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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.

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Related in: MedlinePlus

Raft association of Us9 is critical to anterograde spread of infection in vitro.(A) Non-differentiated and (B) differentiated PC12 Cells were infected with PRV 322 for 12 hours, and then lysed with cold 1% Triton X-100. Lysates were separated on a discontinuous Optiprep™ density gradient, and analyzed by SDS-PAGE. Western blot analysis was performed using biotinylated cholera toxin B subunit (for detection of GM1), and antiserum specific for Us9 and TfR. (C) Trichamber diagram illustrating the system used to measure PRV anterograde spread of infection from neurons to PK15 cells. SCG neurons were plated in the S chamber and allowed to extend neurites into the N chamber. The neurites were guided into the N compartment by a series of grooves. A monolayer of indicator PK15 cells were then plated on top of the axon termini in the N chamber. Cell bodies in the S chamber were infected, virus particles sorted into axons in a Us9-dependent manner, and these particles subsequently infected the PK15 cells that amplified the infection. Cultured neurons in the S chamber were infected at a high MOI with Becker (wild-type), PRV 160 (Us9-), PRV 322 (Us9-TfR), or both Becker and PRV 322. 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 N chambers. Total plaque-forming units (PFU)/ml were determined for each chamber. The median value for the four samples is denoted by the offset open symbol. The P value (p*) was determined using the Wilcoxon two-sample test.
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ppat-1000065-g007: Raft association of Us9 is critical to anterograde spread of infection in vitro.(A) Non-differentiated and (B) differentiated PC12 Cells were infected with PRV 322 for 12 hours, and then lysed with cold 1% Triton X-100. Lysates were separated on a discontinuous Optiprep™ density gradient, and analyzed by SDS-PAGE. Western blot analysis was performed using biotinylated cholera toxin B subunit (for detection of GM1), and antiserum specific for Us9 and TfR. (C) Trichamber diagram illustrating the system used to measure PRV anterograde spread of infection from neurons to PK15 cells. SCG neurons were plated in the S chamber and allowed to extend neurites into the N chamber. The neurites were guided into the N compartment by a series of grooves. A monolayer of indicator PK15 cells were then plated on top of the axon termini in the N chamber. Cell bodies in the S chamber were infected, virus particles sorted into axons in a Us9-dependent manner, and these particles subsequently infected the PK15 cells that amplified the infection. Cultured neurons in the S chamber were infected at a high MOI with Becker (wild-type), PRV 160 (Us9-), PRV 322 (Us9-TfR), or both Becker and PRV 322. 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 N chambers. Total plaque-forming units (PFU)/ml were determined for each chamber. The median value for the four samples is denoted by the offset open symbol. The P value (p*) was determined using the Wilcoxon two-sample test.

Mentions: Next we tested whether the replacement of the wild-type Us9 TMD with that of transferrin receptor affected the affinity of Us9 for DRMs. We infected undifferentiated and differentiated PC12 cells for 12 hours, solubilized cells with 1% TX-100, and performed flotation analysis as done previously. Instead of Us9 being heavily enriched in the raft fraction as observed in Becker infected cells (see Figures 2 and 3), Us9-TfR was predominantly found in the soluble fraction, especially in differentiated PC12 cells (Figure 7A and 7B). To assess whether this impacted Us9 function in primary neurons, we took advantage of a trichamber neuronal culturing system [17],[37]. Dissociated SCG neurons are plated in the soma (S) chamber and allowed to mature for two weeks (Figure 7C). During this period, axons are directed between a series of grooves across the methocellulose (M) chamber to the neurite (N) chamber. A monolayer of indicator PK15 cells are then plated on top of the neurites in the N chamber. Cell bodies in the S chamber are infected, virus particles sort into axons in a Us9-dependent manner, and subsequently infect the PK15 cells that amplify the infection. The initial infection is confined to the S chamber via silicone vacuum grease and a methocellulose barrier. Therefore, infection spreads to the N chamber solely through axons that emanate from neuronal cell bodies and extend to PK15 cells [17]. We compared the anterograde transport and spread capabilities of PRV Becker (wild-type), PRV 160 (Us9-), PRV 322 (Us9-TfR), and a co-infection of Becker and PRV 322 (Figure 7C, lower panel). Though all of the infections produced a comparable number of infectious virus in the S chamber, spread to second order PK15 cells in the N chamber was dramatically different. PRV Becker spread efficiently from neurons to PK15 cells, producing a median titer of 1.2×107 PFU in the N chamber after 24 hours post-infection (Figure 7C). By contrast, the Us9- mutant (PRV 160) did not spread to PK15 cells and no detectable infectious virus was produced in most dishes. However, in one dish, we detected a low number of infectious particles (1.5×103). We interpret a low yield of amplified virus as a single neuron-to-cell spread event (the burst size of an infected PK15 cell is roughly 1000 PFU). Nevertheless, the neuron-to-cell spread capability of PRV 160 is extremely low compared to wild-type PRV Becker. PRV 322 (Us9-TfR) was completely defective in anterograde spread and was indistinguishable from the Us9- mutant (no infectious virus detected in the N-compartment). This phenotype strongly correlated with the inability of Us9-TfR to target to lipid rafts/DRMs. When neurons were co-infected with both Becker and PRV 322, titers were virtually identical to those seen with Becker alone, indicating that PRV 322 does not have a trans-dominant effect on anterograde spread of infection.


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)

Raft association of Us9 is critical to anterograde spread of infection in vitro.(A) Non-differentiated and (B) differentiated PC12 Cells were infected with PRV 322 for 12 hours, and then lysed with cold 1% Triton X-100. Lysates were separated on a discontinuous Optiprep™ density gradient, and analyzed by SDS-PAGE. Western blot analysis was performed using biotinylated cholera toxin B subunit (for detection of GM1), and antiserum specific for Us9 and TfR. (C) Trichamber diagram illustrating the system used to measure PRV anterograde spread of infection from neurons to PK15 cells. SCG neurons were plated in the S chamber and allowed to extend neurites into the N chamber. The neurites were guided into the N compartment by a series of grooves. A monolayer of indicator PK15 cells were then plated on top of the axon termini in the N chamber. Cell bodies in the S chamber were infected, virus particles sorted into axons in a Us9-dependent manner, and these particles subsequently infected the PK15 cells that amplified the infection. Cultured neurons in the S chamber were infected at a high MOI with Becker (wild-type), PRV 160 (Us9-), PRV 322 (Us9-TfR), or both Becker and PRV 322. 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 N chambers. Total plaque-forming units (PFU)/ml were determined for each chamber. The median value for the four samples is denoted by the offset open symbol. The P value (p*) was determined using the Wilcoxon two-sample test.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000065-g007: Raft association of Us9 is critical to anterograde spread of infection in vitro.(A) Non-differentiated and (B) differentiated PC12 Cells were infected with PRV 322 for 12 hours, and then lysed with cold 1% Triton X-100. Lysates were separated on a discontinuous Optiprep™ density gradient, and analyzed by SDS-PAGE. Western blot analysis was performed using biotinylated cholera toxin B subunit (for detection of GM1), and antiserum specific for Us9 and TfR. (C) Trichamber diagram illustrating the system used to measure PRV anterograde spread of infection from neurons to PK15 cells. SCG neurons were plated in the S chamber and allowed to extend neurites into the N chamber. The neurites were guided into the N compartment by a series of grooves. A monolayer of indicator PK15 cells were then plated on top of the axon termini in the N chamber. Cell bodies in the S chamber were infected, virus particles sorted into axons in a Us9-dependent manner, and these particles subsequently infected the PK15 cells that amplified the infection. Cultured neurons in the S chamber were infected at a high MOI with Becker (wild-type), PRV 160 (Us9-), PRV 322 (Us9-TfR), or both Becker and PRV 322. 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 N chambers. Total plaque-forming units (PFU)/ml were determined for each chamber. The median value for the four samples is denoted by the offset open symbol. The P value (p*) was determined using the Wilcoxon two-sample test.
Mentions: Next we tested whether the replacement of the wild-type Us9 TMD with that of transferrin receptor affected the affinity of Us9 for DRMs. We infected undifferentiated and differentiated PC12 cells for 12 hours, solubilized cells with 1% TX-100, and performed flotation analysis as done previously. Instead of Us9 being heavily enriched in the raft fraction as observed in Becker infected cells (see Figures 2 and 3), Us9-TfR was predominantly found in the soluble fraction, especially in differentiated PC12 cells (Figure 7A and 7B). To assess whether this impacted Us9 function in primary neurons, we took advantage of a trichamber neuronal culturing system [17],[37]. Dissociated SCG neurons are plated in the soma (S) chamber and allowed to mature for two weeks (Figure 7C). During this period, axons are directed between a series of grooves across the methocellulose (M) chamber to the neurite (N) chamber. A monolayer of indicator PK15 cells are then plated on top of the neurites in the N chamber. Cell bodies in the S chamber are infected, virus particles sort into axons in a Us9-dependent manner, and subsequently infect the PK15 cells that amplify the infection. The initial infection is confined to the S chamber via silicone vacuum grease and a methocellulose barrier. Therefore, infection spreads to the N chamber solely through axons that emanate from neuronal cell bodies and extend to PK15 cells [17]. We compared the anterograde transport and spread capabilities of PRV Becker (wild-type), PRV 160 (Us9-), PRV 322 (Us9-TfR), and a co-infection of Becker and PRV 322 (Figure 7C, lower panel). Though all of the infections produced a comparable number of infectious virus in the S chamber, spread to second order PK15 cells in the N chamber was dramatically different. PRV Becker spread efficiently from neurons to PK15 cells, producing a median titer of 1.2×107 PFU in the N chamber after 24 hours post-infection (Figure 7C). By contrast, the Us9- mutant (PRV 160) did not spread to PK15 cells and no detectable infectious virus was produced in most dishes. However, in one dish, we detected a low number of infectious particles (1.5×103). We interpret a low yield of amplified virus as a single neuron-to-cell spread event (the burst size of an infected PK15 cell is roughly 1000 PFU). Nevertheless, the neuron-to-cell spread capability of PRV 160 is extremely low compared to wild-type PRV Becker. PRV 322 (Us9-TfR) was completely defective in anterograde spread and was indistinguishable from the Us9- mutant (no infectious virus detected in the N-compartment). This phenotype strongly correlated with the inability of Us9-TfR to target to lipid rafts/DRMs. When neurons were co-infected with both Becker and PRV 322, titers were virtually identical to those seen with Becker alone, indicating that PRV 322 does not have a trans-dominant effect on anterograde spread of infection.

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