Abstract
H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.
Highlights
Vaccinia protein H5 is a multifunctional protein involved in several aspects of viral replication
Circular dichroism, sedimentation equilibrium, and small-angle x-ray scattering (SAXS),2 we have demonstrated that the basic structure of unphosphorylated H5 protein is an elongated rodlike tetramer with the intrinsically disordered N terminus of each subunit contributing to its rod-like structure
We have shown that vaccinia virus protein, H5 is associated with endo-ribonucleolytic cleavage of at least two homogenous late transcripts (ATI and F17) in vitro [22]
Summary
Vaccinia protein H5 is a multifunctional protein involved in several aspects of viral replication. Several studies [23, 26, 27] have shown that this protein interacts with viral late transcription initiation (A2 and G8), elongation (G2), and termination (A18) factors. We have characterized the vaccinia H5 protein as being associated with this activity, suggesting a role for H5 in late transcription termination [22]. This cleavage was characterized to be possibly RNA sequence or structure specific, and phosphorylation was determined to be essential for cleavage activity. Phosphorylation of the protein likely causes aggregation of H5 tetramers to form higher order complexes
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