Limits...
Targeting of pseudorabies virus structural proteins to axons requires association of the viral Us9 protein with lipid rafts.

Lyman MG, Curanovic D, Enquist LW - PLoS Pathog. (2008)

Bottom Line: In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins.In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs).We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.

ABSTRACT
The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.

Show MeSH

Related in: MedlinePlus

Live-cell imaging of GFP-tagged capsid viruses in differentiated PC12 cells.Cells were grown on glass-bottom MatTek dishes coated with poly-DL-ornithine and natural mouse laminin prior to imaging on a Leica SP5 confocal microscope. Each frame of the movie, a 2D projection representing a stack of 15 images that are 0.5 µm apart, contains a scale bar (in microns) and timestamp from the movie sequence. (A) A plate of differentiated PC12 cells was infected with PRV GS443 (green capsid) at a low MOI (0.1) and imaged at 12 hours post-infection (hpi). Numerous green capsid puncta were observed in neurites moving in the anterograde direction, i.e. away from the cell body (see Video S1 in supplemental material). This cell was chosen because it had several axon projections to emphasize the sorting phenotypes. Most differentiated PC12 cells have fewer axon projections (1–3). (B) Differentiated PC12 cells were infected with PRV 368 (green capsid, Us9-) at low MOI and imaged at 12 hpi. The cell body shows robust green fluorescence, but virus capsids were not observed in neurites emanating from the cell body (see Video S2). (C) DIC image showing three neurites extending from the PRV 368-infected cell body (highlighted by the white arrowheads). (D) A differentiated PC12 infected with PRV 368 for 12 hours. No capsids are observed moving in the anterograde direction in two neurites emanating from the cell body (white arrowheads, see Video S3). Note that capsids are not present beyond the proximal segment of the axon. (E and F) Though no green capsid puncta are moving in the anterograde direction (white arrowheads), capsids can be observed moving in a transneuronal, retrograde manner from the infected PC12 cell (red arrowheads, see Video S3) to an uninfected cell above it (panel F, see Video S4). Despite an abundance of moving capsid puncta within the cell body, no other egress events are visible. The brightness in panel F has been increased to better visualize green capsid puncta moving into the uninfected cell body (highlighted by red arrowheads). A white asterisk denotes the accumulation of capsid puncta in the cell body of the uninfected cell.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2361720&req=5

ppat-1000065-g001: Live-cell imaging of GFP-tagged capsid viruses in differentiated PC12 cells.Cells were grown on glass-bottom MatTek dishes coated with poly-DL-ornithine and natural mouse laminin prior to imaging on a Leica SP5 confocal microscope. Each frame of the movie, a 2D projection representing a stack of 15 images that are 0.5 µm apart, contains a scale bar (in microns) and timestamp from the movie sequence. (A) A plate of differentiated PC12 cells was infected with PRV GS443 (green capsid) at a low MOI (0.1) and imaged at 12 hours post-infection (hpi). Numerous green capsid puncta were observed in neurites moving in the anterograde direction, i.e. away from the cell body (see Video S1 in supplemental material). This cell was chosen because it had several axon projections to emphasize the sorting phenotypes. Most differentiated PC12 cells have fewer axon projections (1–3). (B) Differentiated PC12 cells were infected with PRV 368 (green capsid, Us9-) at low MOI and imaged at 12 hpi. The cell body shows robust green fluorescence, but virus capsids were not observed in neurites emanating from the cell body (see Video S2). (C) DIC image showing three neurites extending from the PRV 368-infected cell body (highlighted by the white arrowheads). (D) A differentiated PC12 infected with PRV 368 for 12 hours. No capsids are observed moving in the anterograde direction in two neurites emanating from the cell body (white arrowheads, see Video S3). Note that capsids are not present beyond the proximal segment of the axon. (E and F) Though no green capsid puncta are moving in the anterograde direction (white arrowheads), capsids can be observed moving in a transneuronal, retrograde manner from the infected PC12 cell (red arrowheads, see Video S3) to an uninfected cell above it (panel F, see Video S4). Despite an abundance of moving capsid puncta within the cell body, no other egress events are visible. The brightness in panel F has been increased to better visualize green capsid puncta moving into the uninfected cell body (highlighted by red arrowheads). A white asterisk denotes the accumulation of capsid puncta in the cell body of the uninfected cell.

Mentions: It is difficult to culture a sufficient number of primary rat neurons to perform large biochemical analyses. Therefore, we used PC12 cells, a widely used rat pheochromocytoma cell line that responds to nerve growth factor (NGF) and acquires many of the characteristics of sympathetic neurons [46]. Differentiated PC12 undergo polarized protein sorting, and cell bodies stain for nonphosphorylated neurofilament H (a somatodendritic marker) while axons stain exclusively for phosphorylated neurofilament H (an axonal marker) [32]. This is consistent with mature, sympathetic SCG neurons [47]. It has been reported that PC12 cell are susceptible and permissive to PRV infection [48], and that a PRV GFP-VP22 fusion protein moves inside neurites with fast axonal kinetics [49]. However, it was unclear whether the Us9- phenotype in SCG neurons, i.e. a complete block to axonal sorting of viral structural proteins [15],[16], could be recapitulated in this neuron-like cell line (a critical experiment to ensure that PC12 cells could be used to study Us9 biology). We recently reported that in the absence of Us9, GFP-tagged capsids were unable to sort into axons of live SCG neurons [15]. Therefore, we utilized a similar live-cell imaging approach to examine the axonal sorting of GFP-tagged capsids in differentiated PC12 cells. Cells were infected with PRV GS443, a recombinant PRV strain that expresses GFP fused to VP26, a capsid protein [50]. After 12 h, capsid puncta were readily observed trafficking in the anterograde direction within neurites of PC12 cells (n = 20) (Figure 1A, Video S1). Importantly, when differentiated PC12 cells were infected with PRV 368, a GFP-tagged capsid mutant deleted for Us9, no green puncta were observed moving in the anterograde direction (Figure 1B and 1C, Video S2). These findings were consistent with our Us9 studies in dissociated SCG neurons [15]. Interestingly, we also observed the retrograde trafficking of capsid puncta from cells infected with PRV 368 to uninfected, neighboring cells (in the absence of any anterograde sorting of virus particles in the same field of view) (Figure 1D–1F, Videos S3 and S4). This had been described previously in transneuronal spread studies on Us9 mutants in the rat visual system [11],[26], but had not been observed in tissue culture cells. It is noteworthy that we did not visualize “random” egress of GFP-tagged capsids from infected cell bodies. Capsids either sorted into axons (in the presence of Us9), or to sites of synaptic contact with other axons (transneuronal, retrograde transport). The import of this observation is unclear at present, but may suggest that alpha herpesviruses undergo directed egress from neuronal cell bodies. Overall, our findings suggest that differentiated PC12 cells recapitulate the Us9 sorting phenotypes previously observed in primary sympathetic neurons, and are an efficacious cell line to study Us9 biology (specifically that of Us9 and lipid rafts).


Targeting of pseudorabies virus structural proteins to axons requires association of the viral Us9 protein with lipid rafts.

Lyman MG, Curanovic D, Enquist LW - PLoS Pathog. (2008)

Live-cell imaging of GFP-tagged capsid viruses in differentiated PC12 cells.Cells were grown on glass-bottom MatTek dishes coated with poly-DL-ornithine and natural mouse laminin prior to imaging on a Leica SP5 confocal microscope. Each frame of the movie, a 2D projection representing a stack of 15 images that are 0.5 µm apart, contains a scale bar (in microns) and timestamp from the movie sequence. (A) A plate of differentiated PC12 cells was infected with PRV GS443 (green capsid) at a low MOI (0.1) and imaged at 12 hours post-infection (hpi). Numerous green capsid puncta were observed in neurites moving in the anterograde direction, i.e. away from the cell body (see Video S1 in supplemental material). This cell was chosen because it had several axon projections to emphasize the sorting phenotypes. Most differentiated PC12 cells have fewer axon projections (1–3). (B) Differentiated PC12 cells were infected with PRV 368 (green capsid, Us9-) at low MOI and imaged at 12 hpi. The cell body shows robust green fluorescence, but virus capsids were not observed in neurites emanating from the cell body (see Video S2). (C) DIC image showing three neurites extending from the PRV 368-infected cell body (highlighted by the white arrowheads). (D) A differentiated PC12 infected with PRV 368 for 12 hours. No capsids are observed moving in the anterograde direction in two neurites emanating from the cell body (white arrowheads, see Video S3). Note that capsids are not present beyond the proximal segment of the axon. (E and F) Though no green capsid puncta are moving in the anterograde direction (white arrowheads), capsids can be observed moving in a transneuronal, retrograde manner from the infected PC12 cell (red arrowheads, see Video S3) to an uninfected cell above it (panel F, see Video S4). Despite an abundance of moving capsid puncta within the cell body, no other egress events are visible. The brightness in panel F has been increased to better visualize green capsid puncta moving into the uninfected cell body (highlighted by red arrowheads). A white asterisk denotes the accumulation of capsid puncta in the cell body of the uninfected cell.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361720&req=5

ppat-1000065-g001: Live-cell imaging of GFP-tagged capsid viruses in differentiated PC12 cells.Cells were grown on glass-bottom MatTek dishes coated with poly-DL-ornithine and natural mouse laminin prior to imaging on a Leica SP5 confocal microscope. Each frame of the movie, a 2D projection representing a stack of 15 images that are 0.5 µm apart, contains a scale bar (in microns) and timestamp from the movie sequence. (A) A plate of differentiated PC12 cells was infected with PRV GS443 (green capsid) at a low MOI (0.1) and imaged at 12 hours post-infection (hpi). Numerous green capsid puncta were observed in neurites moving in the anterograde direction, i.e. away from the cell body (see Video S1 in supplemental material). This cell was chosen because it had several axon projections to emphasize the sorting phenotypes. Most differentiated PC12 cells have fewer axon projections (1–3). (B) Differentiated PC12 cells were infected with PRV 368 (green capsid, Us9-) at low MOI and imaged at 12 hpi. The cell body shows robust green fluorescence, but virus capsids were not observed in neurites emanating from the cell body (see Video S2). (C) DIC image showing three neurites extending from the PRV 368-infected cell body (highlighted by the white arrowheads). (D) A differentiated PC12 infected with PRV 368 for 12 hours. No capsids are observed moving in the anterograde direction in two neurites emanating from the cell body (white arrowheads, see Video S3). Note that capsids are not present beyond the proximal segment of the axon. (E and F) Though no green capsid puncta are moving in the anterograde direction (white arrowheads), capsids can be observed moving in a transneuronal, retrograde manner from the infected PC12 cell (red arrowheads, see Video S3) to an uninfected cell above it (panel F, see Video S4). Despite an abundance of moving capsid puncta within the cell body, no other egress events are visible. The brightness in panel F has been increased to better visualize green capsid puncta moving into the uninfected cell body (highlighted by red arrowheads). A white asterisk denotes the accumulation of capsid puncta in the cell body of the uninfected cell.
Mentions: It is difficult to culture a sufficient number of primary rat neurons to perform large biochemical analyses. Therefore, we used PC12 cells, a widely used rat pheochromocytoma cell line that responds to nerve growth factor (NGF) and acquires many of the characteristics of sympathetic neurons [46]. Differentiated PC12 undergo polarized protein sorting, and cell bodies stain for nonphosphorylated neurofilament H (a somatodendritic marker) while axons stain exclusively for phosphorylated neurofilament H (an axonal marker) [32]. This is consistent with mature, sympathetic SCG neurons [47]. It has been reported that PC12 cell are susceptible and permissive to PRV infection [48], and that a PRV GFP-VP22 fusion protein moves inside neurites with fast axonal kinetics [49]. However, it was unclear whether the Us9- phenotype in SCG neurons, i.e. a complete block to axonal sorting of viral structural proteins [15],[16], could be recapitulated in this neuron-like cell line (a critical experiment to ensure that PC12 cells could be used to study Us9 biology). We recently reported that in the absence of Us9, GFP-tagged capsids were unable to sort into axons of live SCG neurons [15]. Therefore, we utilized a similar live-cell imaging approach to examine the axonal sorting of GFP-tagged capsids in differentiated PC12 cells. Cells were infected with PRV GS443, a recombinant PRV strain that expresses GFP fused to VP26, a capsid protein [50]. After 12 h, capsid puncta were readily observed trafficking in the anterograde direction within neurites of PC12 cells (n = 20) (Figure 1A, Video S1). Importantly, when differentiated PC12 cells were infected with PRV 368, a GFP-tagged capsid mutant deleted for Us9, no green puncta were observed moving in the anterograde direction (Figure 1B and 1C, Video S2). These findings were consistent with our Us9 studies in dissociated SCG neurons [15]. Interestingly, we also observed the retrograde trafficking of capsid puncta from cells infected with PRV 368 to uninfected, neighboring cells (in the absence of any anterograde sorting of virus particles in the same field of view) (Figure 1D–1F, Videos S3 and S4). This had been described previously in transneuronal spread studies on Us9 mutants in the rat visual system [11],[26], but had not been observed in tissue culture cells. It is noteworthy that we did not visualize “random” egress of GFP-tagged capsids from infected cell bodies. Capsids either sorted into axons (in the presence of Us9), or to sites of synaptic contact with other axons (transneuronal, retrograde transport). The import of this observation is unclear at present, but may suggest that alpha herpesviruses undergo directed egress from neuronal cell bodies. Overall, our findings suggest that differentiated PC12 cells recapitulate the Us9 sorting phenotypes previously observed in primary sympathetic neurons, and are an efficacious cell line to study Us9 biology (specifically that of Us9 and lipid rafts).

Bottom Line: In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins.In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs).We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.

ABSTRACT
The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.

Show MeSH
Related in: MedlinePlus