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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 NH2 terminus of p32 is exposed on the IMV surface and towards the cytoplasm of infected cells. (A) Purified  IMV was treated for 30 min at 30°C with the indicated concentrations of trypsin. Digested IMV was pelleted by a 30-min centrifugation at 15,000 g. The pelleted virus (pellet) and the remaining  supernatant were analyzed on 10% SDS-PAGE followed by  Western blots using antibodies to p32, 4a, and p14. P14 and 4a  were analyzed after reduction while p32 was analyzed under nonreducing conditions only. The position of the monomer of p32 is  indicated with one and that of the dimer with two asterisks. (B)  Post nuclear supernatants of rifampicin-treated cells infected  with a vv recombinant expressing the MHV-M protein were prepared at 7 h after infection. PNS was treated with the indicated  concentrations of trypsin at 30°C for 30 min. Supernatant was  separated from the membranes (pellet) by a centrifugation for 30  min at 150,000 g and both fractions were analyzed on 15% SDS-PAGE. The proteins were detected by Western blots using antibodies to p32, the NH2 (M-N) or COOH terminus (M-C) of the  MHV-M protein. The unglycosylated (M0) and glycosylated  forms (M3 and M4) of the M protein are indicated (see also 33).  Note that the COOH-terminal specific peptide serum recognizes  only the M0 and M4 forms.
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Figure 5: The NH2 terminus of p32 is exposed on the IMV surface and towards the cytoplasm of infected cells. (A) Purified IMV was treated for 30 min at 30°C with the indicated concentrations of trypsin. Digested IMV was pelleted by a 30-min centrifugation at 15,000 g. The pelleted virus (pellet) and the remaining supernatant were analyzed on 10% SDS-PAGE followed by Western blots using antibodies to p32, 4a, and p14. P14 and 4a were analyzed after reduction while p32 was analyzed under nonreducing conditions only. The position of the monomer of p32 is indicated with one and that of the dimer with two asterisks. (B) Post nuclear supernatants of rifampicin-treated cells infected with a vv recombinant expressing the MHV-M protein were prepared at 7 h after infection. PNS was treated with the indicated concentrations of trypsin at 30°C for 30 min. Supernatant was separated from the membranes (pellet) by a centrifugation for 30 min at 150,000 g and both fractions were analyzed on 15% SDS-PAGE. The proteins were detected by Western blots using antibodies to p32, the NH2 (M-N) or COOH terminus (M-C) of the MHV-M protein. The unglycosylated (M0) and glycosylated forms (M3 and M4) of the M protein are indicated (see also 33). Note that the COOH-terminal specific peptide serum recognizes only the M0 and M4 forms.

Mentions: Hydrophobicity plots of p32 revealed a single hydrophobic segment of 20 amino acids in its extreme COOH terminus that could serve as membrane-anchor (38). Thus, the protein seems to belong to a recently described class of membrane proteins with COOH-terminal hydrophobic domains that insert posttranslationally, thereby exposing most of their NH2 terminus towards the cytoplasm (35). If p32 inserts into the membrane accordingly, the protein would expose its single cysteine in the NH2 terminus at position 262 towards the cytoplasm as well as on the outer surface of the IMV (see also 54). To test this possibility, we carried out two different set of experiments. First, purified IMV was treated with increasing concentrations of trypsin and the digestion of p32 was assayed by Western blot using an antibody recognizing amino acid 77 to 294 (43). Under conditions in which the internally located core protein 4a was completely protease-protected, the entire population of both the dimer as well as the monomer of p32 were digested (Fig. 5 a). In fact, after pelleting the IMV from the digestion mixture, a new 28-kD fragment that was recognized by the p32 antibody was now detected in the supernatant, while the digested virion was totally devoid of immunoreactive p32 protein (Fig. 5 a). The well characterized peripheral membrane protein p14 was completely digested even at the lowest protease concentration tested and no protected fragment was detected in the supernatant.


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

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

The NH2 terminus of p32 is exposed on the IMV surface and towards the cytoplasm of infected cells. (A) Purified  IMV was treated for 30 min at 30°C with the indicated concentrations of trypsin. Digested IMV was pelleted by a 30-min centrifugation at 15,000 g. The pelleted virus (pellet) and the remaining  supernatant were analyzed on 10% SDS-PAGE followed by  Western blots using antibodies to p32, 4a, and p14. P14 and 4a  were analyzed after reduction while p32 was analyzed under nonreducing conditions only. The position of the monomer of p32 is  indicated with one and that of the dimer with two asterisks. (B)  Post nuclear supernatants of rifampicin-treated cells infected  with a vv recombinant expressing the MHV-M protein were prepared at 7 h after infection. PNS was treated with the indicated  concentrations of trypsin at 30°C for 30 min. Supernatant was  separated from the membranes (pellet) by a centrifugation for 30  min at 150,000 g and both fractions were analyzed on 15% SDS-PAGE. The proteins were detected by Western blots using antibodies to p32, the NH2 (M-N) or COOH terminus (M-C) of the  MHV-M protein. The unglycosylated (M0) and glycosylated  forms (M3 and M4) of the M protein are indicated (see also 33).  Note that the COOH-terminal specific peptide serum recognizes  only the M0 and M4 forms.
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Related In: Results  -  Collection

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Figure 5: The NH2 terminus of p32 is exposed on the IMV surface and towards the cytoplasm of infected cells. (A) Purified IMV was treated for 30 min at 30°C with the indicated concentrations of trypsin. Digested IMV was pelleted by a 30-min centrifugation at 15,000 g. The pelleted virus (pellet) and the remaining supernatant were analyzed on 10% SDS-PAGE followed by Western blots using antibodies to p32, 4a, and p14. P14 and 4a were analyzed after reduction while p32 was analyzed under nonreducing conditions only. The position of the monomer of p32 is indicated with one and that of the dimer with two asterisks. (B) Post nuclear supernatants of rifampicin-treated cells infected with a vv recombinant expressing the MHV-M protein were prepared at 7 h after infection. PNS was treated with the indicated concentrations of trypsin at 30°C for 30 min. Supernatant was separated from the membranes (pellet) by a centrifugation for 30 min at 150,000 g and both fractions were analyzed on 15% SDS-PAGE. The proteins were detected by Western blots using antibodies to p32, the NH2 (M-N) or COOH terminus (M-C) of the MHV-M protein. The unglycosylated (M0) and glycosylated forms (M3 and M4) of the M protein are indicated (see also 33). Note that the COOH-terminal specific peptide serum recognizes only the M0 and M4 forms.
Mentions: Hydrophobicity plots of p32 revealed a single hydrophobic segment of 20 amino acids in its extreme COOH terminus that could serve as membrane-anchor (38). Thus, the protein seems to belong to a recently described class of membrane proteins with COOH-terminal hydrophobic domains that insert posttranslationally, thereby exposing most of their NH2 terminus towards the cytoplasm (35). If p32 inserts into the membrane accordingly, the protein would expose its single cysteine in the NH2 terminus at position 262 towards the cytoplasm as well as on the outer surface of the IMV (see also 54). To test this possibility, we carried out two different set of experiments. First, purified IMV was treated with increasing concentrations of trypsin and the digestion of p32 was assayed by Western blot using an antibody recognizing amino acid 77 to 294 (43). Under conditions in which the internally located core protein 4a was completely protease-protected, the entire population of both the dimer as well as the monomer of p32 were digested (Fig. 5 a). In fact, after pelleting the IMV from the digestion mixture, a new 28-kD fragment that was recognized by the p32 antibody was now detected in the supernatant, while the digested virion was totally devoid of immunoreactive p32 protein (Fig. 5 a). The well characterized peripheral membrane protein p14 was completely digested even at the lowest protease concentration tested and no protected fragment was detected in the supernatant.

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