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Moloney murine leukemia virus envelope protein subunits, gp70 and Pr15E, form a stable disulfide-linked complex
D J Opstelten1, M Wallin, H Garoff
1Department for Biosciences at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden. Dirk-Jan.Opstelten@cbt.ki.se
Abstract:
The nature and stability of the interactions between the gp70 and Pr15E/p15E molecules of murine leukemia virus (MLV) have been disputed extensively. To resolve this controversy, we have performed quantitative biochemical analyses on gp70-Pr15E complexes formed after independent expression of the amphotropic and ecotropic Moloney MLV env genes in BHK-21 cells. We found that all cell-associated gp70 molecules are disulfide linked to Pr15E whereas only a small amount of free gp70 is released by the cells. The complexes were resistant to treatment with reducing agents in vivo, indicating that the presence and stability of the disulfide interaction between gp70 and Pr15E are not dependent on the cellular redox state. However, disulfide-bonded Env complexes were disrupted in lysates of nonalkylated cells in a time-, temperature-, and pH-dependent fashion. Disruption seemed not to be caused by a cellular factor but is probably due to a thiol-disulfide exchange reaction occurring within the Env complex after solubilization. The possibility that alkylating agents induce the formation of the intersubunit disulfide linkage was excluded by showing that disulfide-linked gp70-Pr15E complexes exist in freshly made lysates of nonalkylated cells and that disruption of the complexes can be prevented by lowering the pH. Together, these data establish that gp70 and Pr15E form a stable disulfide-linked complex in vivo.
Insights
Murine leukemia virus (MLV) gp70 and Pr15E proteins form a stable disulfide-linked complex in vivo. This interaction is independent of cellular redox state but can be disrupted after cell lysis.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- The interaction between gp70 and Pr15E/p15E subunits of murine leukemia virus (MLV) has been a subject of extensive debate.
- Understanding this interaction is crucial for comprehending MLV assembly and infectivity.
Purpose of the Study:
- To elucidate the precise nature and stability of the gp70-Pr15E complex in murine leukemia virus.
- To resolve controversies regarding the disulfide linkage and its dependence on cellular conditions.
Main Methods:
- Quantitative biochemical analyses were performed on gp70-Pr15E complexes.
- Independent expression of Moloney MLV env genes (amphotropic and ecotropic) in BHK-21 cells.
- Analysis of complex stability under various conditions, including reducing agents, cell lysis, and pH changes.
Main Results:
- All cell-associated gp70 molecules are disulfide-linked to Pr15E; free gp70 release is minimal.
- The disulfide linkage is stable in vivo and resistant to reducing agents, independent of cellular redox state.
- Disulfide-bonded Env complexes dissociate in nonalkylated cell lysates via a time-, temperature-, and pH-dependent process, likely due to thiol-disulfide exchange.
Conclusions:
- gp70 and Pr15E form a stable disulfide-linked complex in vivo.
- The stability of this complex is not reliant on the cellular redox environment.
- Complex dissociation post-solubilization is attributed to thiol-disulfide exchange rather than cellular factors.