Abstract

Retroviral integration is executed by the preintegration complex (PIC), which contains viral DNA together with a number of proteins. Barrier-to-autointegration factor (BAF), a cellular component of Moloney murine leukemia virus (MMLV) PICs, has been demonstrated to protect viral DNA from autointegration and stimulate the intermolecular integration activity of the PIC by its DNA binding activity. Recent studies reveal that the functions of BAF are regulated by phosphorylation via a family of cellular serine/threonine kinases called vaccinia-related kinases (VRK), and VRK-mediated phosphorylation causes a loss of the DNA binding activity of BAF. These results raise the possibility that BAF phosphorylation may influence the integration activities of the PIC through removal of BAF from viral DNA. In the present study, we report that VRK1 was able to abolish the intermolecular integration activity of MMLV PICs in vitro. This was accompanied by an enhancement of autointegration activity and dissociation of BAF from the PICs. In addition, in vitro phosphorylation of BAF by VRK1 abrogated the activity of BAF in PIC function. Among the VRK family members, VRK1 as well as VRK2, which catalyze hyperphosphorylation of BAF, could abolish PIC function. We also found that treatment of PICs with certain nucleotides such as ATP resulted in the inhibition of the intermolecular integration activity of PICs through the dissociation of BAF. More importantly, the ATP-induced disruption was not observed with the PICs from VRK1 knockdown cells. Our in vitro results therefore suggest the presence of cellular kinases including VRKs that can inactivate the retroviral integration complex via BAF phosphorylation.

Highlights

  • The core of an infecting retroviral virion is composed of a nucleoprotein complex in which genomic RNA is associated with enzymes and many proteins that are required for completion of the early phase of infection

  • We obtained evidence that Barrier-to-autointegration factor (BAF) phosphorylation adversely affects the function of another type of virus; VRK1 and VRK2 were capable of disrupting the Moloney murine leukemia virus (MMLV) preintegration complex (PIC) activity by the phosphorylation and dissociation of BAF in vitro

  • We addressed the question of whether, once BAF forms a complex with DNA, VRK1 treatment could release BAF from the DNA complex

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Summary

Dysfunction of Retroviral PIC by Cellular Kinases

BAF causes defects of chromatin segregation in Caenorhabditis elegans and nuclear envelope assembly in mammalian cells [13, 17, 19]. In subcellular fractionation studies of human cells, the unphosphorylated form of BAF was highly enriched in an insoluble pool that contained nuclear matrix and chromatin, whereas equal amounts of phosphorylated BAF were found in the soluble cytoplasmic and nucleoplasmic pool [26]. In contrast to the inhibitory action of BAF during vaccinia virus replication, BAF and its associated DNA binding activity are beneficial to the integration activity of the retroviral PIC [8, 10, 11, 34] These findings raise the possibility that a cellular homologue of B1 kinase, VRK, would adversely affect the function of retroviral PICs by phosphorylation and subsequent dissociation of BAF. We report that murine VRK1 and VRK2 do induce the dissociation of BAF from MMLV PICs, resulting in abrogation of intermolecular integration activity in vitro

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