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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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Upon viral entry  the core proteins become reduced while the membrane  proteins do not. HeLa cells  were infected with purified  virus preparations for 60 min  at 37°C and cell lysates were  prepared as described in Materials and Methods. The cell  lysates (L) or purified virions (V) that were not exposed to cells,  were run on 15% SDS-PAGE after boiling in LSB with (DTT +)  or without (DTT −) 5% β-ME and 100 mM DTT. The membrane proteins p21 and p16 and the core proteins 4a and p25  were detected by Western blot.
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Figure 10: Upon viral entry the core proteins become reduced while the membrane proteins do not. HeLa cells were infected with purified virus preparations for 60 min at 37°C and cell lysates were prepared as described in Materials and Methods. The cell lysates (L) or purified virions (V) that were not exposed to cells, were run on 15% SDS-PAGE after boiling in LSB with (DTT +) or without (DTT −) 5% β-ME and 100 mM DTT. The membrane proteins p21 and p16 and the core proteins 4a and p25 were detected by Western blot.

Mentions: Since reduction of the IMV in vitro, as well as in vivo led to a form of uncoating of the virus, we were curious to know whether reduction of vv proteins was also required for disassembly in vivo, that is during the infection process. Highly purified IMV stocks were therefore bound to cells on ice and subsequently shifted to 37°C for 1 h. Bound (but nonpenetrated) particles were removed by trypsin treatment on ice (see Materials and Methods). Subsequently, the infected cells were lysed and the vv proteins analyzed under reducing and nonreducing condition using Western blots. Under these conditions the two core proteins 4a and p25 migrated exclusively at the position of the reduced form (Fig. 10). To our surprise, the membrane proteins p16 and p21 did not appear to become reduced during early times of infection. Instead both proteins migrated with a slightly higher molecular mass than their respective dimers seen under nonreducing conditions (Fig. 10). When analyzed after reduction these forms of both p16 and p21 migrated slightly slower than their respective monomers (Fig. 10). Since by 2-D gel analysis, these newly generated forms of p21 and p16 appeared to have the same pI as the proteins in isolated virions (not shown), we presume that this slower migration is due to a (unconventional) conformational change and not the consequence of their binding to some other (unknown) protein.


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

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

Upon viral entry  the core proteins become reduced while the membrane  proteins do not. HeLa cells  were infected with purified  virus preparations for 60 min  at 37°C and cell lysates were  prepared as described in Materials and Methods. The cell  lysates (L) or purified virions (V) that were not exposed to cells,  were run on 15% SDS-PAGE after boiling in LSB with (DTT +)  or without (DTT −) 5% β-ME and 100 mM DTT. The membrane proteins p21 and p16 and the core proteins 4a and p25  were detected by Western blot.
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Related In: Results  -  Collection

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

Figure 10: Upon viral entry the core proteins become reduced while the membrane proteins do not. HeLa cells were infected with purified virus preparations for 60 min at 37°C and cell lysates were prepared as described in Materials and Methods. The cell lysates (L) or purified virions (V) that were not exposed to cells, were run on 15% SDS-PAGE after boiling in LSB with (DTT +) or without (DTT −) 5% β-ME and 100 mM DTT. The membrane proteins p21 and p16 and the core proteins 4a and p25 were detected by Western blot.
Mentions: Since reduction of the IMV in vitro, as well as in vivo led to a form of uncoating of the virus, we were curious to know whether reduction of vv proteins was also required for disassembly in vivo, that is during the infection process. Highly purified IMV stocks were therefore bound to cells on ice and subsequently shifted to 37°C for 1 h. Bound (but nonpenetrated) particles were removed by trypsin treatment on ice (see Materials and Methods). Subsequently, the infected cells were lysed and the vv proteins analyzed under reducing and nonreducing condition using Western blots. Under these conditions the two core proteins 4a and p25 migrated exclusively at the position of the reduced form (Fig. 10). To our surprise, the membrane proteins p16 and p21 did not appear to become reduced during early times of infection. Instead both proteins migrated with a slightly higher molecular mass than their respective dimers seen under nonreducing conditions (Fig. 10). When analyzed after reduction these forms of both p16 and p21 migrated slightly slower than their respective monomers (Fig. 10). Since by 2-D gel analysis, these newly generated forms of p21 and p16 appeared to have the same pI as the proteins in isolated virions (not shown), we presume that this slower migration is due to a (unconventional) conformational change and not the consequence of their binding to some other (unknown) protein.

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