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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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The axonal localization of the essential viral membrane protein gB requires Us9 but not gE. Less than 10% of the neurons in a culture were infected for 12 h with the wild-type (A and D), the gE- mutant (B and E), or the Us9- mutant (C and F) and then were fixed and permeabilized. Infected neurons were labeled with antibodies that recognize gB. A–C have image planes at the level of the cell bodies, whereas D–F have image planes at the substrate so that axons can be visualized. Arrows point to axons of infected neurons, and N indicates the nucleus of infected neurons. Bar, 25 μm.
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fig2: The axonal localization of the essential viral membrane protein gB requires Us9 but not gE. Less than 10% of the neurons in a culture were infected for 12 h with the wild-type (A and D), the gE- mutant (B and E), or the Us9- mutant (C and F) and then were fixed and permeabilized. Infected neurons were labeled with antibodies that recognize gB. A–C have image planes at the level of the cell bodies, whereas D–F have image planes at the substrate so that axons can be visualized. Arrows point to axons of infected neurons, and N indicates the nucleus of infected neurons. Bar, 25 μm.

Mentions: Predictions from previous work were that gE and Us9 mutants would be defective either in the transport of intracellular virions into axons or that mutant virions could not exit from axon terminals (Husak et al., 2000). Accordingly, we determined the localization of viral structural components in neurons infected with wild-type or mutant viruses. Initially, we examined the subcellular distribution of the viral glycoprotein gB in neurons that were infected with wild-type, gE-, and Us9- viruses. gB is an abundant viral membrane protein that is required for virus entry and cell-to-cell spread in all cells (Rauh and Mettenleiter, 1991). The kinetics of expression and the cellular localization of gB were indistinguishable in the cell body of neurons infected with all three viruses (Fig. 2 A compared with C). In neurons infected with the wild-type and gE- mutants, puncta of gB immunoreactivity appeared in the axons at ∼11 h after infection (unpublished data). At 16 h after infection, gB puncta could be found throughout the extent of axons of neurons infected with either virus (Fig. 2, D and E, respectively). However, in Us9- infections very little gB could be detected in the axons of infected neurons even at the latest times after infection (Fig. 2 F). Occasionally, puncta of gB immunoreactivity appeared in axons after Us9- mutant infection but only in the proximal portion of the axon. These experiments provided the first direct evidence that Us9 and gE may have different functions in infected neurons leading to transsynaptic spread.


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

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

The axonal localization of the essential viral membrane protein gB requires Us9 but not gE. Less than 10% of the neurons in a culture were infected for 12 h with the wild-type (A and D), the gE- mutant (B and E), or the Us9- mutant (C and F) and then were fixed and permeabilized. Infected neurons were labeled with antibodies that recognize gB. A–C have image planes at the level of the cell bodies, whereas D–F have image planes at the substrate so that axons can be visualized. Arrows point to axons of infected neurons, and N indicates the nucleus of infected neurons. Bar, 25 μm.
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Related In: Results  -  Collection

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fig2: The axonal localization of the essential viral membrane protein gB requires Us9 but not gE. Less than 10% of the neurons in a culture were infected for 12 h with the wild-type (A and D), the gE- mutant (B and E), or the Us9- mutant (C and F) and then were fixed and permeabilized. Infected neurons were labeled with antibodies that recognize gB. A–C have image planes at the level of the cell bodies, whereas D–F have image planes at the substrate so that axons can be visualized. Arrows point to axons of infected neurons, and N indicates the nucleus of infected neurons. Bar, 25 μm.
Mentions: Predictions from previous work were that gE and Us9 mutants would be defective either in the transport of intracellular virions into axons or that mutant virions could not exit from axon terminals (Husak et al., 2000). Accordingly, we determined the localization of viral structural components in neurons infected with wild-type or mutant viruses. Initially, we examined the subcellular distribution of the viral glycoprotein gB in neurons that were infected with wild-type, gE-, and Us9- viruses. gB is an abundant viral membrane protein that is required for virus entry and cell-to-cell spread in all cells (Rauh and Mettenleiter, 1991). The kinetics of expression and the cellular localization of gB were indistinguishable in the cell body of neurons infected with all three viruses (Fig. 2 A compared with C). In neurons infected with the wild-type and gE- mutants, puncta of gB immunoreactivity appeared in the axons at ∼11 h after infection (unpublished data). At 16 h after infection, gB puncta could be found throughout the extent of axons of neurons infected with either virus (Fig. 2, D and E, respectively). However, in Us9- infections very little gB could be detected in the axons of infected neurons even at the latest times after infection (Fig. 2 F). Occasionally, puncta of gB immunoreactivity appeared in axons after Us9- mutant infection but only in the proximal portion of the axon. These experiments provided the first direct evidence that Us9 and gE may have different functions in infected neurons leading to transsynaptic spread.

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