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A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.

Tomishima MJ, Enquist LW - J. Cell Biol. (2001)

Bottom Line: We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins.The data support a model where virion subassemblies but not complete virions are transported in the axon.Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Pseudorabies virus, an alpha-herpesvirus, is capable of infecting the nervous system and spreading between synaptically connected neurons in diverse hosts. At least three viral membrane proteins (gE, gI, and Us9) are necessary for the spread of infection from presynaptic to postsynaptic neurons (anterograde spread) in infected rodents. To understand how these proteins effect anterograde spread between neurons, we analyzed the subcellular localization of viral proteins after infection of cultured rat sympathetic neurons with wild-type or mutant viruses. After Us9- mutant infections but not gE- mutant infections, only a subset of the viral structural proteins had entered axons. Surprisingly, capsid and tegument proteins but not viral membrane proteins were detected in axons. The spread of Us9 missense mutants in the rodent nervous system correlated with the amount of viral membrane proteins localized to axons. We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins. The data support a model where virion subassemblies but not complete virions are transported in the axon. Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

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Analysis of the neuronal secretory system during PRV infection. (A) Cell surface gE during wild-type or Us9- infections. Neurons were infected such that every neuron was infected for 9 h with the wild-type (a and b) or Us9- virus (c and d). Antibodies that recognize gE were placed directly in the culture medium for 5 min, and then unbound antibodies were removed and the infected neurons were fixed. (B) The steady-state localization of the synaptic vesicle marker SV2. Neurons were mock infected (a) or infected with the wild-type virus (b) or the Us9- virus (c) such that every neuron was infected for 16 h, and then the neurons were fixed and permeabilized. An antibody that recognizes SV2 was then used in indirect immunofluorescence experiments. (C) The subcellular localization of a cellular protein (NgCAM) expressed during PRV infection. A replication- defective virus expressing the cellular protein NgCAM was used to infect cultured neurons. After 1 h, the medium was removed, and cultures were mock infected (a–c) or infected with the wild-type (d–f) or Us9- virus (g–i). a–c show NgCAM localization in the absence of PRV infection. NgCAM appears on the surface of the cell body (a), in vesicles within the axon (b; note that this is an image of a large fasiculation, and therefore many axons are present within the bundle), and on the surface of the axon (c). NgCAM labeled the surface of neurons infected with wild-type virus (d) and Us9- virus (g). The expression of gE during the wild-type (e) and Us9- (h) infections demonstrates that they are infected with PRV. Merged images of the wild-type infection (f) and Us9- infection (i) are shown with NgCAM in green and gE in red. Bars: (A, a and b, B, and C) 25 μm; (A, c and d) 150 μm.
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fig4: Analysis of the neuronal secretory system during PRV infection. (A) Cell surface gE during wild-type or Us9- infections. Neurons were infected such that every neuron was infected for 9 h with the wild-type (a and b) or Us9- virus (c and d). Antibodies that recognize gE were placed directly in the culture medium for 5 min, and then unbound antibodies were removed and the infected neurons were fixed. (B) The steady-state localization of the synaptic vesicle marker SV2. Neurons were mock infected (a) or infected with the wild-type virus (b) or the Us9- virus (c) such that every neuron was infected for 16 h, and then the neurons were fixed and permeabilized. An antibody that recognizes SV2 was then used in indirect immunofluorescence experiments. (C) The subcellular localization of a cellular protein (NgCAM) expressed during PRV infection. A replication- defective virus expressing the cellular protein NgCAM was used to infect cultured neurons. After 1 h, the medium was removed, and cultures were mock infected (a–c) or infected with the wild-type (d–f) or Us9- virus (g–i). a–c show NgCAM localization in the absence of PRV infection. NgCAM appears on the surface of the cell body (a), in vesicles within the axon (b; note that this is an image of a large fasiculation, and therefore many axons are present within the bundle), and on the surface of the axon (c). NgCAM labeled the surface of neurons infected with wild-type virus (d) and Us9- virus (g). The expression of gE during the wild-type (e) and Us9- (h) infections demonstrates that they are infected with PRV. Merged images of the wild-type infection (f) and Us9- infection (i) are shown with NgCAM in green and gE in red. Bars: (A, a and b, B, and C) 25 μm; (A, c and d) 150 μm.

Mentions: The absence of viral membrane proteins in axons during Us9- infections may reflect aberrant trafficking of viral membrane proteins through the secretory system. Accordingly, we examined the kinetics of cell surface expression of a viral membrane protein during infection. If Us9- infections lead to retention of viral membrane proteins in the secretory system, viral membrane proteins should not be found on the surface of the infected cell. Neurons were infected with the wild-type or the Us9- virus for varying lengths of time, and the cell surface localization of gE was examined. gE appeared on the neuronal surface after infection with either virus (Fig. 4) . The kinetics of surface expression were similar for both viruses: detectable levels of gE appeared ∼7 h after infection (during experiments where all neurons in a culture were infected). Despite similar kinetics, gE initially surfaced on the axons of neurons infected with the wild-type virus, but little to no gE was detected on the surface of axons infected with the Us9- virus (unpublished data). By 9 h after infection, markedly different surface-staining patterns were evident: gE covered the surface of neurons during the wild-type infection, both on the cell body and the axon (Fig. 4 A, a and b). During Us9- infections, little to no detectable staining appeared on the axons (Fig. 4 A, c and d), despite significant staining on the surface of the cell body. These results demonstrate that gE is able to traffic through the secretory system to the cell body surface during Us9- mutant infections yet is not targeted to the axon.


A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.

Tomishima MJ, Enquist LW - J. Cell Biol. (2001)

Analysis of the neuronal secretory system during PRV infection. (A) Cell surface gE during wild-type or Us9- infections. Neurons were infected such that every neuron was infected for 9 h with the wild-type (a and b) or Us9- virus (c and d). Antibodies that recognize gE were placed directly in the culture medium for 5 min, and then unbound antibodies were removed and the infected neurons were fixed. (B) The steady-state localization of the synaptic vesicle marker SV2. Neurons were mock infected (a) or infected with the wild-type virus (b) or the Us9- virus (c) such that every neuron was infected for 16 h, and then the neurons were fixed and permeabilized. An antibody that recognizes SV2 was then used in indirect immunofluorescence experiments. (C) The subcellular localization of a cellular protein (NgCAM) expressed during PRV infection. A replication- defective virus expressing the cellular protein NgCAM was used to infect cultured neurons. After 1 h, the medium was removed, and cultures were mock infected (a–c) or infected with the wild-type (d–f) or Us9- virus (g–i). a–c show NgCAM localization in the absence of PRV infection. NgCAM appears on the surface of the cell body (a), in vesicles within the axon (b; note that this is an image of a large fasiculation, and therefore many axons are present within the bundle), and on the surface of the axon (c). NgCAM labeled the surface of neurons infected with wild-type virus (d) and Us9- virus (g). The expression of gE during the wild-type (e) and Us9- (h) infections demonstrates that they are infected with PRV. Merged images of the wild-type infection (f) and Us9- infection (i) are shown with NgCAM in green and gE in red. Bars: (A, a and b, B, and C) 25 μm; (A, c and d) 150 μm.
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fig4: Analysis of the neuronal secretory system during PRV infection. (A) Cell surface gE during wild-type or Us9- infections. Neurons were infected such that every neuron was infected for 9 h with the wild-type (a and b) or Us9- virus (c and d). Antibodies that recognize gE were placed directly in the culture medium for 5 min, and then unbound antibodies were removed and the infected neurons were fixed. (B) The steady-state localization of the synaptic vesicle marker SV2. Neurons were mock infected (a) or infected with the wild-type virus (b) or the Us9- virus (c) such that every neuron was infected for 16 h, and then the neurons were fixed and permeabilized. An antibody that recognizes SV2 was then used in indirect immunofluorescence experiments. (C) The subcellular localization of a cellular protein (NgCAM) expressed during PRV infection. A replication- defective virus expressing the cellular protein NgCAM was used to infect cultured neurons. After 1 h, the medium was removed, and cultures were mock infected (a–c) or infected with the wild-type (d–f) or Us9- virus (g–i). a–c show NgCAM localization in the absence of PRV infection. NgCAM appears on the surface of the cell body (a), in vesicles within the axon (b; note that this is an image of a large fasiculation, and therefore many axons are present within the bundle), and on the surface of the axon (c). NgCAM labeled the surface of neurons infected with wild-type virus (d) and Us9- virus (g). The expression of gE during the wild-type (e) and Us9- (h) infections demonstrates that they are infected with PRV. Merged images of the wild-type infection (f) and Us9- infection (i) are shown with NgCAM in green and gE in red. Bars: (A, a and b, B, and C) 25 μm; (A, c and d) 150 μm.
Mentions: The absence of viral membrane proteins in axons during Us9- infections may reflect aberrant trafficking of viral membrane proteins through the secretory system. Accordingly, we examined the kinetics of cell surface expression of a viral membrane protein during infection. If Us9- infections lead to retention of viral membrane proteins in the secretory system, viral membrane proteins should not be found on the surface of the infected cell. Neurons were infected with the wild-type or the Us9- virus for varying lengths of time, and the cell surface localization of gE was examined. gE appeared on the neuronal surface after infection with either virus (Fig. 4) . The kinetics of surface expression were similar for both viruses: detectable levels of gE appeared ∼7 h after infection (during experiments where all neurons in a culture were infected). Despite similar kinetics, gE initially surfaced on the axons of neurons infected with the wild-type virus, but little to no gE was detected on the surface of axons infected with the Us9- virus (unpublished data). By 9 h after infection, markedly different surface-staining patterns were evident: gE covered the surface of neurons during the wild-type infection, both on the cell body and the axon (Fig. 4 A, a and b). During Us9- infections, little to no detectable staining appeared on the axons (Fig. 4 A, c and d), despite significant staining on the surface of the cell body. These results demonstrate that gE is able to traffic through the secretory system to the cell body surface during Us9- mutant infections yet is not targeted to the axon.

Bottom Line: We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins.The data support a model where virion subassemblies but not complete virions are transported in the axon.Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
Pseudorabies virus, an alpha-herpesvirus, is capable of infecting the nervous system and spreading between synaptically connected neurons in diverse hosts. At least three viral membrane proteins (gE, gI, and Us9) are necessary for the spread of infection from presynaptic to postsynaptic neurons (anterograde spread) in infected rodents. To understand how these proteins effect anterograde spread between neurons, we analyzed the subcellular localization of viral proteins after infection of cultured rat sympathetic neurons with wild-type or mutant viruses. After Us9- mutant infections but not gE- mutant infections, only a subset of the viral structural proteins had entered axons. Surprisingly, capsid and tegument proteins but not viral membrane proteins were detected in axons. The spread of Us9 missense mutants in the rodent nervous system correlated with the amount of viral membrane proteins localized to axons. We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins. The data support a model where virion subassemblies but not complete virions are transported in the axon. Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

Show MeSH
Related in: MedlinePlus