Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions.

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.

Adenovirus ADP protein (E3-11.6K), which is required for efficient cell lysis and virus release, interacts with human MAD2B.

The human subgroup C adenovirus (Ad) protein named adenovirus death protein (ADP) (previously named E3-11.6K) is synthesized at very late stages of infection when it mediates efficient lysis of cells and release of adenovirus to infect other cells. ADP is an integral membrane N-linked, O-linked palmitoylated glycoprotein of 101 amino acids (aa) that localizes to the nuclear membrane, endoplasmic reticulum (ER), and Golgi. It has a single membrane spanning region (roughly aa 40-60) and is oriented with aa 1-40 in the lumen and aa 61-101 in the nucleoplasm and cytoplasm. Using aa 61-101 of Ad2 ADP as bait in a yeast two-hybrid screen, we isolated a cDNA for a 211-aa protein that initially was not in the database but has now been published by others with the names human MAD2B, MAD2L2, and REV7. ADP binds strongly to human MAD2B not only in yeast but also in GST pull-down experiments and in coimmunoprecipitations of ADP and MAD2B synthesized in vitro or in vivo. ADP mutants with deletions throughout the bait region do not interact with human MAD2B, whereas a Pro69Pro70 to Ala69Ala70 mutant in the "basic-proline" domain of ADP does interact. Northern blot analyses indicate that human MAD2B is expressed ubiquitously. Human MAD2B is about 25% identical to human MAD2, a spindle assembly checkpoint protein. Two human A549 cell lines were made that constitutively overexpress MAD2B. Wild-type adenovirus lyses these cells significantly more slowly than it lyses parental A549 cells, raising the possibility that ADP and MAD2B act in opposition and suggesting that the ADP-MAD2B interaction is biologically relevant.

The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model.

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.