Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Oct;82(20):10308-11.
doi: 10.1128/JVI.01204-08. Epub 2008 Aug 6.

Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons

M G Lyman  1 , D Curanovic, A D Brideau, L W Enquist
Affiliations

Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons

M G Lyman et al. J Virol. 2008 Oct.

Abstract

Pseudorabies virus encodes a membrane protein (Us9) that is essential for the axonal sorting of virus particles within neurons and anterograde spread in the mammalian nervous system. Enhanced green fluorescent protein (GFP)-tagged Us9 mimicked the trafficking properties of the wild-type protein in nonneuronal cells. We constructed a pseudorabies virus strain that expressed Us9-GFP and tested its spread capabilities in the rat visual system and in primary neuronal cultures. We report that Us9-EGFP does not promote anterograde spread of infection and may disrupt packing of viral membrane proteins in lipid rafts, an essential step for Us9-mediated axonal sorting.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Analysis of Us9-EGFP mediated anterograde spread in the rat visual system. (A) Schematic representation of the genome of PRV 164. A CMV Us9-GFP expression cassette was inserted into the gG locus (striped box upstream of gD), and the endogenous Us9 open reading frame was deleted (black box between gE and Us2). IR, inverted repeat. (B) Approximately 2.5 × 105 PFU of either PRV Becker or PRV 164 was injected into the vitreous humor of Sprague-Dawley male rats. At the time of imminent death, the animals were sacrificed and the brains were fixed and sliced into 35-μm-thick coronal sections with a freezing microtome. Infected tissue was stained for PRV antigen or Us9-EGFP (with Rb133 or Us9 rabbit polyvalent antiserum, respectively). Areas reached by anterograde, transneuronal spread are highlighted in red. D, dorsal; V, ventral.
FIG. 2.
FIG. 2.
Dropping the EGFP tag from Us9 restores anterograde, neuron-to-cell spread in vitro. (A) Schematic of the PRV strains 151, 164, and 328. IR, inverted repeat. (B) SCG neurons were plated in the S compartment and allowed to extend neurites into the N compartment for 2 weeks. Axons were guided by a series of grooves scratched into the surface of the tissue culture dish. After 2 weeks, a monolayer of indicator PK15 cells was plated on top of the axon termini in the N compartment. Cell bodies in the S compartment were then infected at an MOI of 10 with PRV 151, 164, or 328. Four chambers were used for each type of infection (closed symbols). At 24 hpi, medium and infected cells were harvested together from either the S or the N compartment. Total PFU/ml were determined for each chamber. The mean value for the four samples is denoted by the offset open symbol. Black arrowheads denote the two plates infected with PRV 164 that showed no anterograde, neuron-to-cell spread. M, methocellulose compartment.
FIG. 3.
FIG. 3.
Targeting of Us9-EGFP to detergent-resistant microdomains. Differentiated PC12 cells were infected with PRV 328 (A) or PRV 164 (B) for 12 h and then lysed with cold 1% Triton X-100. Lysates were separated on a discontinuous Optiprep density gradient (5%, 30%, and 40%) by ultracentrifugation at 4°C for 20 h (13). Detergent-insoluble complexes (i.e., lipid rafts) floated to the 5%/30% interface, while detergent-soluble proteins remained at the bottom of the gradient. Fractions were collected from the top to the bottom of the tube (1 ml each). Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting analysis was performed using biotinylated cholera toxin B subunit (for GM1) and antiserum to PRV Us9 and transferrin receptor (TfR). Numbers at right of each panel are molecular masses in kilodaltons.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Banfield, B. W., G. S. Yap, A. C. Knapp, and L. W. Enquist. 1998. A chicken embryo eye model for the analysis of alphaherpesvirus neuronal spread and virulence. J. Virol. 724580-4588. - PMC - PubMed
    1. Brideau, A. D., B. W. Banfield, and L. W. Enquist. 1998. The Us9 gene product of pseudorabies virus, an alphaherpesvirus, is a phosphorylated, tail-anchored type II membrane protein. J. Virol. 724560-4570. - PMC - PubMed
    1. Brideau, A. D., J. P. Card, and L. W. Enquist. 2000. Role of pseudorabies virus Us9, a type II membrane protein, in infection of tissue culture cells and the rat nervous system. J. Virol. 74834-845. - PMC - PubMed
    1. Brideau, A. D., T. del Rio, E. J. Wolffe, and L. W. Enquist. 1999. Intracellular trafficking and localization of the pseudorabies virus Us9 type II envelope protein to host and viral membranes. J. Virol. 734372-4384. - PMC - PubMed
    1. Brideau, A. D., M. G. Eldridge, and L. W. Enquist. 2000. Directional transneuronal infection by pseudorabies virus is dependent on an acidic internalization motif in the Us9 cytoplasmic tail. J. Virol. 744549-4561. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources