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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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The proteins from viruses  isolated from DTT-treated cells are  not disulfide bonded. Infected HeLa  cells were treated (DTT +) or not  (DTT −) with 5mM DTT from 4 h  after infection and the medium replaced each hour as described in the  Results. 9 h after infection, virions  were isolated from the cells as described in Materials and Methods.  Isolated virions were analyzed on  10% SDS-PAGE and the viral proteins p21, p16, 4a, and p25 were detected by Western blotting. Virions  isolated from untreated cells (V−)  were analyzed after boiling in sample  buffer with (DTT +) or without  (DTT −) 5% β-ME and 100 mM DTT, while virions isolated  from treated (V +) cells were only analyzed after boiling in sample buffer without (DTT −) these reducing agents. On the right  the positions of the 34-, 38-, 54-, 84-, 116-, and 180-kD marker  proteins are indicated.
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Figure 8: The proteins from viruses isolated from DTT-treated cells are not disulfide bonded. Infected HeLa cells were treated (DTT +) or not (DTT −) with 5mM DTT from 4 h after infection and the medium replaced each hour as described in the Results. 9 h after infection, virions were isolated from the cells as described in Materials and Methods. Isolated virions were analyzed on 10% SDS-PAGE and the viral proteins p21, p16, 4a, and p25 were detected by Western blotting. Virions isolated from untreated cells (V−) were analyzed after boiling in sample buffer with (DTT +) or without (DTT −) 5% β-ME and 100 mM DTT, while virions isolated from treated (V +) cells were only analyzed after boiling in sample buffer without (DTT −) these reducing agents. On the right the positions of the 34-, 38-, 54-, 84-, 116-, and 180-kD marker proteins are indicated.

Mentions: In infected cells both p25 and 4a are initially made as precursors of 28 and 110 kD, respectively, which are subsequently cleaved upon IMV formation into their mature forms of 25 and 65 kD, respectively (57, 58). Indeed, in rifampicin treated cell lysates both proteins migrated at the position of their precursor form and the migration of both proteins was the same when analyzed with or without prior reduction. However, in the IMV the mature forms of these two proteins showed additional bands when analyzed without reduction. The protein 4a occurred both as a monomer of 65 kD, as well as a dimer of ∼130 kD when β-ME and DTT were omitted from the sample buffer (Fig. 2 b). That the dimer indeed ran at ∼130 kD can be better appreciated in Fig. 8, where 4a was analyzed in a 10% SDS-PAGE (see below). After reduction 4a migrated predominantly in its (reduced) mature form of 65 kD, while the 130 kD band was clearly absent. Two additional bands were seen in this reduced sample, the upper one represents the uncleaved precursor, p4a (note the comigration of this band with the form made in rifampicin-blocked cell lysates), of which small amounts can be detected in mature virions, while the identity of the lower of the two remained open. When analyzing p25 in isolated virions under nonreducing conditions the protein showed a very unusual pattern and appeared as several (disulfide bonded) species some of which migrated as a smear rather than discrete bands (Fig. 2 b). When analyzed after reduction, the protein migrated predominantly at the position expected for its mature 25-kD form, but also, as for 4a, some of the uncleaved precursor could still be detected.


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

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

The proteins from viruses  isolated from DTT-treated cells are  not disulfide bonded. Infected HeLa  cells were treated (DTT +) or not  (DTT −) with 5mM DTT from 4 h  after infection and the medium replaced each hour as described in the  Results. 9 h after infection, virions  were isolated from the cells as described in Materials and Methods.  Isolated virions were analyzed on  10% SDS-PAGE and the viral proteins p21, p16, 4a, and p25 were detected by Western blotting. Virions  isolated from untreated cells (V−)  were analyzed after boiling in sample  buffer with (DTT +) or without  (DTT −) 5% β-ME and 100 mM DTT, while virions isolated  from treated (V +) cells were only analyzed after boiling in sample buffer without (DTT −) these reducing agents. On the right  the positions of the 34-, 38-, 54-, 84-, 116-, and 180-kD marker  proteins are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132897&req=5

Figure 8: The proteins from viruses isolated from DTT-treated cells are not disulfide bonded. Infected HeLa cells were treated (DTT +) or not (DTT −) with 5mM DTT from 4 h after infection and the medium replaced each hour as described in the Results. 9 h after infection, virions were isolated from the cells as described in Materials and Methods. Isolated virions were analyzed on 10% SDS-PAGE and the viral proteins p21, p16, 4a, and p25 were detected by Western blotting. Virions isolated from untreated cells (V−) were analyzed after boiling in sample buffer with (DTT +) or without (DTT −) 5% β-ME and 100 mM DTT, while virions isolated from treated (V +) cells were only analyzed after boiling in sample buffer without (DTT −) these reducing agents. On the right the positions of the 34-, 38-, 54-, 84-, 116-, and 180-kD marker proteins are indicated.
Mentions: In infected cells both p25 and 4a are initially made as precursors of 28 and 110 kD, respectively, which are subsequently cleaved upon IMV formation into their mature forms of 25 and 65 kD, respectively (57, 58). Indeed, in rifampicin treated cell lysates both proteins migrated at the position of their precursor form and the migration of both proteins was the same when analyzed with or without prior reduction. However, in the IMV the mature forms of these two proteins showed additional bands when analyzed without reduction. The protein 4a occurred both as a monomer of 65 kD, as well as a dimer of ∼130 kD when β-ME and DTT were omitted from the sample buffer (Fig. 2 b). That the dimer indeed ran at ∼130 kD can be better appreciated in Fig. 8, where 4a was analyzed in a 10% SDS-PAGE (see below). After reduction 4a migrated predominantly in its (reduced) mature form of 65 kD, while the 130 kD band was clearly absent. Two additional bands were seen in this reduced sample, the upper one represents the uncleaved precursor, p4a (note the comigration of this band with the form made in rifampicin-blocked cell lysates), of which small amounts can be detected in mature virions, while the identity of the lower of the two remained open. When analyzing p25 in isolated virions under nonreducing conditions the protein showed a very unusual pattern and appeared as several (disulfide bonded) species some of which migrated as a smear rather than discrete bands (Fig. 2 b). When analyzed after reduction, the protein migrated predominantly at the position expected for its mature 25-kD form, but also, as for 4a, some of the uncleaved precursor could still be detected.

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