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DNA-binding proteins in the cytoplasm of vaccinia virus-infected mouse L-cells
Abstract:
Mouse L cell fibroblasts were infected with vaccinia virus and labeled 2 to 3 h postinfection with [35S]methionine. Labeled proteins were fractionated on native and denatured DNA-cellulose columns and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Twenty-four 90,000 to 12,500, were detected. VDP-12A (molecular weight, 29,750) had affinity for denatured but not native DNA, and its synthesis was dependent on viral DNA replication. VDP-20 (molecular weight, 41,000) bound very tightly to native and denatured DNA and was displaced only after boiling the protein-DNA-cellulose matrix in 1% sodium dodecyl sulfate. VDP-8,-11,-12,-13, -and-14 behaved electrophoretically like the polypeptide species previously shown to be present in DNA-protein complexes prepared from infected cells. The molecular weights of VDP-10 (50,000), VDP-11 (36,000), and VDP-8 (67,000) were similar to the polypeptide subunits of polyadenylate polymerase and phosphohydrolase I, enzymes purified from virions which have also been shown to have affinity for DNA.
Insights
Researchers identified vaccinia virus proteins that bind to DNA. These viral DNA-binding proteins (VDPs) are crucial for viral DNA replication and may include enzymes like polyadenylate polymerase.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Vaccinia virus infection involves complex interactions between viral and host cell machinery.
- Understanding viral protein functions, particularly DNA-binding proteins, is essential for deciphering viral replication mechanisms.
Purpose of the Study:
- To identify and characterize vaccinia virus-encoded proteins that exhibit affinity for DNA.
- To investigate the role of these DNA-binding proteins in viral DNA replication.
Main Methods:
- Infection of mouse L cell fibroblasts with vaccinia virus.
- Metabolic labeling of infected cells with [35S]methionine.
- Fractionation of labeled proteins using native and denatured DNA-cellulose columns.
- Analysis of protein fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Main Results:
- Twenty-four vaccinia virus-induced proteins (VDPs) with molecular weights ranging from 12,500 to 90,000 were detected.
- VDP-12A showed affinity for denatured DNA and its synthesis was dependent on viral DNA replication.
- VDP-20 exhibited very tight binding to both native and denatured DNA.
- Several VDPs (VDP-8, -11, -12, -13, -14) showed electrophoretic similarities to known DNA-protein complex components.
- The molecular weights of VDP-10, VDP-11, and VDP-8 were comparable to subunits of polyadenylate polymerase and phosphohydrolase I, enzymes with known DNA affinity.
Conclusions:
- Vaccinia virus infection induces the synthesis of multiple DNA-binding proteins.
- Some of these VDPs are likely involved in viral DNA replication.
- Potential candidates for DNA-binding proteins include polyadenylate polymerase and phosphohydrolase I subunits.