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Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection.

ABSTRACT: In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP-pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP-pUL25 and FP-pUL36 preserved wild-type properties better than traditional FP-pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

SUBMITTER: Bohannon KP

PROVIDER: S-EPMC3352333 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection.

Bohannon Kevin P KP Sollars Patricia J PJ Pickard Gary E GE Smith Gregory A GA

The Journal of general virology 20111005 Pt 1

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP-pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The proper ...[more]

PMID: 21976610

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Project description:Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.

Dataset Information

Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection.

Publications

Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection.

Similar Datasets

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