Adenovirus encodes for the pVII protein, which interacts and modulates virus DNA structure in the infected cells. The pVII protein is synthesized as the precursor protein and undergoes proteolytic processing by viral proteinase Avp, leading to release of a propeptide sequence and accumulation of the mature VII protein. Here we elucidate the molecular functions of the propeptide sequence present in the precursor pVII protein. The results show that the propeptide is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. Our data further indicate that the propeptide sequence and the lysine residues K26 and K27 regulate the precursor pVII protein stability in a co-dependent manner. We also provide evidence that the Cullin-3 E3 ubiquitin ligase complex alters the precursor pVII protein stability by association with the propeptide sequence. In addition, we show that inactivation of the Cullin-3 protein activity reduces adenovirus E1A gene expression during early phase of virus infection. Collectively, our results indicate a novel function of the adenovirus propeptide sequence and involvement of Cullin-3 in adenovirus gene expression.