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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 apparently normal looking  IMVs are formed but in some of them the viral membranes detach from the underlying core. A shows Epon sections of control,  untreated HeLa cells infected with vv for 8 h. IMVs and immature viruses (IVs) are evident. In the inset on the left the outer  and inner membrane profiles of the IMV are indicated by arrows.  The inset on the right shows an intermediate stage between the  IV and IMV (arrowhead) next to a IV. B and C show vv-infected  cells treated with 5 mM DTT from 4 h after infection onwards  and fixed at 8 h after infection (see Results for details). In B, one  sees both normal looking IMVs (star) as well as aberrant IMVs  (arrowheads), where the viral core is seen as a distinct entity separated from the outer membranes. This is more evident at higher  magnifications in C–F; in C a normal IV is apparent. The arrowheads indicate the viral core that in all these examples appears as  a distinct entity. Bars, 100 nm.
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Figure 6: In the presence of DTT apparently normal looking IMVs are formed but in some of them the viral membranes detach from the underlying core. A shows Epon sections of control, untreated HeLa cells infected with vv for 8 h. IMVs and immature viruses (IVs) are evident. In the inset on the left the outer and inner membrane profiles of the IMV are indicated by arrows. The inset on the right shows an intermediate stage between the IV and IMV (arrowhead) next to a IV. B and C show vv-infected cells treated with 5 mM DTT from 4 h after infection onwards and fixed at 8 h after infection (see Results for details). In B, one sees both normal looking IMVs (star) as well as aberrant IMVs (arrowheads), where the viral core is seen as a distinct entity separated from the outer membranes. This is more evident at higher magnifications in C–F; in C a normal IV is apparent. The arrowheads indicate the viral core that in all these examples appears as a distinct entity. Bars, 100 nm.

Mentions: At first glance, the addition of DTT had no obvious effect on IMV assembly, since by thin section EM we readily observed many apparently normal looking immature viruses (IVs), intermediates and IMVs (Fig. 6; see 53). However, upon closer examination a substantial fraction of IMVs could be detected that resembled virions in various stages of apparent disassembly (Fig. 6, b–f). Profiles were seen of particles in which the membranes had become less rigid and detaching from the underlying core. Occasionally we also observed particles indistinguishable from viral cores (not shown). Compared with the control cells, IVs and intermediates (53) appeared totally normal. None of these aberrant particles with detaching membranes or naked cores could be detected in the untreated control cells, implying that they resulted from the prolonged DTT incubation.


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

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

In the presence of DTT apparently normal looking  IMVs are formed but in some of them the viral membranes detach from the underlying core. A shows Epon sections of control,  untreated HeLa cells infected with vv for 8 h. IMVs and immature viruses (IVs) are evident. In the inset on the left the outer  and inner membrane profiles of the IMV are indicated by arrows.  The inset on the right shows an intermediate stage between the  IV and IMV (arrowhead) next to a IV. B and C show vv-infected  cells treated with 5 mM DTT from 4 h after infection onwards  and fixed at 8 h after infection (see Results for details). In B, one  sees both normal looking IMVs (star) as well as aberrant IMVs  (arrowheads), where the viral core is seen as a distinct entity separated from the outer membranes. This is more evident at higher  magnifications in C–F; in C a normal IV is apparent. The arrowheads indicate the viral core that in all these examples appears as  a distinct entity. Bars, 100 nm.
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Related In: Results  -  Collection

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Figure 6: In the presence of DTT apparently normal looking IMVs are formed but in some of them the viral membranes detach from the underlying core. A shows Epon sections of control, untreated HeLa cells infected with vv for 8 h. IMVs and immature viruses (IVs) are evident. In the inset on the left the outer and inner membrane profiles of the IMV are indicated by arrows. The inset on the right shows an intermediate stage between the IV and IMV (arrowhead) next to a IV. B and C show vv-infected cells treated with 5 mM DTT from 4 h after infection onwards and fixed at 8 h after infection (see Results for details). In B, one sees both normal looking IMVs (star) as well as aberrant IMVs (arrowheads), where the viral core is seen as a distinct entity separated from the outer membranes. This is more evident at higher magnifications in C–F; in C a normal IV is apparent. The arrowheads indicate the viral core that in all these examples appears as a distinct entity. Bars, 100 nm.
Mentions: At first glance, the addition of DTT had no obvious effect on IMV assembly, since by thin section EM we readily observed many apparently normal looking immature viruses (IVs), intermediates and IMVs (Fig. 6; see 53). However, upon closer examination a substantial fraction of IMVs could be detected that resembled virions in various stages of apparent disassembly (Fig. 6, b–f). Profiles were seen of particles in which the membranes had become less rigid and detaching from the underlying core. Occasionally we also observed particles indistinguishable from viral cores (not shown). Compared with the control cells, IVs and intermediates (53) appeared totally normal. None of these aberrant particles with detaching membranes or naked cores could be detected in the untreated control cells, implying that they resulted from the prolonged DTT incubation.

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