New 3D imaging approach reveals intricate steps of virus assembly

A new combination of microscopy methods has revealed exquisite detail of the virus assembly process used by herpes simplex virus during replication.

The research, published today as a Reviewed Preprint in eLife, is described by the editors 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. Additionally, by integrating cryo-light microscopy and soft X-ray tomography, it presents an innovative approach to investigating viral assembly within cells that will be of broad interest to virologists, cellular biologists and structural biologists.

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.

“HSV-1 mutants that cannot make certain proteins have been used to study the role of viral genes in virus assembly, using a method called thin section transmission electron microscopy, or TEM,” says lead author Kamal Nahas, Beamline Scientist at Beamline B24, Diamond Light Source, Harwell Science & Innovation Campus, Didcot, UK. “However, the extensive sample processing required for TEM can distort the microscopic structure and complicate the interpretation of features in viral assembly.”

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.

Using their CLXT approach, they were able to rank the relative importance of five of the mutant viral proteins in the process of nuclear egress. They were also able to reveal the role of a further five viral proteins in the cytoplasmic envelopment stage. For example, a protein called VP16 was found to be important in delivering the capsid to envelopment compartments and is now thought to have a larger role in nuclear egress than previously thought. In addition, the new method revealed that the absence of four other proteins caused virus particles to build up in the cytoplasm where assembly had stalled.

“Our multi-modal imaging strategy has provided novel ultrastructural insight into HSV-1 assembly, allowing the assembly trajectory of normal and mutant viruses to be observed in 3D,” concludes senior author Colin Crump, Professor of Molecular Virology at the University of Cambridge, UK. “Our data underscore the power of correlative fluorescence and X-ray tomography cryo-imaging for interrogating and conducting further studies on the process of virus assembly.”

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