Search

It is being assumed that human papillomavirus (HPV) utilizes three different modes of replication during its life cycle: initial amplification during the establishment phase, genome maintenance, and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model.

Supplementary Methods PDF file - 98K, Supplementary materials and methods

Supplementary Table S2 PDF file - 90K, Ingenuity transcription factor analysis of CIP2A depleted cells

Supplementary Figure 5 PDF file - 82K, Prognostic value of CIP2A in advanced human breast cancer patient material and characterization of vinorelbine effects

Supplementary Figure 4 PDF file - 269K, Characterization of hypomorphic CIP2A HOZ mice

Supplementary Figure 3 PDF file - 114K, RB expression is not needed for CIP2A-depletion induced senescence

Supplementary Figure 2 PDF file - 96K, Transcriptional regulation and functional role for CIP2A in response to p53 reactivation

HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes.

Even though replication and transcription of human papillomavirus type 16 (HPV16) has been intensively studied, little is known about immediate early events of the viral life cycle due to the lack of an efficient infection model allowing genetic dissection of viral factors. We employed the recently developed infection model (Bienkowska-Haba et al. PLOS Pathogen 2018) to investigate genome amplification and transcription immediately after infectious delivery of viral genome to nuclei of primary keratinocytes. Using EdU pulse labeling and highly sensitive fluorescence in situ hybridization, we observed that the HPV16 genome is replicated and amplified in an E1 and E2 dependent manner. Knockout of E1 resulted in failure of the viral genome to replicate and amplify. In contrast, knockout of the E8^E2 repressor led to increased viral genome copy number confirming previous reports. Genome copy control by E8^E2 was confirmed for differentiation-induced genome amplification. Lack of functional E1 had no effect on transcription from the early promoter, suggesting that viral genome replication is not required for p97 promoter activity. However, infection with an HPV16 mutant virus defective for E2 transcriptional function revealed a requirement of E2 for efficient transcription from the early promoter. In absence of the E8^E2 protein, early transcript levels are unaltered and even decreased when normalized to genome copy number. Surprisingly, lack of functional E8^E2 repressor did not affect E8^E2 transcript levels when normalized to genome copy number. These data suggest that the main function of E8^E2 in the viral life cycle is to control genome copy number.IMPORTANCEIt is being assumed that HPV utilizes three different modes of replication during its lifecycle: initial amplification during the establishment phase, genome maintenance and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model. Using our recently established infection model (Bienkowska-Haba et al. PLOS Pathogen 2018), we demonstrate herein that viral genome is indeed amplified in an E1 and E2 dependent manner. Furthermore, we find that the main function of the viral repressor E8^E2 is to control viral genome copy number. We did not find evidence that it regulates its own promoter in a negative feedback loop. Our data also suggest that the E2 transactivator function is required for stimulation of early promoter activity, which has been debated in the literature. Overall, this report confirms the usefulness of the infection model for studying early events of the HPV lifecycle using mutational approaches.

The current model for human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. HPV genome copy number remains constant during the maintenance stage. Differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. Nuclear viral genome copy number is reset to pre-S phase levels during mitosis. The majority of viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genome gradually decreases during cell cycle progression. Loss of cytosolic genome is blocked in presence of NH4Cl or other drugs interfering with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genome was not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genome as well. We conclude that HPV maintains viral genome copy number by balancing viral genome amplification during S phase with loss of viral genome that is lost to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes resets genome copy number in each cell cycle.IMPORTANCEHPV genome maintenance is currently being thought to be achieved by regulating expression and activity of viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy number by competing with E1 and E2 for binding to the viral origin of replication and by recruitment of repressor complexes. Herein, we demonstrate that HPV viral genome is amplified in each S phase. Nuclear genome copy number is reset during mitosis by a failure of the majority of genomes to tether to mitotic chromosomes. Rather, they accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is quickly degraded in G1 in a lysosome dependent manner contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contributes to viral genome maintenance.

Beginning in 2018, a new papaya disease occurred in Ibadan, Oyo State, Nigeria. Affected papaya trees exhibited symptoms including excessive shoot proliferation at the top of the crown, leaf mosaic and crinkling, and dieback. The disease was designated as Nigerian papaya bunchy top (NGPBT) disease. Since excessive shoot proliferation was the most characteristic symptom of the disease and it was indicative of phytoplasma infection, a polymerase chain reaction (PCR)-based phytoplasma molecular diagnostic method was employed in this study. Phytoplasmas were detected in diseased papaya samples by semi-nested PCR using phytoplasma 16 S rRNA gene-specific primers. Subsequent determination and analysis of the amplified 16 S rRNA gene sequences and phylogenetic analysis revealed that NGPBT phytoplasmas were most closely related to ‘Candidatus Phytoplasma convolvuli’ in the Stolbur (16SrXII) group. Virtual restriction fragment length polymorphism (RFLP) analysis based on the 16 S rRNA gene F2nR2 fragment showed that NGPBT strains represented a novel subgroup in the Stolbur phytoplasma group, designated as 16SrXII-O. This is the first report of a phytoplasma disease in papaya in Nigeria. Identification of the NGPBT strains not only broadened the genetic diversity of the Stolbur phytoplasma group but also signaled an expansion of the phytoplasma group's geographic distribution. Notably, two previously reported phytoplasmas associated with papaya diseases in Australia and Taiwan also belong to the 16SrXII group and exhibit symptoms similar to NGPBT disease. It would be interesting to learn how these three diseases relate to each other. Further in-depth study on weed reservoirs and insect vectors of NGPBT phytoplasmas will contribute to a better understanding of pathosystem and epidemiology of the NGPBT disease.