The dual-specificity phosphatase encoded by vaccinia virus, VH1, is essential for viral transcription in vivo and in vitro

Abstract

The genetic complexity of vaccinia virus is such that as well as encoding its own transcription and replication machinery, it encodes two protein kinases and a protein phosphatase. The latter enzyme, designated VH1, is a prototype for the dual-specificity class of phosphatases. Here we report that the H1 phosphatase is encapsidated within vaccinia virions and describe the construction of a viral recombinant in which expression of the H1 gene is regulated by the presence or absence of isopropylthiogalactopyranoside (IPTG) in the culture medium. When expression of H1 is repressed, the number of viral particles produced is not compromised but the fraction of these particles which is infectious is significantly reduced. The lack of infectivity of the H1-deficient particles is specifically correlated with their inability to direct the transcription of early genes either in vitro or in vivo. A proximal role for the viral phosphatase in regulating the onset of viral gene expression is implied. Prominent among the encapsidated proteins found to be hyperphosphorylated in H1-deficient virions is the 11-kDa product of the F18 gene; this protein is the major DNA-binding component of the viral nucleoprotein complex. The ability of recombinant H1 phosphatase to reverse this hyperphosphorylation in permeabilized virions strengthens the conclusion that the F18 protein is a bona fide substrate for the H1 phosphatase.

STAT1 and pathogens, not a friendly relationship

“…The vaccinia virus, a Poxviridae family virus, encodes for several proteins which neutralize the IFN host defence system at different levels, including inhibition of PKR, and the release of cytokine homologues which block the IFNa/b and g Receptors. In addition to this, the virus encodes a protein with dual tyrosine/serine phosphatase activity whose expression is required for virus viability in tissue culture [241,242]. The dual phosphatase can both prevent STAT1 phosphorylation in infected cells and induce its dephosphorylation, thereby preventing the nuclear translocation of STAT1 and the induction of gene targets [243].…”

Aurintricarboxylic Acid Inhibits the Early Stage of Vaccinia Virus Replication byTargeting both Cellular and Viral Factors

“…We also show that ATA can inhibit the activity of the vaccinia virus phosphatase H1L in vitro. H1L plays an essential role in initiating viral early gene transcription and has also been implicated in evasion of the host immune response (19,24).…”

“…ATA has previously been shown to inhibit dual-specificity phosphatases, including those from both eukaryotic and prokaryotic sources (9,18). H1L plays an essential role in initiating early viral gene transcription (19). To further characterize the mechanism behind the inhibition of vaccinia virus replication by ATA, we performed enzyme inhibition assays to determine if ATA could inhibit the H1L phosphatase.…”

“…Two possible mechanisms through which ATA may exert this effect are the down-regulation of ERK signaling and the inhibition of vaccinia virus H1L phosphatase activity. The activity of both of these factors is required during the early phase of vaccinia virus replication (1,19). Treatment of cells with ATA strongly inhibited virus replication.…”

“…The gene encoding H1L is well conserved among the poxviruses, with homologues identified in all chordopoxviruses studied (29). H1L is necessary for initiating viral early gene transcription, and its immediate role in infection is highlighted by the finding that it is packaged in mature virions (19). Therefore, the inhi- Although it has been reported that H1L dephosphorylates Stat1 in vaccinia virus-infected, gamma interferon-treated cells (24), this is unlikely to be related to the transcriptional downregulation of vaccinia virus early genes observed.…”