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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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In the presence of  DTT p4a is cleaved to its mature (4a) form. Infected  HeLa cells were treated  (DTT +) or not treated  (DTT +) from 4 h after infection onwards with 5 mM DTT. In  each case, when medium with DTT was applied to the cells,  freshly prepared DTT was added to the medium just before use.  At 5 h after infection the medium was replaced with medium  with or without DTT. At 5.5 h after infection the cells were  starved in methionine-free DMEM with or without DTT. Pulse-labeling for 15 min (chase 0 h) was at 6 h after infection and consisted of replacing the methionine-free medium by the same medium containing 50 μCi of [35S]methionine with or without DTT.  After the pulse, cells were chased for 2 and 3 h (chase 2 and 3 h)  in medium with or without DTT that was replaced each hour. At  the end of the pulse or chase cell lysates were prepared and 4a  and its precursor form (p4a; both indicated) were immunoprecipitated from them. Immunoprecipitates were run on 10% SDS-PAGE and the proteins detected by autoradiography. M-14C-labeled marker protein of 93 kD.
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Figure 7: In the presence of DTT p4a is cleaved to its mature (4a) form. Infected HeLa cells were treated (DTT +) or not treated (DTT +) from 4 h after infection onwards with 5 mM DTT. In each case, when medium with DTT was applied to the cells, freshly prepared DTT was added to the medium just before use. At 5 h after infection the medium was replaced with medium with or without DTT. At 5.5 h after infection the cells were starved in methionine-free DMEM with or without DTT. Pulse-labeling for 15 min (chase 0 h) was at 6 h after infection and consisted of replacing the methionine-free medium by the same medium containing 50 μCi of [35S]methionine with or without DTT. After the pulse, cells were chased for 2 and 3 h (chase 2 and 3 h) in medium with or without DTT that was replaced each hour. At the end of the pulse or chase cell lysates were prepared and 4a and its precursor form (p4a; both indicated) were immunoprecipitated from them. Immunoprecipitates were run on 10% SDS-PAGE and the proteins detected by autoradiography. M-14C-labeled marker protein of 93 kD.

Mentions: In the final stage of IMV assembly, just before the IMV closure, at least three core proteins are proteolytically cleaved (30, 41). Estimation of the cleavage of the precursor of a protein such as the core protein 4a using pulse– chase experiments can thus be used as an indirect measure of the extent of IMV formation. To test the effect of DTT on IMV assembly using this assay, infected cells were left untreated or treated with DTT from 4 h after infection onwards. Subsequently, they were pulse labeled for 15 min at 6 h after infection, chased for 2 or 3 h and cell lysates were then processed for immunoprecipitation, using antibodies to 4a and its precursor. Again, during the course of this experiment the medium was replaced several times with medium containing freshly prepared DTT (see legend to Fig. 7). As shown in Fig. 7 both in the presence and absence of DTT the precursor p4a of 110 kD was efficiently cleaved into its 65-kD mature form (4a) during the chase. PhosphorImager analyses showed that, consistent with previously published results (41; Fig. 7), after 2 h of chase 35% of 4a was cleaved in the absence and 40% in the presence of DTT, while these percentages amounted to 48 and 57%, respectively, after 3 h of chase (not shown). These same analyses also showed that the overall synthesis of 4a was reduced by ∼40% in the presence of DTT, which is most likely the result of the long incubations with this drug. Importantly, however, 4a and p4a did not seem to undergo substantial degradation, suggesting that the newly formed cores were quite stable, both with and without DTT.


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

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

In the presence of  DTT p4a is cleaved to its mature (4a) form. Infected  HeLa cells were treated  (DTT +) or not treated  (DTT +) from 4 h after infection onwards with 5 mM DTT. In  each case, when medium with DTT was applied to the cells,  freshly prepared DTT was added to the medium just before use.  At 5 h after infection the medium was replaced with medium  with or without DTT. At 5.5 h after infection the cells were  starved in methionine-free DMEM with or without DTT. Pulse-labeling for 15 min (chase 0 h) was at 6 h after infection and consisted of replacing the methionine-free medium by the same medium containing 50 μCi of [35S]methionine with or without DTT.  After the pulse, cells were chased for 2 and 3 h (chase 2 and 3 h)  in medium with or without DTT that was replaced each hour. At  the end of the pulse or chase cell lysates were prepared and 4a  and its precursor form (p4a; both indicated) were immunoprecipitated from them. Immunoprecipitates were run on 10% SDS-PAGE and the proteins detected by autoradiography. M-14C-labeled marker protein of 93 kD.
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Related In: Results  -  Collection

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Figure 7: In the presence of DTT p4a is cleaved to its mature (4a) form. Infected HeLa cells were treated (DTT +) or not treated (DTT +) from 4 h after infection onwards with 5 mM DTT. In each case, when medium with DTT was applied to the cells, freshly prepared DTT was added to the medium just before use. At 5 h after infection the medium was replaced with medium with or without DTT. At 5.5 h after infection the cells were starved in methionine-free DMEM with or without DTT. Pulse-labeling for 15 min (chase 0 h) was at 6 h after infection and consisted of replacing the methionine-free medium by the same medium containing 50 μCi of [35S]methionine with or without DTT. After the pulse, cells were chased for 2 and 3 h (chase 2 and 3 h) in medium with or without DTT that was replaced each hour. At the end of the pulse or chase cell lysates were prepared and 4a and its precursor form (p4a; both indicated) were immunoprecipitated from them. Immunoprecipitates were run on 10% SDS-PAGE and the proteins detected by autoradiography. M-14C-labeled marker protein of 93 kD.
Mentions: In the final stage of IMV assembly, just before the IMV closure, at least three core proteins are proteolytically cleaved (30, 41). Estimation of the cleavage of the precursor of a protein such as the core protein 4a using pulse– chase experiments can thus be used as an indirect measure of the extent of IMV formation. To test the effect of DTT on IMV assembly using this assay, infected cells were left untreated or treated with DTT from 4 h after infection onwards. Subsequently, they were pulse labeled for 15 min at 6 h after infection, chased for 2 or 3 h and cell lysates were then processed for immunoprecipitation, using antibodies to 4a and its precursor. Again, during the course of this experiment the medium was replaced several times with medium containing freshly prepared DTT (see legend to Fig. 7). As shown in Fig. 7 both in the presence and absence of DTT the precursor p4a of 110 kD was efficiently cleaved into its 65-kD mature form (4a) during the chase. PhosphorImager analyses showed that, consistent with previously published results (41; Fig. 7), after 2 h of chase 35% of 4a was cleaved in the absence and 40% in the presence of DTT, while these percentages amounted to 48 and 57%, respectively, after 3 h of chase (not shown). These same analyses also showed that the overall synthesis of 4a was reduced by ∼40% in the presence of DTT, which is most likely the result of the long incubations with this drug. Importantly, however, 4a and p4a did not seem to undergo substantial degradation, suggesting that the newly formed cores were quite stable, both with and without DTT.

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