Phosphorylation of E2 Serine Residue 402 Is Required for the Transcription and Replication of the HPV5 Genome.

Cutaneous human papillomavirus type 5 (HPV5) belongs to the supposedly oncogenic ß-HPVs associated with specific types of skin and oral cavity cancers. Three viral proteins, namely, helicase E1 and transcription factors E2 and E8^E2, are master regulators of the viral life cycle. HPV5 E2 is a transcriptional activator that also participates in the E1-dependent replication and nuclear retention of the viral genome, whereas E8^E2 counterbalances the activity of E2 and inhibits HPV transcription and replication. In the present study, we demonstrate that the HPV5 E2 protein is extensively phosphorylated by cellular protein kinases, and serine residue 402 (S402) is the highest scoring phosphoacceptor site. This residue is located within a motif conserved among many ß-HPVs and in the oncogenic HPV31 α-type. Using the nonphosphorylatable and phosphomimetic mutants, we demonstrate that phosphorylation of the E2 S402 residue is required for the transcription and replication of the HPV5 genome in U2OS cells and human primary keratinocytes. Mechanistically, the E2-S402-phopshodeficient protein is unable to trigger viral gene transcription and has an impaired ability to support E1-dependent replication, but the respective E8^E2-S213 mutant displays no phenotype. However, phosphorylation of the E2 S402 residue has no impact on the E2 stability, subcellular localization, self-assembly, DNA-binding capacity, and affinity to the E1 and BRD4 proteins. Further studies are needed to identify the protein kinase(s) responsible for this phosphorylation. IMPORTANCE Human papillomavirus type 5 (HPV5) may play a role in the development of specific types of cutaneous and head and neck cancers. The persistence of the HPV genome in host cells depends on the activity of its proteins, namely, a helicase E1 and transcription/replication factor E2. The latter also facilitates the attachment of episomal viral genomes to host cell chromosomes. In the present study, we show that the HPV5 E2 protein is extensively phosphorylated by host cell protein kinases, and we identify serine residue 402 as the highest scoring phosphoacceptor site of E2. We demonstrate that the replication of the HPV5 genome may be blocked by a single point mutation that prevents phosphorylation of this serine residue and switches off the transcriptional activity of the E2 protein. The present study contributes to a better understanding of ß-HPV5 replication and its regulation by host cell protein kinases.

Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes.

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

E2 protein is the major determinant of specificity at the human papillomavirus origin of replication.

The replication of human papillomavirus (HPV) genomes requires E1 and E2 proteins as the viral trans-factors and the replication origin, located in the URR, as a cis-element. The minimal requirements for an HPV replication origin vary among different virus types but always include one or more binding sites for the E2 protein. The requirements for an E1 binding site seem to vary among different HPV genera, with alpha-HPV11 and -18 minimal origins able to replicate without E1 binding site in contrast to beta-HPV8. In the present article, we analysed the sequence requirements for the beta-HPV5 minimal origin of replication. We show that the HPV5 URR is able to replicate in U2OS cells without the sequence proposed as an E1 binding site, albeit at lower levels than wt URR, given that three E2 binding sites are intact and both viral replication proteins are present. The lack of an absolute requirement of the E1 binding site for the origin of replication of HPV5 led us to analyse whether the viral E1 and E2 proteins from other HPV types are competent to support replication from this origin. Surprisingly, the E1 and E2 proteins from beta-HPV types support replication from the origin in contrast to proteins from alpha-HPV types 11, -16, or -18. Furthermore, the replication proteins E1 and E2 of these alpha-HPV types are unable to support the replication of HPV5 URR, even if the E1 binding site is intact. In light of these results, we performed a detailed analysis of the ability of different combinations of E1 and E2 proteins from various alpha- and beta-HPV types to support the replication of URR sequences from the respective HPV types in the U2OS cell line.