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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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Western blots of vv  proteins analyzed under reducing and nonreducing conditions. In A analysis of four  IMV membrane proteins,  p21 (A17L), p16 (A14L), p35  (H3R), and p32 (D8L). Cell  lysates of infected HeLa cells  treated (F) or not treated (L)  with rifampicin or purified  virus (V) were run on 15%  SDS-PAGE. Before electrophoresis the samples were  boiled for 3 min in LSB with  (R) or without (NR) 5% β-ME  and 100 mM DTT. The proteins were detected by Western blotting using the respective antibodies. On the right  side of the figure the positions of the 54-, 38-, and 34-kD marker proteins is indicated. In B, three vv core  proteins 4a (A10L), p39  (A4L), and p25 (L4R) were  analyzed as described in A.  Note that in rifampicin-blocked cell lysates for both  4a and p25 only their precursor form (of 110 and 28 kD, respectively) can be detected that is  subsequently cleaved to the 65- and 25-kD mature forms, respectively, in the IMV. Some of the uncleaved form, can however,  still be detected in the isolated virions. The positions of the 130-,  66-, 54-, 38-, and 34-kD marker proteins are indicated on the  right.
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Figure 2: Western blots of vv proteins analyzed under reducing and nonreducing conditions. In A analysis of four IMV membrane proteins, p21 (A17L), p16 (A14L), p35 (H3R), and p32 (D8L). Cell lysates of infected HeLa cells treated (F) or not treated (L) with rifampicin or purified virus (V) were run on 15% SDS-PAGE. Before electrophoresis the samples were boiled for 3 min in LSB with (R) or without (NR) 5% β-ME and 100 mM DTT. The proteins were detected by Western blotting using the respective antibodies. On the right side of the figure the positions of the 54-, 38-, and 34-kD marker proteins is indicated. In B, three vv core proteins 4a (A10L), p39 (A4L), and p25 (L4R) were analyzed as described in A. Note that in rifampicin-blocked cell lysates for both 4a and p25 only their precursor form (of 110 and 28 kD, respectively) can be detected that is subsequently cleaved to the 65- and 25-kD mature forms, respectively, in the IMV. Some of the uncleaved form, can however, still be detected in the isolated virions. The positions of the 130-, 66-, 54-, 38-, and 34-kD marker proteins are indicated on the right.

Mentions: The membrane proteins p21, p16, and p32 (see Fig. 1 and Table I) appeared to form disulfide-bonded dimers under all three conditions, since they occurred in monomer and dimer forms under nonreducing conditions (Fig. 2 a). The amount of dimer formation was somewhat variable; in the case of p21 and p16 ∼50% of the protein dimerized (see also below) while of p32 no more than 30% of the protein was in the dimer form. As noted before (48, 49, 55), the dimers of both p16 and p21 migrated at molecular masses that were smaller than the sum of two monomers. We presume that this is a consequence of a folded conformation of the dimer and not due to heterodimerization. By two-dimensional (2-D) gel analysis we have shown before that the dimers of p21 and p16 run at the same pI as their respective monomers (26). Since heterodimer formation would result in a shift of the pI relative to the monomeric protein, these 2-D gel data show that both proteins form homodimers.


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

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

Western blots of vv  proteins analyzed under reducing and nonreducing conditions. In A analysis of four  IMV membrane proteins,  p21 (A17L), p16 (A14L), p35  (H3R), and p32 (D8L). Cell  lysates of infected HeLa cells  treated (F) or not treated (L)  with rifampicin or purified  virus (V) were run on 15%  SDS-PAGE. Before electrophoresis the samples were  boiled for 3 min in LSB with  (R) or without (NR) 5% β-ME  and 100 mM DTT. The proteins were detected by Western blotting using the respective antibodies. On the right  side of the figure the positions of the 54-, 38-, and 34-kD marker proteins is indicated. In B, three vv core  proteins 4a (A10L), p39  (A4L), and p25 (L4R) were  analyzed as described in A.  Note that in rifampicin-blocked cell lysates for both  4a and p25 only their precursor form (of 110 and 28 kD, respectively) can be detected that is  subsequently cleaved to the 65- and 25-kD mature forms, respectively, in the IMV. Some of the uncleaved form, can however,  still be detected in the isolated virions. The positions of the 130-,  66-, 54-, 38-, and 34-kD marker proteins are indicated on the  right.
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Related In: Results  -  Collection

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Figure 2: Western blots of vv proteins analyzed under reducing and nonreducing conditions. In A analysis of four IMV membrane proteins, p21 (A17L), p16 (A14L), p35 (H3R), and p32 (D8L). Cell lysates of infected HeLa cells treated (F) or not treated (L) with rifampicin or purified virus (V) were run on 15% SDS-PAGE. Before electrophoresis the samples were boiled for 3 min in LSB with (R) or without (NR) 5% β-ME and 100 mM DTT. The proteins were detected by Western blotting using the respective antibodies. On the right side of the figure the positions of the 54-, 38-, and 34-kD marker proteins is indicated. In B, three vv core proteins 4a (A10L), p39 (A4L), and p25 (L4R) were analyzed as described in A. Note that in rifampicin-blocked cell lysates for both 4a and p25 only their precursor form (of 110 and 28 kD, respectively) can be detected that is subsequently cleaved to the 65- and 25-kD mature forms, respectively, in the IMV. Some of the uncleaved form, can however, still be detected in the isolated virions. The positions of the 130-, 66-, 54-, 38-, and 34-kD marker proteins are indicated on the right.
Mentions: The membrane proteins p21, p16, and p32 (see Fig. 1 and Table I) appeared to form disulfide-bonded dimers under all three conditions, since they occurred in monomer and dimer forms under nonreducing conditions (Fig. 2 a). The amount of dimer formation was somewhat variable; in the case of p21 and p16 ∼50% of the protein dimerized (see also below) while of p32 no more than 30% of the protein was in the dimer form. As noted before (48, 49, 55), the dimers of both p16 and p21 migrated at molecular masses that were smaller than the sum of two monomers. We presume that this is a consequence of a folded conformation of the dimer and not due to heterodimerization. By two-dimensional (2-D) gel analysis we have shown before that the dimers of p21 and p16 run at the same pI as their respective monomers (26). Since heterodimer formation would result in a shift of the pI relative to the monomeric protein, these 2-D gel data show that both proteins form homodimers.

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