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Characterization of subcellular localization of duck enteritis virus UL51 protein.

Shen C, Guo Y, Cheng A, Wang M, Zhou Y, Lin D, Xin H, Zhang N - Virol. J. (2009)

Bottom Line: Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i.TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism.From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument.

View Article: PubMed Central - HTML - PubMed

Affiliation: Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Ya'an, Sichuan, PR China. vorber@163.com

ABSTRACT

Background: Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells.

Results: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism.

Conclusion: In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.

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Intracellular location and distribution of DEV pUL51 analyzed by IIF. Mock-infected (A) and DEV-infected (B to F) DEF were fixed as described in Materials and Methods. The samples were stained with the UL51 antiserum (A, C to F) or pre-immune serum (B), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). The merged fluorescence microscopy images of DEF are shown in panels A to F with high magnification (600×).
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Figure 2: Intracellular location and distribution of DEV pUL51 analyzed by IIF. Mock-infected (A) and DEV-infected (B to F) DEF were fixed as described in Materials and Methods. The samples were stained with the UL51 antiserum (A, C to F) or pre-immune serum (B), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). The merged fluorescence microscopy images of DEF are shown in panels A to F with high magnification (600×).

Mentions: A detailed analysis of the intracellular localization of DEV pUL51 was investigated using the purified UL51 antiserum or pre-immune serum by IIF staining of mock- and DEV-infected cells. As shown in Fig 2, a faint pUL51-specific fluorescence was first detected in the cytoplasm of DEV-infected cells at 9 h p.i. (Fig 2C), and then a strong fluorescence was observed mainly in the juxtanuclear region at 12 h p.i. (Fig 2D). After that, the pUL51-specific fluorescence in the juxtanuclear region was dense and localized on wide areas of the cytoplasm. At 36 h p.i. (Fig 2E), the pUL51-specific fluorescence was found widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region; meanwhile, the nucleus of some DEV-infected cells also contained little fluorescence granular. Following by a series of morphological changes, the cytoplasm disintegration and nuclear fragmentation in DEV-infected cells, the intensity of the reaction increased at 48 and 60 h p.i. (Fig 2F), while the pUL51-specific fluorescence was mainly detected in the cytoplasm of infected cells and that one localized in the nuclear was faint. No pUL51-specific fluorescence could be detected in mock-infected cells reacted with the UL51 antiserum (Fig 2A) and in DEV-infected cells reacted with the pre-immune serum (Fig 2B).


Characterization of subcellular localization of duck enteritis virus UL51 protein.

Shen C, Guo Y, Cheng A, Wang M, Zhou Y, Lin D, Xin H, Zhang N - Virol. J. (2009)

Intracellular location and distribution of DEV pUL51 analyzed by IIF. Mock-infected (A) and DEV-infected (B to F) DEF were fixed as described in Materials and Methods. The samples were stained with the UL51 antiserum (A, C to F) or pre-immune serum (B), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). The merged fluorescence microscopy images of DEF are shown in panels A to F with high magnification (600×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2714536&req=5

Figure 2: Intracellular location and distribution of DEV pUL51 analyzed by IIF. Mock-infected (A) and DEV-infected (B to F) DEF were fixed as described in Materials and Methods. The samples were stained with the UL51 antiserum (A, C to F) or pre-immune serum (B), and reacted with anti-rabbit IgG-conjugated FITC, and then counter-stained with DAPI (blue is representative the cell nuclei). The merged fluorescence microscopy images of DEF are shown in panels A to F with high magnification (600×).
Mentions: A detailed analysis of the intracellular localization of DEV pUL51 was investigated using the purified UL51 antiserum or pre-immune serum by IIF staining of mock- and DEV-infected cells. As shown in Fig 2, a faint pUL51-specific fluorescence was first detected in the cytoplasm of DEV-infected cells at 9 h p.i. (Fig 2C), and then a strong fluorescence was observed mainly in the juxtanuclear region at 12 h p.i. (Fig 2D). After that, the pUL51-specific fluorescence in the juxtanuclear region was dense and localized on wide areas of the cytoplasm. At 36 h p.i. (Fig 2E), the pUL51-specific fluorescence was found widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region; meanwhile, the nucleus of some DEV-infected cells also contained little fluorescence granular. Following by a series of morphological changes, the cytoplasm disintegration and nuclear fragmentation in DEV-infected cells, the intensity of the reaction increased at 48 and 60 h p.i. (Fig 2F), while the pUL51-specific fluorescence was mainly detected in the cytoplasm of infected cells and that one localized in the nuclear was faint. No pUL51-specific fluorescence could be detected in mock-infected cells reacted with the UL51 antiserum (Fig 2A) and in DEV-infected cells reacted with the pre-immune serum (Fig 2B).

Bottom Line: Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i.TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism.From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument.

View Article: PubMed Central - HTML - PubMed

Affiliation: Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Ya'an, Sichuan, PR China. vorber@163.com

ABSTRACT

Background: Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells.

Results: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism.

Conclusion: In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.

Show MeSH
Related in: MedlinePlus