Establishment Of A Saccharomyces Cerevisiae System For Human Papillomavirus Type 58 Research And Studies On The Functions Of HPV58 E2 Gene In The Yeast System

Human Papillomaviruses(HPVs) are DNA viruses that infect keratinocytes in differentiating epithelia and induce a series of benign and malignant hyperproliferative lesions.The viruses are classified as either high risk or low risk viruses.Low-risk HPV genotypes,such as HPV1, 2,6,11,induce benign warts such as common warts and condyloma acuminata.In contrast, infections by high-risk HPV types including HPV 16,18,31,58 can lead to the development of cervical cancer.HPVs are a family of approximately 8kb,non-enveloped and double-stranded DNA viruses.The genome of HPV can be divided into three regions:the long control region,the early and late regions.The productive life cycle of HPVs is intimately linked to epithelial growth and cell differentiation.The link has posed a substantial barrier to the study of HPV in the laboratory because HPVs cannot be propagated in conventional cell lines.HPV 58 is an important HPV type tightly associated with cervical cancer.Several studies have suggested that HPV58 plays a prominent role in cervical cancer in Asia,and it is also the second most prevalent HPV type found in cervical cancer patients in China except HPV16.So far,cell lines containing the DNA of high-risk HPVs such asHPV16,18 and 31 in an episomal state are available for biological studies,neither cell lines nor model system containing HPV58 have been established for the study of HPV58.S.cerevisiae is a single-cell eukaryotic organism.The cellular mechanism required for DNA replication in human cells is sufficiently similar to those in yeast.In the present study,we inserted a yeast selectable marker Ura into the HPV58 genome to generate a recombinant HPV58-Ura construct and transformed the circular HPV58-Ura construct into yeast.We discovered that HPV58 genome can replicate stably as an episome and transcribe its early and late open-reading frames(ORFs) in transformed yeast cells. However,when the E2 gene of HPV58-ura was interrupted by stop codons in the 5' of the ORF to construct the recombinant HPV58-Ura-E2mt,the HPV 58 genome can not replicate stably in the yeast system and the transcription pattern of early genes(E6 and E7) and late genes(L1 and L2) was also changed,which means that E2 protein can not only facilitate the replication and stability of HPV DNA but also regulate the gene transcription level.The HPV58/Yeast system described here provides us with a powerful model to further study HPV genome replication and viral gene transcription in future.Furthermore,we have developed a simple and efficient method for the preparation of total RNA from Saccharomyces cerevisiae.Yeast cells were incubated at 65℃for 5 min in yeast RNA isolation buffer(10 mM EDTA,50 mM Tris-HCl,5%SDS,pH 6.0),and the RNA was isolated and purified.The yield and quality of the isolated RNA was consistently high,and the isolated RNA was suitable for downstream applications,such as Northern blot hybridization and reverse transcription PCR(RT-PCR).1.Establishment of HPV58/Yeast systemA 1.1kb yeast selective marker Ura3 was subcloned into the L2 ORF of HPV58 genome to construct the recombinant HPV58-Ura.The HPV58-Ura was recircularized and transformed into yeast.The HPV58-Ura transformed yeast was cultured in selective medium for 5 passages,then yeast DNA and total RNA was isolated.The result of real time quantitative PCR indicated that HPV58 can replicate its genome in yeast cells,and Southern blot analysis revealed that viral genome can replicate episomally in yeast cells without integration or recombinantion.The DNA stability assay indicated that HPV58-Ura was very stable in the yeast cells with a loss rate of 1.9±0.31%per cell generation.RT-PCR analysis reveals that HPV58-ura can transcribe its early(E1,E2,E6,E7) and late genes(L1 and L2) in yeast cells. HPV58/yeast system described here was able to direct the stable replication of HPV58 genome and induce the transcriptions of both early late genes.Therefore,the established system provides us with the opportunity to study the mechanisms that HPVs can complete their multiplication life cycle in such a simple eukaryote.2.Studies of HPV58 E2 gene functions in yeast systemA recombinant HPV58-Ura-E2mt was constructed by introducing three stop codons in the 5' of E2 ORF through a PCR based cassette mutagenesis.The HPV58 E2 expression plasmid pDBLeu-E2 was constructed at the same time.HPV58-Ura-E2mt transformed yeast cells grow poorly in the selective plates.When the pDBLeu-E2 plasmid was transformed into yeast cells harboring HPV58-Ura-E2mt.Then the HPV58-Ura-E2mt/ pDBLeu-E2 transformed yeast cells can grow stably in selective medium.E2 protein is a auxiliary replication protein and mitotic stability factor.HPV58 E2 protein can not only facilitate the replication of HPV58 genome but also help to distribute the viral genome to daughter cells' nucleuses during cell division.We also found the different mechisms of viral genome maintenance between HPV58 and HPV16 in the yeast system.In order to explore the regulation function of E2 on viral early promoter which promote the E6 and E7 gene transcription and late promoter which promote the L1 and L2 gene transcription.The E6,E7,L1 and L2 gene transcription level was compared through qRT-PCR.The results of qRT-PCR indicated E2 protein can repress the early gene transcription of episomal HPV58 genome in yeast.However,we found that E2 protein can stimulate the late gene transcription of episomal HPV58 genome in S.cerevisiae,the detailed mechanism needs further research..3.A waterbath method for preparation of RNA from Saccharomyces cerevisiaeYeast RNA has traditionally been extracted by tedious vortex mixing with glass beads or with hot acid/phenol.Although the glass beads method results in efficient breakage of the cells,the procedure itself is complicated,especially for multiple samples.The hot acid/phenol method is simpler than the glass beads method,but it requires the equilibration of phenol with AE buffer(50 mM sodium acetate,pH 5.3,10 mM EDTA).Here,we describe a novel,simple and reliable method for preparation of total RNA from baker's yeast.The yeast cells were incubated at 65℃for 5 min in yeast RNA isolation buffer(10 mM EDTA,50 mM Tris-HCl, 5%SDS,pH 6.0),and then the SDS and cell debris were precipitated by the addition of KCl. The RNA was isolated by extraction with phenol/chloroform and precipitation in ethanol.The RNA waterbath extraction method gave good yields of high-quality RNA from S.cerevisiae within 1 h without any vortex mixing or expensive Trizol reagent.This method is very suitable for dealing with large quantities of yeast cultures.The RNA is suitable for downstream applications,such as RT-PCR and Northem blot hybridization.To conclude,we are the first to establish a HPV58/yeast system,which is potential for the studies on the HPV58 life cycle;secondly,we studied the HPV58 E2 gene function in the yeast system and found its important roles in DNA replication,maintenance and transcription regulation;thirdly,we have developed a novel method for preparation of intact yeast RNA.