Tyrosine kinase-dependent release of an adenovirus preterminal protein complex from the nuclear matrix

Abstract

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) through the interaction of Ad preterminal protein (pTP). The NM is a highly salt-resistant fibrillar network which is known to anchor transcription, mRNA splicing, and DNA replication complexes. Incubation of rATP with NM to which pTP was bound caused the release of pTP as a pTP-NM complex with a size of 220 to 230 kDa; incubation with 5 adenylylimidodiphosphate (rAMP-PNP) showed no significant release, indicating that rATP hydrolysis was required. With NM extracts, it was shown that a pTP-NM complex which was capable of binding Ad origin DNA could be reconstituted in vitro. A number of high-molecular-weight NM proteins ranging in size from 120 to 200 kDa were identified on Far Western blots for their ability to bind pTP. rATP-dependent release of pTP from the NM was inhibited in a dose-dependent fashion by the addition of tyrosine kinase inhibitors, such as quercetin, methyl-2,5-dihydroxycinnamate, or genistein. NM-mediated phosphorylation of a poly(Glu, Tyr) substrate was also significantly abrogated by the addition of these compounds. rATP-dependent release of Ad DNA termini bound to the NM via pTP was also blocked by the addition of these inhibitors. These results indicate that a tyrosine kinase mechanism controls the release of pTP from its binding sites on the NM. These data support the concept that phosphorylation may play a key role in the modulation of pTP binding sites on the NM.

Role of preterminal protein processing in adenovirus replication

“…No difference in efficiency of pTP-DNA complex was observed in vitro, but this does not rule out that TP-DNA is required in vivo for the first round of DNA replication, where there is a low abundance of template. Perhaps the most attractive explanation for processing of pTP relates to the matrix association properties of TP-and pTP-DNA (2,15,36). Both TP-DNA and pTP have been shown to bind tightly to the nuclear matrix, but the identity of their respective sites of attachment is unknown, meaning that a combination of proteolysis and phosphorylation could serve to guide the viral DNA though the nucleus to appropriate sites for early transcription, DNA replication, late transcription, and packaging into the virus (2,15,28,36).…”

“…Perhaps the most attractive explanation for processing of pTP relates to the matrix association properties of TP-and pTP-DNA (2,15,36). Both TP-DNA and pTP have been shown to bind tightly to the nuclear matrix, but the identity of their respective sites of attachment is unknown, meaning that a combination of proteolysis and phosphorylation could serve to guide the viral DNA though the nucleus to appropriate sites for early transcription, DNA replication, late transcription, and packaging into the virus (2,15,28,36). Supporting evidence for this hypothesis comes from results of immunofluorescence experiments, which show that monoclonal antibodies recognizing epitopes spanning the TP site bind to pTP only in the foci of viral DNA replication (Fig.…”

“…The significance of the two templates in terms of defining their roles in virus infection remains to be established. In particular, it has been shown that both pTP and TP bind strongly to the nuclear matrix, in a manner assumed analogous to that of matrix attachment regions of eukaryotic chromosomes (2,15,36). The identities of the proteins in the nuclear matrix to which pTP and TP bind, and indeed whether they bind to the same sites, are not known (2).…”

“…In particular, it has been shown that both pTP and TP bind strongly to the nuclear matrix, in a manner assumed analogous to that of matrix attachment regions of eukaryotic chromosomes (2,15,36). The identities of the proteins in the nuclear matrix to which pTP and TP bind, and indeed whether they bind to the same sites, are not known (2). Immunofluorescence studies reveal that although sites of viral DNA replication and transcription are proximal in infected nuclei, they are distinct (4,14,28).…”

“…Immunofluorescence studies reveal that although sites of viral DNA replication and transcription are proximal in infected nuclei, they are distinct (4,14,28). Consensus appears to be that the terminal protein moiety, be it TP or pTP, is at least in part responsible for directing adenovirus DNA to appropriate sites in the nucleus during the course of virus infection (2,15,36). Extensive mutagenesis of pTP has been carried out and the conclusions from such studies are that regions spanning the molecule are required for matrix attachment, DNA replication, and virus viability (15-17, 27, 33, 34).…”