Generation of the HPV18 marker genomes expressing fluorescent proteins

Abstract

Nearly 4.5% of cancers worldwide are caused by human papillomaviruses (HPVs). The oncogenic HPVs are attributed to anogenital, head and neck, and cervical cancers, with HPV16 and HPV18 being responsible for 73% of these cancers. Among the challenges in studying HPV is the rapid and efficient quantification of HPV genome copy numbers in the host cells. An ideal system would eliminate the need for cellular lysis, DNA extraction, and other downstream processing steps needed in the currently used methods, allowing researchers to measure viral copy numbers in living cells. In the current study, I generated the HPV18 marker genomes bearing different fluorescent genes in the early region that is known to be transcriptionally active during the initial stages of the viral genome replication in keratinocytes and in the U2OS cells used in the this study as an HPV model host cells. The early region encodes two proteins required for viral genome replication, the helicase E1 and transcription factor E2, as well as oncoproteins E6 and E7. Also, two early proteins, E1^E4, and E8^E2, are translated from alternatively spliced mRNAs, with the latter being a potent repressor of viral transcription and replication. The generated marker genomes were: (1) the HPV18 genome with the sequences encoding for the enhanced green fluorescent protein (EGFP) and foot-and-mouth disease virus 2A self-processing peptide (2AP) inserted immediately after the E1 stop codon; (2) the HPV18-E1-RFP genome with the red fluorescent protein (RFP) encoding sequence inserted after the first 15 nucleotides of the E1 open reading frame (ORF), in the E1^E4 splice site which theoretically yielded the wild-type (WT) E1^E4 and E1-RFP fusion protein; (3) the HPV-E7-EGFP-2AP genome with EGFP with 2AP encoding sequences inserted after E7 ORF. The marker genomes were transfected into U2OS cells, and their replication efficiency was detected. Effectively replicated HPV18-E1-RFP and HPV-E7-EGFP-2AP genomes were further processed to knock out the E8^E2 repressor protein. The E8^E2-deficient marker genomes exhibited even better replication efficiency than their WT counterparts. However, it was not possible to quantify their replication using fluorescence flow cytometry or fluorescence microscopy. Therefore, the marker genomes deficient in E8^E2 expression are candidates for further optimization required for successful quantification of the viral genome copy numbers in the transfected or infected cells using fluorescence-based techniques. To increase the amount of the fluorescence in the cells, the following options may be applied: to introduce stability motifs to increase the stability of mRNA, to modify the FPs to enhance their brightness, to use the brighter FPs, or to use several FPs in tandem.