Functional analysis of adenovirus protein IX identifies domains involved in capsid stability, transcriptional activity, and nuclear reorganization

The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively pa rticipate in the stability of the viral icosahedron, acting as a capsid cem ent. We have previously demonstrated that pIX is also a transcriptional act ivator of several viral and cellular TATA-containing promoters, likely cont ributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-termina l domain are crucial for incorporation of the protein into the virion; spec ific residues of the C-terminal leucine repeat are responsible for pIX inte ractions with itself and possibly other proteins, a property that is critic al for pIX transcriptional activity. We also show that pIX takes part in th e virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nucle ar structures which appear as dispersed nuclear globules by immunofluoresce nce staining and as clear amorphous spherical inclusions by electron micros copy, The integrity of the leucine repeat appears to be essential for the f ormation and nuclear retention of these inclusions. Together, our results d emonstrate the multifunctional nature of pIX and provide new insights into Ad biology.