Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase

Abstract

Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1␣2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates.Vaccinia virus, the prototypic member of the Poxvirus family, encodes a 192-kb double-stranded DNA genome that encodes ϳ200 proteins. This expansive coding capacity enables vaccinia virus to replicate in the cytoplasm of host cells with significant autonomy from cellular factors within the nucleus. A requirement for this compartmentalization is that vaccinia virus must encode and synthesize the majority of components required for productive viral infection, including those involved in transcription and genome replication. Through genetic and biochemical analysis of complementation groups of temperaturesensitive (ts) mutants, five viral open reading frames (ORFs) essential for genome replication have been described. These include the viral DNA polymerase (E9) (6, 7, 26-28, 38, 40, 41, 44), a component of the processive DNA polymerase complex (A20) (17, 32), a DNA-independent nucleotide triphosphatase (D5) (10, 11), uracil DNA glycosylase (D4) (39), and a serine/ threonine protein kinase (B1) (33,34,43). B1 is an essential 34-kDa serine/threonine protein kinase which is made at early times postinfection, prior to the onset of viral DNA synthesis. Phenotypic analysis of two ts mutants with defined lesions in the B1 ORF (ts2 and ts25) has demonstrated that, in the absence of B1, the virus is deficient in its ability to synthesize viral DNA at high temperature (33). The severity of this defect varies with cell type and multiplici...

Viral Serine/Threonine Protein Kinases

“…Since H5 is also involved in transcription elongation (via interaction with A18 and G2), DNA replication (via interaction with A20), and virion morphogenesis, phosphorylation by B1 may also affect these aspects of the virus cycle (23,50,131). However, arguing against a direct involvement of B1-mediated H5 phosphorylation in DNA replication is the finding that rescue of DNA replication in the ts2 mutant by expression of the B1-like VRK1 was not associated with H5 phosphorylation (24). An alternative role for the B1/H5 interaction may lie in evasion of the innate antiviral response, since both proteins appear to be involved in inhibiting CD1d-mediated antigen presentation to natural killer T (NKT) cells (228).…”

“…Although not proven as of yet, it is possible that the molluscum contagiosum virus is able to compensate for the lack of B1 by usurping related host kinases, such as VRKs (108). In line with this idea, expression of the VRK1 genes of humans and, to a lesser extent, mice in the B1-defective ts2 mutant of vaccinia virus rescues the inhibition of DNA replication, virus production, and plaque formation observed with this mutant (24).…”

Molecular Characterization of the Host Defense Activity of the Barrier to Autointegration Factor against Vaccinia Virus

“…An understanding of the virus-host interactions occurring during vaccinia virus infection therefore provides insights into how critical aspects of the host immune system can be targeted by a human pathogen. It was recently discovered that the B1 kinase, which is highly homologous to the cellular VRK proteins (3,25), should be counted among the subversion measures of vaccinia virus against antiviral responses. The studies supporting this conclusion began with the discovery that, like VRKs, B1 can phosphorylate the N terminus of BAF both in vitro and in cultured cells.…”

VRK1 phosphorylates CREB and mediates CCND1 expression

“…Vaccinia virus B1 (vvB1) kinase plays a critical role in viral DNA replication (Rempel et al, 1990) by regulating intermediate viral gene expression (Kovacs et al, 2001). Human vaccinia-related kinase 1 (VRK1) can functionally rescue the DNA replication deficiency of vaccinia virus harboring a temperature-sensitive mutation of vvB1 under nonpermissive temperature conditions (Boyle and Traktman, 2004). This capacity requires the kinase activity of VRK1 because a catalytically inactive allele of VRK1 fails to rescue viral replication and suggests that VRK1 mimics the function of vvB1 kinase (Boyle and Traktman, 2004).…”