Mechanism of vaccinia virus assembly in infected-cells

In a recent article posted to the bioRxiv* preprint server, researchers explored the driving factors of the vaccinia virus assembly across infected cells.

Study: Cryo-ET of infected cells reveals that a succession of two lattices drives vaccinia virus assembly. Image Credit: Kateryna Kon/Shutterstock
Study: Cryo-ET of infected cells reveals that a succession of two lattices drives vaccinia virus assembly. Image Credit: Kateryna Kon/Shutterstock

Background

Variola virus causing smallpox and monkeypox are examples of poxviruses inducing illness among humans. Other poxviruses, such as the modified vaccinia Ankara (MVA) and vaccinia virus, are employed as vaccine vectors. 

Additionally, these extensive double-stranded deoxyribonucleic acid (DNA) viruses replicate and assemble their virions across perinuclear viral factories in the cytoplasm. The cytoplasm of infected cells is where the vaccinia virus and other poxviruses, including monkeypox, first gather spherical non-infectious immature virions (IV). The viral D13 lattice covers the IV membrane entirely, enclosing the DNA genome and the viral proteins. Following the loss of the D13 lattice, IV develops into the brick-shaped intracellular mature virus (IMV), which has a well-organized core encasing the genome.

Despite their significance, it is still unclear how infectious poxvirus virions congregate in the infected-cells cytoplasm. The primary structural factors that control the alterations appearing during the IV to IMV conversion and those that characterize the IMV are yet uncertain.

About the study

In the present study, the investigators used cryo-electron tomography (cryo-ET) of frozen-hydrated vaccinia-infected cells to structurally define the maturation procedure of spherical IVs into the infectious brick-shaped IMV in situ.

The authors created recombinant Western Reserve (WR) vaccinia strains missing F11 since this stops infected cells from rounding up early in infection to conduct cryo-electron microscopy (cryo-EM) investigations. Further, cryo-tomograms of IMV and IV-containing thin cellular areas were obtained from the edges of HeLa cells exposed to the vaccinia virus for eight hours. Recombinant D13 in isolation was structurally analyzed to gain a thorough molecular comprehension of the D13 trimer.

In addition, the team evaluated the structure of the IMV in infected cells. They carried out subtomogram averaging independently with intracellular enveloped virions (IEV), IMV, extracellular enveloped virions (EEV), and cell-associated enveloped virus (CEV) particles to examine the structure of the palisade. To better understand the corrugation mechanism during IV maturation to IMV, the researchers assessed the middle-plane perimeter of the viral membrane among IV and IMV, following the viral membrane's wrinkles to determine its contour length.

Results

The team discovered that D13-coated IV have identical lattice dimensions and diameters to spherical D13 ensembles generated in vitro, proving that D13 controls IV size. They found that a part of the D13-coated IV exhibits an extended membrane invagination despite having a relatively consistent diameter. Although IV membrane distortions were seen in some studies, this characteristic has gone largely unreported in earlier EM experiments imaging thin sections.

A novel pseudohexagonal lattice develops within IV during IMV generation, producing the viral core. This lattice consisted of trimeric pillars bearing outward protrusions resembling a palisade when viewed in cross-section. Remarkably, the IV diameter accurately matches the long axis of IMV, indicating core length. Hence, the authors noted that the D13 lattice regulates the IMV. 

The scientists discovered that spherical IV shed their D13-coat and shrink their volume by about 50% during maturation to IMV. Meanwhile, the viral membrane develops corrugations as it conforms to the shape of the lateral bodies and capsid-like palisade layer that make up the core. This inference shows that membrane removal was non-essential to accommodate the 50% volume reduction that occurs when switching from IV to IMV.

Additionally, the palisade was a continuous regular lattice featuring pseudohexagonal symmetry covering the viral core completely and delineating its boundaries. Furthermore, this palisade, which does not change during subsequent virion morphogenesis, was primarily made up of A4 and p4A. This novel viral lattice also decides IMV's dimensions and shape.

Collectively, the study results implied that the sequential D13 and palisade lattices structurally defined vaccinia assembly and maturation.

Conclusions

In the present work, the team applied cryo-ET to image the thin periphery of plunge-frozen, vaccinia virus-infected cells, displaying virus structure in situ. The study gives a broad picture of the vaccinia virus's maturation from IV to IMV, but structural knowledge is still sketchy.

The current high-resolution in situ observations uncovered novel traits regarding the inner core of mature virions and the viral membrane of the vaccinia virus. The emerging hypothesis proposes that the IMV assembly was driven by a sequence of two distinct viral lattices: D13 molds the IV membrane, which later coalesces onto the pseudohexagonal palisade lattice, resembling a viral capsid.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Shanet Susan Alex

Written by

Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.

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