Progeny Release of Species B Human Adenoviruses Is Not Mediated By Early Region 3 Proteins 20.1K, 20.5k, and 10.9k

The early region 3 (E3) of the human adenovirus (HAdV) genome encodes proteins that regulate the host immune response to viral infection. The E3 region also exhibits the highest level of genetic diversity among the genomes of the six (A-F) species of HAdV. This diversity in genetic content is found primarily between the highly conserved E3-gp19K and RIDα open reading frames (ORFs). It has been previously shown that HAdV-C encodes the adenovirus death protein (ADP) in this location. ADP is an 11.6kDa, transmembrane protein that localizes to the nuclear membrane, and facilitates the efficient release of progeny virions. HAdV-B1 encodes three ORFs, E3-20.1K, E3-20.5K and E3-10.9K, in the analogous region of the viral genome. Since ADP and the three novel HAdV-B1 proteins share several structural characteristics and location in the E3 region, we hypothesized that one or more of these unique ORFs play a similar role to ADP in facilitating viral progeny egress. Reverse transcriptase PCR showed that transcripts of the novel ORFs are expressed at both early and late time points post infection. Through the examination of ectopically expressed EGFP fusion proteins, we demonstrated that E3-20.1K, E3-20.5K and E3-10.9K localize to the plasma membrane and intracellular vesicle-like structures. Localization to intracellular vesicle-like structures was also observed when the novel HAdV-B1 E3 proteins fused with short, C-terminal, epitope tags were expressed from the viral genome. We generated a HAdV-3 knock out mutant virus which lacks the ability to express the E3-20.1K, E3-20.5K, and E3-10.9K proteins to examine whether the products of these ORFs are required for viral replication or play a role in facilitating the release of viral progeny from infected polarized or non-polarized lung epithelial cells. The knock out virus did not show any impaired growth in non-polarized A549 cells or polarized Calu3 cells when compared to wild type virus. The knockout HAdV-B1 mutant also did not show any significant changes in the development of plaques, size of plaques, or dissemination of the virus on cell monolayers. Even though the novel E3-20.1K, E3-20.5K, and 10.9K proteins of HAdV-B1 are encoded in the analogous region of E3 and they share several structural characteristics with ADP, our experimental data show that they do not play a role in the release of progeny virions from infected cells.Biomedical SciencesMastersUniversity of New Mexico. Biomedical Sciences Graduate ProgramKajon, AdrianaOzbun, MichellePeabody, DavidKajon, Adrian