Analysis of gene expression from the adenovirus type 35 immunoregulatory early region 3

Abstract

Adenovirus type 35 (Ad35), a subgroup B Ad, has a unique association with immunocompromised individuals such as bone marrow transplant recipients and AIDS patients. In order to begin to characterize how the immunoregulatory early region 3 (E3) may influence Ad35 pathogenesis, analysis of Ad35 E3 mRNA and protein expression was undertaken. Also, DNA sequencing of the Ad35 pVIII, E3 and fiber genes was carried out.;Sequence data from the Ad35 E3 demonstrates that the Ad35 E3 has several differences compared with the E3s of the previously studied subgroup C E3s. These differences include the presence of unique open reading frames (ORFs) two of which potentially encode glycoproteins of 20.3 and 20.6 kilodaltons (k).;In order to begin to characterize the unique proteins encoded by the subgroup B E3s, fusion proteins containing the Ad35 E3 20.3k and 20.6k proteins were expressed in bacteria. Polyclonal antibodies were generated against these fusion proteins. The anti-20.6k antiserum was shown to interact with the portion of the fusion protein corresponding to the Ad35 20.6k ORF. Using this antibody, small amounts of a glycoprotein could be specifically immunoprecipitated from {dollar}\sp3{dollar}H-leucine labeled and {dollar}\sp3{dollar}H-mannose labeled, Ad35 infected cells, suggesting that the 20.6k glycoprotein is made during Ad35 infection.;A map of the Ad35 E3 mRNAs was generated. Using RT-PCR, RNAse protection and Northern blotting techniques, six transcripts likely encoding seven proteins were detected. The splice junctions were determined by sequencing of RT-PCR products. Several differences between Ad35 and Ad2 E3 mRNAs were noted, including differences in the relative abundance of mRNAs encoding homologous proteins and differences in the structures of the E3 mRNAs. These differences in mRNA abundance likely reflect differences in how Ad35 regulates E3 pre-mRNA processing compared with the Ad2 E3. These differences may have important consequences in terms of how these viruses are able to evade host immune and cytokine responses.