| The promoter of the IVa2 gene of human adenovirus type 2 is active only during the late phase of infection and is essential for progression through the viral infectious cycle. This promoter is regulated by an uncharacterized cellular repressor (IVa2-RF) which binds upon infection. Titration of IVa2-RF as the concentration of viral genomes increases upon viral DNA synthesis results in activation of IVa2 transcription and production of the IVa2 protein, which subsequently contributes to stimulation of major-late transcription. Identification of IVa2-RF will allow for the investigation of the normal functions of this protein in the cells and the mechanism by which IVa2 transcription is repressed and activated. Furthermore, knowledge of this repressor's role in cells is an important prerequisite for us to understand why this cellular repressor plays such a significant role in adenoviral transcription.;To establish the molecular identity of IVa2-RF, we have employed multiple biochemical approaches. Using IVa2-RF-specific activity assays, we were able to follow the purification of IVa2-RF and identify IVa2-RF candidate proteins by mass spectroscopy. Multi-step column chromatography, ultra violet cross-linking of IVa2 DNA to a semi-purified protein fraction containing IVa2-RF followed by 2-dimensional gel electrophoresis and a multi-step DNA-affinity scheme have all lead to significant purification of IVa2-RF. Mass spectroscopy has led to the identification of multiple IVa2-RF candidate proteins, many of which were tested for IVa2-RF activity.;As yet, none of the identified IVa2-RF candidate proteins have proven to be a sequence specific repressor of the IVa2 promoter. However two proteins, an RNA-Helicase, Dead Box Healicase I (DDX1) and the uncharacterized human niban-like protein 1/MEG3 were identified through multiple biochemical approaches. Tested separately, DDX1 and human niban-like protein 1/MEG3 did not show any IVa2-RF repressor activity. However, our data indicate that IVa2-RF may be a complex of more than one protein. Further testing of a recombinant human niban-like protein 1/MEG3 alone and together with DDX1 is needed and in progress, but if the human niban-like protein 1/MEG3 alone or together with DDX1 is shown to not be IVa2-RF, then different non-biochemical purification experimental approaches will have to be employed to indentify the elusive IVa2-RF. |
