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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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Disulfide bonding occurs posttranslationally. Infected  HeLa cells were pulse-labeled (0 min chase) for 2 min at 6 h after  infection and chased for the indicated time. Cell lysates were prepared and the p16 protein immunoprecipitated from them. The  samples were run on 15% SDS-PAGE after boiling for 3 min in  LSB with or without β-ME and DTT. The proteins were detected  by autoradiography. After the pulse only the monomeric p16 is  seen. After 60 min of chase the disulfide bonded dimer is detected as assessed by the absence of the 25-kD band when analyzed after reduction. The band migrating with a molecular mass  between the monomer and the dimer is a N-glycosylated form of  p16 that will be described elsewhere (Krijnse Locker, J., manuscript in preparation). M-14C-labeled marker proteins, of which  the 14- and 30-kD proteins are indicated.
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Figure 4: Disulfide bonding occurs posttranslationally. Infected HeLa cells were pulse-labeled (0 min chase) for 2 min at 6 h after infection and chased for the indicated time. Cell lysates were prepared and the p16 protein immunoprecipitated from them. The samples were run on 15% SDS-PAGE after boiling for 3 min in LSB with or without β-ME and DTT. The proteins were detected by autoradiography. After the pulse only the monomeric p16 is seen. After 60 min of chase the disulfide bonded dimer is detected as assessed by the absence of the 25-kD band when analyzed after reduction. The band migrating with a molecular mass between the monomer and the dimer is a N-glycosylated form of p16 that will be described elsewhere (Krijnse Locker, J., manuscript in preparation). M-14C-labeled marker proteins, of which the 14- and 30-kD proteins are indicated.

Mentions: Infected cells were pulse-labeled at 6 h after infection and chased for up to 4 h. Small amounts of the dimer of p16 were first detected after 60 min, but was seen more clearly at 2 h of chase. At 4 h of chase ∼50% of p16 was in the dimer form (Fig. 4). The band migrating intermediate between the monomer and the dimer of p16 is due to the addition to some of N-linked glycosylation to a fraction of the molecules (Krijnse Locker, J., manuscript in preparation). A similar analysis of the core protein p39 showed that its disulfide bonding also occurred posttranslationally. The half time of its formation was ∼2 h, similar to the half time of IMV formation (data not shown).


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

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

Disulfide bonding occurs posttranslationally. Infected  HeLa cells were pulse-labeled (0 min chase) for 2 min at 6 h after  infection and chased for the indicated time. Cell lysates were prepared and the p16 protein immunoprecipitated from them. The  samples were run on 15% SDS-PAGE after boiling for 3 min in  LSB with or without β-ME and DTT. The proteins were detected  by autoradiography. After the pulse only the monomeric p16 is  seen. After 60 min of chase the disulfide bonded dimer is detected as assessed by the absence of the 25-kD band when analyzed after reduction. The band migrating with a molecular mass  between the monomer and the dimer is a N-glycosylated form of  p16 that will be described elsewhere (Krijnse Locker, J., manuscript in preparation). M-14C-labeled marker proteins, of which  the 14- and 30-kD proteins are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Disulfide bonding occurs posttranslationally. Infected HeLa cells were pulse-labeled (0 min chase) for 2 min at 6 h after infection and chased for the indicated time. Cell lysates were prepared and the p16 protein immunoprecipitated from them. The samples were run on 15% SDS-PAGE after boiling for 3 min in LSB with or without β-ME and DTT. The proteins were detected by autoradiography. After the pulse only the monomeric p16 is seen. After 60 min of chase the disulfide bonded dimer is detected as assessed by the absence of the 25-kD band when analyzed after reduction. The band migrating with a molecular mass between the monomer and the dimer is a N-glycosylated form of p16 that will be described elsewhere (Krijnse Locker, J., manuscript in preparation). M-14C-labeled marker proteins, of which the 14- and 30-kD proteins are indicated.
Mentions: Infected cells were pulse-labeled at 6 h after infection and chased for up to 4 h. Small amounts of the dimer of p16 were first detected after 60 min, but was seen more clearly at 2 h of chase. At 4 h of chase ∼50% of p16 was in the dimer form (Fig. 4). The band migrating intermediate between the monomer and the dimer of p16 is due to the addition to some of N-linked glycosylation to a fraction of the molecules (Krijnse Locker, J., manuscript in preparation). A similar analysis of the core protein p39 showed that its disulfide bonding also occurred posttranslationally. The half time of its formation was ∼2 h, similar to the half time of IMV formation (data not shown).

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