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Tagging of the vaccinia virus protein F13 with mCherry causes aberrant virion morphogenesis
David C J Carpentier1, Michael S Hollinshead1, Helen A Ewles1
1Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
Abstract:
Vaccinia virus produces two distinct infectious virions; the single-enveloped intracellular mature virus (IMV), which remains in the cell until cell lysis, and the double-enveloped extracellular enveloped virus (EEV), which mediates virus spread. The latter is derived from a triple-enveloped intracellular enveloped virus (IEV) precursor, which is transported to the cell periphery by the kinesin-1 motor complex. This transport involves the viral protein A36 as well as F12 and E2. A36 is an integral membrane protein associated with the outer virus envelope and is the only known direct link between virion and kinesin-1 complex. Yet in the absence of A36 virion egress still occurs on microtubules, albeit at reduced efficiency. In this paper double-fluorescent labelling of the capsid protein A5 and outer-envelope protein F13 was exploited to visualize IEV transport by live-cell imaging in the absence of either A36 or F12. During the generation of recombinant viruses expressing both A5-GFP and F13-mCherry a plaque size defect was identified that was particularly severe in viruses lacking A36. Electron microscopy showed that this phenotype was caused by abnormal wrapping of IMV to form IEV, and this resulted in reduced virus egress to the cell surface. The aberrant wrapping phenotype suggests that the fluorescent fusion protein interferes with an interaction of F13 with the IMV surface that is required for tight association between IMVs and wrapping membranes. The severity of this defect suggests that these viruses are imperfect tools for characterizing virus egress.
Insights
Vaccinia virus protein A36 is crucial for efficient wrapping of intracellular mature virus (IMV) into intracellular enveloped virus (IEV). Fluorescent labeling revealed defects in IMV wrapping and reduced virus egress when A36 is absent.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Vaccinia virus produces two infectious forms: intracellular mature virus (IMV) and extracellular enveloped virus (EEV).
- EEV is derived from the intracellular enveloped virus (IEV) precursor, transported via kinesin-1 motor complex involving viral proteins A36, F12, and E2.
- A36 is the sole known direct link between the virion and the kinesin-1 complex, though virion egress occurs without it.
Purpose of the Study:
- To investigate the role of viral proteins A36 and F12 in intracellular enveloped virus (IEV) transport and formation.
- To visualize IEV transport using live-cell imaging with double-fluorescently labeled viral proteins.
Main Methods:
- Generation of recombinant vaccinia viruses expressing capsid protein A5-GFP and outer-envelope protein F13-mCherry.
- Live-cell imaging to visualize IEV transport in the absence of A36 or F12.
- Plaque assays to assess virus infectivity and replication.
- Electron microscopy to examine virion structure and wrapping defects.
Main Results:
- Viruses lacking A36 exhibited a severe plaque size defect.
- Electron microscopy revealed abnormal wrapping of IMV to form IEV in A36-deficient viruses.
- This aberrant wrapping led to reduced virus egress to the cell surface.
- Fluorescent fusion proteins (A5-GFP, F13-mCherry) interfered with F13 interaction with IMV surface, impacting wrapping.
Conclusions:
- Protein A36 plays a significant role in the efficient wrapping of IMV into IEV.
- The observed aberrant wrapping phenotype suggests that fluorescently tagged proteins are imperfect tools for studying vaccinia virus egress.
- Further research is needed to understand the precise mechanisms of IEV formation and transport.