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The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment.

Melancon JM, Fulmer PA, Kousoulas KG - Virol. J. (2007)

Bottom Line: In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization.In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites.These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biotechnology and Molecular Medicine, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA. jmelan@lsuhsc.edu

ABSTRACT
The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20- virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.

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The effect of UL20p amino terminal mutations on UL20p and gK colocalization in TGN cellular compartments. Vero cells were co-transfected with gK tagged with the V5 epitope (D1V5), as well as with plasmids encoding wild-type or different mutant UL20ps tagged with the 3 × FLAG epitope (UL20D1FLAG). Thirty-six hours post-transfection, cells were washed thoroughly, fixed, and processed for confocal microscopy. After permeabilization, rabbit anti-FLAG (α FLAG) mAb was used to detect UL20p, mouse anti-V5 (α V5) epitope was used to detect gK, and sheep aTGN46 mAb was used to identify the TGN. First three rows of the confocal pictures show co-localization of UL20p with gK, while rows 4–6 show colocalization of gK with TGN46.
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Figure 4: The effect of UL20p amino terminal mutations on UL20p and gK colocalization in TGN cellular compartments. Vero cells were co-transfected with gK tagged with the V5 epitope (D1V5), as well as with plasmids encoding wild-type or different mutant UL20ps tagged with the 3 × FLAG epitope (UL20D1FLAG). Thirty-six hours post-transfection, cells were washed thoroughly, fixed, and processed for confocal microscopy. After permeabilization, rabbit anti-FLAG (α FLAG) mAb was used to detect UL20p, mouse anti-V5 (α V5) epitope was used to detect gK, and sheep aTGN46 mAb was used to identify the TGN. First three rows of the confocal pictures show co-localization of UL20p with gK, while rows 4–6 show colocalization of gK with TGN46.

Mentions: Transport and localization of UL20p and gK was further assessed by transient coexpression of gK and UL20p and simultaneous detection of the TGN compartment. We showed previously that in the absence of UL20p, gK was localized exclusively to reticular-like compartments and was absent from the Golgi and TGN. A similar pattern was detected for UL20p in the absence of gK [31]. In contrast, coexpression of gK and UL20p significantly altered the distribution pattern of both gK and UL20p with UL20p and gK colocalized in intracellular compartments that stained for the TGN marker TGN46. Overall, these results showed that gK and UL20p intracellular transport and TGN localization were functionally interdependent strongly suggesting that gK and UL20p physically interacted [31]. Similar confocal colocalization assays were performed to test the ability of each UL20 mutant to facilitate transport and colocalization with gK. The CL38-CL49, Y38-Y49, Y38A and Y49A UL20 mutants produced similar patterns to those of the wild-type UL20 gene, since they effectively colocalized with gK (Fig. 4: rows 1–3). In addition, gK was colocalized with TGN46 (Fig. 4: rows 4–6), indicating that these UL20 mutations did not affect intracellular transport and TGN colocalization of the mutant UL20ps with gK. Similar assays were performed for the UL20p carboxyl terminal truncations 216t, 211t, and 204t (Fig. 5). The UL20p mutants CL153 and CL61 that were previously shown not to complement for either infectious virus production or virus-induced cell fusion [30] were also tested as negative controls, while the wild-type UL20 gene served as the positive control. Both 216t and 211t UL20 truncations enabled efficient colocalization of UL20 and gK in TGN compartments, while the 204t UL20 truncation failed to transport and colocalize with gK in TGN compartments (Fig. 5). Figure 5 represents a three color confocal microscopy experiment, while Figure 4 was a two-color confocal microscopy experiment.


The herpes simplex virus UL20 protein functions in glycoprotein K (gK) intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment.

Melancon JM, Fulmer PA, Kousoulas KG - Virol. J. (2007)

The effect of UL20p amino terminal mutations on UL20p and gK colocalization in TGN cellular compartments. Vero cells were co-transfected with gK tagged with the V5 epitope (D1V5), as well as with plasmids encoding wild-type or different mutant UL20ps tagged with the 3 × FLAG epitope (UL20D1FLAG). Thirty-six hours post-transfection, cells were washed thoroughly, fixed, and processed for confocal microscopy. After permeabilization, rabbit anti-FLAG (α FLAG) mAb was used to detect UL20p, mouse anti-V5 (α V5) epitope was used to detect gK, and sheep aTGN46 mAb was used to identify the TGN. First three rows of the confocal pictures show co-localization of UL20p with gK, while rows 4–6 show colocalization of gK with TGN46.
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Related In: Results  -  Collection

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Figure 4: The effect of UL20p amino terminal mutations on UL20p and gK colocalization in TGN cellular compartments. Vero cells were co-transfected with gK tagged with the V5 epitope (D1V5), as well as with plasmids encoding wild-type or different mutant UL20ps tagged with the 3 × FLAG epitope (UL20D1FLAG). Thirty-six hours post-transfection, cells were washed thoroughly, fixed, and processed for confocal microscopy. After permeabilization, rabbit anti-FLAG (α FLAG) mAb was used to detect UL20p, mouse anti-V5 (α V5) epitope was used to detect gK, and sheep aTGN46 mAb was used to identify the TGN. First three rows of the confocal pictures show co-localization of UL20p with gK, while rows 4–6 show colocalization of gK with TGN46.
Mentions: Transport and localization of UL20p and gK was further assessed by transient coexpression of gK and UL20p and simultaneous detection of the TGN compartment. We showed previously that in the absence of UL20p, gK was localized exclusively to reticular-like compartments and was absent from the Golgi and TGN. A similar pattern was detected for UL20p in the absence of gK [31]. In contrast, coexpression of gK and UL20p significantly altered the distribution pattern of both gK and UL20p with UL20p and gK colocalized in intracellular compartments that stained for the TGN marker TGN46. Overall, these results showed that gK and UL20p intracellular transport and TGN localization were functionally interdependent strongly suggesting that gK and UL20p physically interacted [31]. Similar confocal colocalization assays were performed to test the ability of each UL20 mutant to facilitate transport and colocalization with gK. The CL38-CL49, Y38-Y49, Y38A and Y49A UL20 mutants produced similar patterns to those of the wild-type UL20 gene, since they effectively colocalized with gK (Fig. 4: rows 1–3). In addition, gK was colocalized with TGN46 (Fig. 4: rows 4–6), indicating that these UL20 mutations did not affect intracellular transport and TGN colocalization of the mutant UL20ps with gK. Similar assays were performed for the UL20p carboxyl terminal truncations 216t, 211t, and 204t (Fig. 5). The UL20p mutants CL153 and CL61 that were previously shown not to complement for either infectious virus production or virus-induced cell fusion [30] were also tested as negative controls, while the wild-type UL20 gene served as the positive control. Both 216t and 211t UL20 truncations enabled efficient colocalization of UL20 and gK in TGN compartments, while the 204t UL20 truncation failed to transport and colocalize with gK in TGN compartments (Fig. 5). Figure 5 represents a three color confocal microscopy experiment, while Figure 4 was a two-color confocal microscopy experiment.

Bottom Line: In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization.In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites.These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biotechnology and Molecular Medicine, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA. jmelan@lsuhsc.edu

ABSTRACT
The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20- virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively) allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.

Show MeSH
Related in: MedlinePlus