic envelopment). Imaging methods that maintain the HSV-1-infected cells as close to physiological conditions as possible are needed to fully understand this complex, three-dimensional (3D) assembly process.

The authors used an emerging 3D imaging approach to study the envelopment mechanism and investigate the importance of different HSV-1 genes for viral assembly by investigating the impact of specific mutation of these viral genes. Their new approach combined two methods – cryo-structured illumination microscopy (cryoSIM) to detect fluorescently labelled capsid or envelope components, and cryo-soft-X-ray tomography (cryoSXT) to identify the cellular substructure in the same infected cells. Together, this ‘correlative light X-ray tomography’ (CLXT) approach makes it possible to identify specific structural components within the viral assembly process, allowing the team to visualise exactly where the assembly process stalls for each mutant virus, and providing insights into the unmutated gene’s usual role in viral assembly.

The authors captured different assembly stages during cytoplasmic envelopment using their mutant viruses and showed that – contrary to previous theories – cytoplasmic envelopment is caused by the budding of a capsid into an intracellular membrane ‘sack’ or vesicle, and not by the capsid being ‘wrapped’ by the vesicle membrane. A further new finding is that this budding is asymmetric; the team observed several instances of stalled viral assembly where groups of capsids were gathered at one region, or side, of a spherical vesicle.
 

For more information, please see: 'New 3D imaging approach reveals intricate steps of virus assembly' – https://elifesciences.org/for-the-press/f0131ff7/new-3d-imaging-approach-reveals-intricate-steps-of-virus-assembly

Reviewed Preprint title: 'Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis'

DOI link to full study: https://doi.org/10.7554/eLife.105209.1 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)