Summary auto-generated
This study investigates the phosphorylation state and cellular localization of H5R, a 36 kDa phosphoprotein produced during vaccinia virus (VV) infection. Using two-dimensional gel electrophoresis and immunofluorescence, the researchers found that H5R exists in two distinct forms: an underphosphorylated form (pI 6.8) comprising approximately 75% of total H5R, which localizes to virosomes (viral factories), and a highly phosphorylated form (pI 5.5) comprising ~25%, which remains soluble in the cytoplasm. An H5R-specific antibody recognized only underphosphorylated forms, suggesting phosphorylation affects the N-terminal epitope. When viral DNA replication was blocked with araC, underphosphorylated H5R distributed diffusely throughout the cytoplasm and accumulated in punctate particles alongside the B1R kinase. However, H5R successfully associated with newly formed virosomes even when late protein synthesis was blocked, indicating viral DNA synthesis alone is sufficient for virosomal targeting. These findings suggest H5R has multiple roles in VV development, with different phosphorylation states and subcellular localizations reflecting distinct functional states during viral replication and assembly.
Key findings
- Vaccinia virus H5R protein exists in two phosphorylation states with different subcellular localizations: underphosphorylated forms (75%) localize to virosomes while highly phosphorylated forms (25%) remain cytoplasmic
- An H5R-specific antibody recognizes only underphosphorylated forms, indicating that threonine residues in the N-terminal region are phosphorylated in more acidic variants
- Viral DNA replication, rather than late gene expression, is sufficient for H5R binding to virosomes, suggesting direct interaction with newly synthesized viral DNA
- When DNA replication is blocked, both H5R and the B1R kinase accumulate at punctate cytoplasmic particles, suggesting a functional relationship between these proteins at early replication sites
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Abstract
The phosphorylation state of vaccinia virus (VV) protein H5R synthesized in infected cells was investigated by two-dimensional gel electrophoresis. Most of the H5R protein was underphosphorylated (pI 5.9 to 6.8) and, on centrifugation of cell lysates, was associated with virosomes sedimenting with nuclei. However, about a quarter of the H5R protein synthesized was highly phosphorylated (pI 5.5), and this was the major form of the H5R protein present in cytoplasmic extracts. Immunofluorescence of VV-infected cells in the absence of DNA replication showed that underphosphorylated H5R protein, specifically recognized by antibody, was abundantly distributed throughout the cytoplasm but also present in punctate particles, whereas most of the B1R protein detected was in the punctate particles. Late gene expression was not required for the H5R protein to accumulate in virosomes--viral DNA synthesis was sufficient. The different phosphorylation states and cytological locations of the H5R protein suggest it has multiple roles in VV development.