Adenoviruses code for a protease that is essential for infectivity and is activated by a disulfide-linked peptide, derived from the C terminus of the virus structural protein pVI (pVI-CT). The protease was synthesized at relatively high levels late in infection and was detected in both cytoplasmic and nuclear fractions of adenovirus-infected cells. DNA was not found to be a cofactor of the protease, as previously proposed (W.

In adenovirus-infected cells, the virus-encoded preterminal protein and DNA polymerase form a heterodimer that is directly involved in initiation of DNA replication. Monoclonal antibodies were raised against preterminal protein, and epitopes recognized by the antibodies were identified by using synthetic peptides. Partial proteolysis of preterminal protein reveals that it has a tripartite structure, with the three domains being separated by two protease-sensitive areas, located at sites processed by adenovirus protease. These areas of protease sensitivity are probably surface-exposed loops, as they are the sites, along with the C-terminal region of preterminal protein, recognized by the monoclonal antibodies. Preterminal protein is protected from proteolytic cleavage when bound to adenovirus DNA polymerase, suggesting either multiple contact points between the proteins or a DNA polymerase-induced conformational change in preterminal protein. Two of the preterminal protein-specific antibodies induced dissociation of the preterminal protein-adenovirus DNA polymerase heterodimer and inhibited initiation of adenovirus DNA replication in vitro. Antibodies binding close to the primary processing sites of adenovirus protease inhibited DNA binding, consistent with UV cross-linking results which reveal that an N-terminal, protease-resistant domain of preterminal protein contacts DNA. Monoclonal antibodies recognizing epitopes within the C-terminal 60 amino acids of preterminal protein stimulate DNA binding, an effect mediated through a decrease in the dissociation rate constant. These results suggest that preterminal protein contains a large, noncontiguous surface required for interaction with DNA polymerase, an N-terminal DNA binding domain, and a C-terminal regulatory domain. by guest http://jvi.asm.org/ Downloaded fromlittle is known about NFI, Adpol, and the subject of this study, pTP. Previous attempts to identify functionally important domains in pTP have been confined to insertion/deletion mutagenesis (10-12, 27, 33, 34), with the general conclusions from such studies being that regions spanning the protein are required for DNA replication and virus viability. Here we describe the domain structure of pTP determined by a combination of approaches, including antibody accessibility experiments, partial proteolysis, and photochemical cross-linking. This information was used to identify regions of pTP participating in interactions with DNA and Adpol. MATERIALS AND METHODSPurification of pTP, Adpol, DBP, NFI, and NFIII. Adenovirus type 2 (Ad2) pTP was expressed in Spodoptera frugiperda sf9 cells by using recombinant baculoviruses as described previously (41). Cells were resuspended in ice-cold 50 mM HEPES-NaOH (pH 8)-5 mM KCl-0.5 mM MgCl 2 supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 g of pepstatin per ml, 1 g of E-64 per ml, and 1 g of pepstatin per ml), incubated on ice for 10 min, then disrupted by 15 strokes in a Dounce homogenizer using a type B pestle. Nuclei were collected by centri...

Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5 end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.Adenovirus DNA replicates by a protein-priming mechanism in which the virus-encoded preterminal protein (pTP) and adenovirus DNA polymerase (Adpol) form a stable heterodimer and pTP serves as the protein primer (20,35,37,39). DNA replication is initiated by the covalent attachment of dCMP to pTP, following the binding of pTP-Adpol to the core origin of replication (20,35,42). Initiation is enhanced by two cellular factors, nuclear factor I (NFI or CTF1) and nuclear factor III (NFIII or Oct1), and by the virus-encoded, singlestranded DNA binding protein (3,5,6,8,10,12,25,26,31,40). In addition to the incoming pTP, the presence of terminal protein (TP), covalently attached to the 5Ј end of the DNA, stimulates initiation in in vitro DNA replication assays, a phenomenon due in part to an increased affinity of the incoming pTP-Adpol for TP-linked DNA (29,30). Initiation of DNA replication is followed by dissociation of NFI from the complex (9), elongation of the daughter strand by Adpol, and strand displacement. The nature of the molecular rearrangements at the origin of replication preceding elongation and the functional contributions of each of the protein components remain to be established. In particular, questions that elude us are whether pTP and Adpol remain associated throughout elongation, and the nature, if any, of the roles of the pTP moieties attached to the displaced and daughter strands (20,43).

No abstract

SUMMARYAdenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA-complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn 2+ ions for Mg 2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6.4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for a least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription. INTRODUCTIONThe virion of adenovirus consists of an icosahedral protein capsid surrounding a nucleoprotein core, which contains a linear double-stranded DNA genome (35937 bp in adenovirus type 2) with inverted terminal repeats. Attached to each of the 5' termini is a viruscoded 55K terminal protein (TP). This protein is synthesized as an 80K precursor (pTP), which is processed by a virus-coded protease to the mature protein at later stages of infection. Closely associated with the genome are the virus-coded basic proteins VII (of Mr 19K) and V (of Mr 46K) and a minor virus protein IVa z (of Mr 52K) (Russell & Precious, 1982). These so called core proteins are made late in infection and at least one of these (VII) is also synthesized as a precursor protein, which is processed by the virus-coded protease during virus maturation. Cores of virus DNA with covalently attached TP and associated proteins can be readily prepared from purified virus preparations by gentle disruption of virus, using a pulse of heat (56 °C) in the presence of 0.5 ~ sodium deoxycholate . In addition, very mild