The mucosotrophic human papillomaviruses (HPVs) are classified as high-risk (HR) or low-risk (LR) genotypes based on their neoplastic properties. We have demonstrated previously that the E7 protein destabilizes p130, a pRb-related pocket protein, thereby promoting S-phase reentry in postmitotic, differentiated keratinocytes of squamous epithelia, and that HR HPV E7 does so more efficiently than LR HPV E7. The E7 proteins of LR HPV-11 and -6b uniquely possess lysine residues following a casein kinase II phosphorylation motif which is critical for the biological function of E7. We now show that mutations of these lysine residues elevated the efficiency of S-phase reentry, independent of their charge. An 11E7 K39,42R mutation moderately increased the association with and the destabilization of p130. Unexpectedly, polyubiquitination on these lysine residues did not attenuate E7 activity, as their mutation caused elevated proteasomal degradation and decreased protein stability. In this regard, the biologically more potent HR HPV E7 proteins were also less stable than the LR HPV E7 proteins. We infer that these lysine residues impede functional protein-protein interactions. A G22D mutation of 11E7 at the pocket protein binding motif possessed augmented efficiency in promoting S-phase reentry and strongly enhanced association with p130 and pRb. The combined effects of these two classes of 11E7 mutations exhibited an efficiency of S-phase reentry comparable to that of HR HPV E7. Thus, these nonconserved residues are primarily responsible for the differential abilities of LR and HR HPV E7 proteins to promote unscheduled DNA replication in organotypic raft cultures. © 2011, American Society for Microbiology.