Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26.

TitleFunctional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26.
Publication TypeJournal Article
Year of Publication2018
AuthorsHogue, IB, Jean, J, Esteves, AD, Tanneti, NS, Scherer, J, Enquist, LW
JournalJ Virol
Date Published2018 Jan 01
KeywordsAxonal Transport, Capsid Proteins, Cells, Cultured, Cryoelectron Microscopy, Herpesvirus 1, Suid, Luminescent Proteins, Microscopy, Fluorescence, Molecular Structure, Neurons, Recombinant Fusion Proteins, Virus Replication

<p>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. 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.</p>

Alternate JournalJ Virol
PubMed ID29046447
PubMed Central IDPMC5730785
Grant ListR01 NS033506 / NS / NINDS NIH HHS / United States
R01 NS060699 / NS / NINDS NIH HHS / United States
K22 AI123159 / AI / NIAID NIH HHS / United States
R37 NS033506 / NS / NINDS NIH HHS / United States
T32 GM007388 / GM / NIGMS NIH HHS / United States
F32 GM112337 / GM / NIGMS NIH HHS / United States