Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus

Abstract

Background: Vaccinia protein H5 is a multifunctional protein involved in several aspects of viral replication. Results: H5 is a nucleic-acid binding, elongated rod-like tetramer with an instrinsically disordered N terminus and an ␣-helical C terminus. Conclusion: H5 functions as a hub protein in vaccinia virus replication linking otherwise unique viral processes. Significance: Understanding the mechanism of H5 function in vaccinia virus replication is crucial for studying vaccinia biology.

Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

“…In support of H5's contribution to transcription, it has also been shown to stimulate late transcription in a semipurified in vitro assay (54). Germane to each of these findings, the work of D'Costa, Condit, and colleagues has confirmed that the purified H5 protein exhibits nucleic acid binding activity, with the highest affinity being observed for dsDNA and double-stranded RNA (dsRNA) (K d s [dissociation constants] ϭ 12 and 14 nM, respectively) (53).…”

“…H5 is an abundant multifunctional protein that has been reported to interact with components of the DNA replication machinery as well as the postreplicative transcriptional machinery (24,25,28,30). In addition, the H5 protein copurifies with and may be associated with an enzymatic activity capable of processing the 3= termini of a subset of postreplicative viral mRNAs (53). In support of H5's contribution to transcription, it has also been shown to stimulate late transcription in a semipurified in vitro assay (54).…”

“…H5 might in fact form a lattice that serves as a scaffold on which the genomes and/or the holoenzyme might become arrayed for efficient synthesis. Modeling of the H5 protein and biophysical analyses suggest that much of the N-terminal 2/3 of the H5 protein is unstructured, whereas the C-terminal region is thought to form an amphipathic helix (53). It will be important to determine which regions of the protein mediate DNA binding, multimerization, and association with A20.…”

“…Alternatively, the high-affinity association of H5 with the viral genome might help to organize the genomes within the factory so that they are readily accessible to the replication machinery. H5 is highly abundant, and we know that it forms higher-order structures, with the tetrameric form being the most stable form in vitro (53). H5 might in fact form a lattice that serves as a scaffold on which the genomes and/or the holoenzyme might become arrayed for efficient synthesis.…”

Cascade regulation of vaccinia virus gene expression is modulated by multistage promoters

“…H5 is a constitutively expressed multifunctional nucleic acid binding protein involved in post-replicative transcription and mRNA processing (Cresawn and Condit, 2007; D’Costa et al, 2008; Kovacs and Moss, 1996), genome replication (D’Costa et al, 2010) and virion morphogenesis (DeMasi and Traktman, 2000). Moreover, biochemical and biophysical properties suggest that the H5 protein is intrinsically disordered and may be a “hub” protein (Kay et al, 2013). Although the late transcription factor cell line was successfully used to express reporter genes with late promoters, we have been unable to use it for isolation of a VACV late transcription factor deletion mutant.…”