Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus.

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5'end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP-host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp ß, and a complex of Imp α/ß but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp ß or Imp α/ß. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.

A Phase 1 Two-Arm, Randomized, Double-Blind, Active-Controlled Study of Live, Oral Plasmid-Derived Adenovirus Type 4 and Type 7 Vaccines in Seronegative Adults.

PXVX0047 is an investigational vaccine developed for active immunization to prevent febrile acute respiratory disease (ARD) caused by adenovirus serotypes 4 (Ad4) and 7 (Ad7). PXVX0047 consists of a modernized, plasmid-derived vaccine that was generated using a virus isolated from Wyeth Ad4 and Ad7 vaccine tablets. A phase 1 two-arm, randomized, double-blind, active-controlled study was conducted to evaluate the safety profile and immunogenicity of the investigational adenovirus vaccines. The two components of PXVX0047 were administered orally together in a single dose to 11 subjects. For comparison, three additional subjects received the Ad4/Ad7 vaccine that is currently in use by the US military. The results of this study show that the tolerability and immunogenicity of the PXVX0047 Ad7 component are comparable with that of the control Ad4/Ad7 vaccine; however, the immunogenicity of the PXVX0047 Ad4 component was lower than expected. Clinical trial number NCT03160339.

Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein.

Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.