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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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[3H]NEM has access to the viral core proteins. In A, isolated and purified IMVs were labeled for  20 min on ice with [3H]NEM  (3H). Infected cells were labeled for 3 d with [35S]methionine and the labeled virions isolated and purified  (35S). 35S-labeled virions  were disrupted by incubation  for 30 min at 37°C with 1%  SDS and subsequently diluted in lysis buffer to a final  concentration of 0.1% SDS.  From these lysed particles  the protein 4a (4a), p25  (p25), and p11 (p11) were  immunoprecipitated. Analysis was on 15% SDS-PAGE  followed by autoradiography. M-14C-labeled marker  proteins of 14, 30, 45, 69, 93,  and 200 kD. In B, infected  cells were treated (NEM +)  or not treated (NEM −) with  20 mM NEM for 20 min on ice before the preparation of PNS in  buffer with or without NEM. The virions were concentrated by  pelleting through a sucrose cushion at 24 krpm in a SW40 rotor  for 30 min. Pellets were resuspended in buffer without NEM.  Equal amounts of pelleted virions were incubated with 5 μCi of  [3H]NEM and the labeled virions analyzed on 15% SDS-PAGE  followed by autoradiography. In the left lane (of + or − NEM) 3  μg of total protein, in the right lane 8 μg of protein was used for  [3H]NEM labeling. 35S virus control lane consisting of [35S]methionine-labeled, purified virions. M-14C-labeled marker proteins  of 69, 45, 30, and 14 kD. In C, the same amounts of virions as used  for the [3H]NEM labeling (3 μg of protein [left] and 8 μg [right],  respectively) isolated from cells that were pretreated (NEM +)  or not pretreated (NEM −) with NEM were run on 15% SDS-PAGE and the proteins were detected by silver staining.
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Figure 3: [3H]NEM has access to the viral core proteins. In A, isolated and purified IMVs were labeled for 20 min on ice with [3H]NEM (3H). Infected cells were labeled for 3 d with [35S]methionine and the labeled virions isolated and purified (35S). 35S-labeled virions were disrupted by incubation for 30 min at 37°C with 1% SDS and subsequently diluted in lysis buffer to a final concentration of 0.1% SDS. From these lysed particles the protein 4a (4a), p25 (p25), and p11 (p11) were immunoprecipitated. Analysis was on 15% SDS-PAGE followed by autoradiography. M-14C-labeled marker proteins of 14, 30, 45, 69, 93, and 200 kD. In B, infected cells were treated (NEM +) or not treated (NEM −) with 20 mM NEM for 20 min on ice before the preparation of PNS in buffer with or without NEM. The virions were concentrated by pelleting through a sucrose cushion at 24 krpm in a SW40 rotor for 30 min. Pellets were resuspended in buffer without NEM. Equal amounts of pelleted virions were incubated with 5 μCi of [3H]NEM and the labeled virions analyzed on 15% SDS-PAGE followed by autoradiography. In the left lane (of + or − NEM) 3 μg of total protein, in the right lane 8 μg of protein was used for [3H]NEM labeling. 35S virus control lane consisting of [35S]methionine-labeled, purified virions. M-14C-labeled marker proteins of 69, 45, 30, and 14 kD. In C, the same amounts of virions as used for the [3H]NEM labeling (3 μg of protein [left] and 8 μg [right], respectively) isolated from cells that were pretreated (NEM +) or not pretreated (NEM −) with NEM were run on 15% SDS-PAGE and the proteins were detected by silver staining.

Mentions: The above results strongly suggest that vv membrane and core proteins form disulfide bonds. Care was taken in each of these experiments to incubate the infected cells at least 20 min with NEM on ice before cell lysis, thus excluding the possibility that the disulfide bonding resulted from post-lysis oxidation. However, the mature IMV is a highly compact structure and we had to rule out that NEM might not have access to vv proteins, especially to those within the viral core. Therefore, we performed two experiments to address this issue. First, isolated and purified IMVs were labeled with [3H]NEM for 20 min on ice and the labeled protein pattern compared with [35S]methionine-labeled virions. [3H]NEM labeling showed a discrete pattern of bands, the most abundant of which comigrated with the core proteins 4a, p11, and p25 (Fig. 3 a). Some of the other minor bands were identified as p21, p16, and p35 (not shown). The [3H]NEM labeling of both p21 and p16 increased significantly when the IMV was treated with DTT before the labeling (not shown).


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

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

[3H]NEM has access to the viral core proteins. In A, isolated and purified IMVs were labeled for  20 min on ice with [3H]NEM  (3H). Infected cells were labeled for 3 d with [35S]methionine and the labeled virions isolated and purified  (35S). 35S-labeled virions  were disrupted by incubation  for 30 min at 37°C with 1%  SDS and subsequently diluted in lysis buffer to a final  concentration of 0.1% SDS.  From these lysed particles  the protein 4a (4a), p25  (p25), and p11 (p11) were  immunoprecipitated. Analysis was on 15% SDS-PAGE  followed by autoradiography. M-14C-labeled marker  proteins of 14, 30, 45, 69, 93,  and 200 kD. In B, infected  cells were treated (NEM +)  or not treated (NEM −) with  20 mM NEM for 20 min on ice before the preparation of PNS in  buffer with or without NEM. The virions were concentrated by  pelleting through a sucrose cushion at 24 krpm in a SW40 rotor  for 30 min. Pellets were resuspended in buffer without NEM.  Equal amounts of pelleted virions were incubated with 5 μCi of  [3H]NEM and the labeled virions analyzed on 15% SDS-PAGE  followed by autoradiography. In the left lane (of + or − NEM) 3  μg of total protein, in the right lane 8 μg of protein was used for  [3H]NEM labeling. 35S virus control lane consisting of [35S]methionine-labeled, purified virions. M-14C-labeled marker proteins  of 69, 45, 30, and 14 kD. In C, the same amounts of virions as used  for the [3H]NEM labeling (3 μg of protein [left] and 8 μg [right],  respectively) isolated from cells that were pretreated (NEM +)  or not pretreated (NEM −) with NEM were run on 15% SDS-PAGE and the proteins were detected by silver staining.
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Related In: Results  -  Collection

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Figure 3: [3H]NEM has access to the viral core proteins. In A, isolated and purified IMVs were labeled for 20 min on ice with [3H]NEM (3H). Infected cells were labeled for 3 d with [35S]methionine and the labeled virions isolated and purified (35S). 35S-labeled virions were disrupted by incubation for 30 min at 37°C with 1% SDS and subsequently diluted in lysis buffer to a final concentration of 0.1% SDS. From these lysed particles the protein 4a (4a), p25 (p25), and p11 (p11) were immunoprecipitated. Analysis was on 15% SDS-PAGE followed by autoradiography. M-14C-labeled marker proteins of 14, 30, 45, 69, 93, and 200 kD. In B, infected cells were treated (NEM +) or not treated (NEM −) with 20 mM NEM for 20 min on ice before the preparation of PNS in buffer with or without NEM. The virions were concentrated by pelleting through a sucrose cushion at 24 krpm in a SW40 rotor for 30 min. Pellets were resuspended in buffer without NEM. Equal amounts of pelleted virions were incubated with 5 μCi of [3H]NEM and the labeled virions analyzed on 15% SDS-PAGE followed by autoradiography. In the left lane (of + or − NEM) 3 μg of total protein, in the right lane 8 μg of protein was used for [3H]NEM labeling. 35S virus control lane consisting of [35S]methionine-labeled, purified virions. M-14C-labeled marker proteins of 69, 45, 30, and 14 kD. In C, the same amounts of virions as used for the [3H]NEM labeling (3 μg of protein [left] and 8 μg [right], respectively) isolated from cells that were pretreated (NEM +) or not pretreated (NEM −) with NEM were run on 15% SDS-PAGE and the proteins were detected by silver staining.
Mentions: The above results strongly suggest that vv membrane and core proteins form disulfide bonds. Care was taken in each of these experiments to incubate the infected cells at least 20 min with NEM on ice before cell lysis, thus excluding the possibility that the disulfide bonding resulted from post-lysis oxidation. However, the mature IMV is a highly compact structure and we had to rule out that NEM might not have access to vv proteins, especially to those within the viral core. Therefore, we performed two experiments to address this issue. First, isolated and purified IMVs were labeled with [3H]NEM for 20 min on ice and the labeled protein pattern compared with [35S]methionine-labeled virions. [3H]NEM labeling showed a discrete pattern of bands, the most abundant of which comigrated with the core proteins 4a, p11, and p25 (Fig. 3 a). Some of the other minor bands were identified as p21, p16, and p35 (not shown). The [3H]NEM labeling of both p21 and p16 increased significantly when the IMV was treated with DTT before the labeling (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