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An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins.

Locker JK, Griffiths G - J. Cell Biol. (1999)

Bottom Line: Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv).Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core.Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology Programme, 69117 Heidelberg, Germany. krijnse@embl-heidelberg.de

ABSTRACT
Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.

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Negative staining EM of particles isolated from untreated (B) or DTT treated cells (A, C, and D). Particles from infected HeLa cells untreated or treated with DTT were isolated as  described in Materials and Methods. The particles were absorbed  to 300 mesh formvar and carbon-coated grids and immuno-labeled with antibodies to p39 (A, B, and D) and to p16 (C), followed by protein A–gold. Whereas virions isolated from untreated cells are not labeled with p39 (B), the particles from  treated cells label heavily with the antibody (A and D). The anti-p16 antibody labels fragments that are still attached to the particles isolated from DTT-treated cells. These fragments often appear as tubular structures (C). Bars, 100 nm.
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Figure 9: Negative staining EM of particles isolated from untreated (B) or DTT treated cells (A, C, and D). Particles from infected HeLa cells untreated or treated with DTT were isolated as described in Materials and Methods. The particles were absorbed to 300 mesh formvar and carbon-coated grids and immuno-labeled with antibodies to p39 (A, B, and D) and to p16 (C), followed by protein A–gold. Whereas virions isolated from untreated cells are not labeled with p39 (B), the particles from treated cells label heavily with the antibody (A and D). The anti-p16 antibody labels fragments that are still attached to the particles isolated from DTT-treated cells. These fragments often appear as tubular structures (C). Bars, 100 nm.

Mentions: When the particles isolated from DTT-treated cells were examined by negative staining EM, we observed that most of them were not intact (in contrast to untreated preparations where most of them were intact). By combining this approach with immunogold labeling using specific antibodies, we found that almost all of the isolated particles appeared to have lost or opened up their membranes. The brick-shaped particles from DTT-treated cells, labeled heavily for p39, an abundant antigen on the surface of the vv core (Fig. 9, a and d; 8, 50), while IMV particles isolated from nontreated cells (as expected; 8) could not be labeled with this antibody (Fig. 9 b). In the particles isolated from DTT-treated cells, the viral membranes often remained attached to the core and were heavily labeled with antibodies to the membrane protein p16. We have shown before that in intact IMVs this protein is located on the inner of the two cisternal membranes and hidden from antibody accessibility (Fig. 9 c; see 51).


An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins.

Locker JK, Griffiths G - J. Cell Biol. (1999)

Negative staining EM of particles isolated from untreated (B) or DTT treated cells (A, C, and D). Particles from infected HeLa cells untreated or treated with DTT were isolated as  described in Materials and Methods. The particles were absorbed  to 300 mesh formvar and carbon-coated grids and immuno-labeled with antibodies to p39 (A, B, and D) and to p16 (C), followed by protein A–gold. Whereas virions isolated from untreated cells are not labeled with p39 (B), the particles from  treated cells label heavily with the antibody (A and D). The anti-p16 antibody labels fragments that are still attached to the particles isolated from DTT-treated cells. These fragments often appear as tubular structures (C). Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132897&req=5

Figure 9: Negative staining EM of particles isolated from untreated (B) or DTT treated cells (A, C, and D). Particles from infected HeLa cells untreated or treated with DTT were isolated as described in Materials and Methods. The particles were absorbed to 300 mesh formvar and carbon-coated grids and immuno-labeled with antibodies to p39 (A, B, and D) and to p16 (C), followed by protein A–gold. Whereas virions isolated from untreated cells are not labeled with p39 (B), the particles from treated cells label heavily with the antibody (A and D). The anti-p16 antibody labels fragments that are still attached to the particles isolated from DTT-treated cells. These fragments often appear as tubular structures (C). Bars, 100 nm.
Mentions: When the particles isolated from DTT-treated cells were examined by negative staining EM, we observed that most of them were not intact (in contrast to untreated preparations where most of them were intact). By combining this approach with immunogold labeling using specific antibodies, we found that almost all of the isolated particles appeared to have lost or opened up their membranes. The brick-shaped particles from DTT-treated cells, labeled heavily for p39, an abundant antigen on the surface of the vv core (Fig. 9, a and d; 8, 50), while IMV particles isolated from nontreated cells (as expected; 8) could not be labeled with this antibody (Fig. 9 b). In the particles isolated from DTT-treated cells, the viral membranes often remained attached to the core and were heavily labeled with antibodies to the membrane protein p16. We have shown before that in intact IMVs this protein is located on the inner of the two cisternal membranes and hidden from antibody accessibility (Fig. 9 c; see 51).

Bottom Line: Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv).Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core.Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Cell Biology Programme, 69117 Heidelberg, Germany. krijnse@embl-heidelberg.de

ABSTRACT
Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.

Show MeSH
Related in: MedlinePlus