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The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome.

Isok-Paas H, Männik A, Ustav E, Ustav M - Virol. J. (2015)

Bottom Line: However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains.Mutating the E8 ORF ATG start codon to ACG eliminated the translation of fusion proteins from the E8 ORF coupled to E1 and E2 proteins C-terminal sequences, leading to the de-repression of gene expression (particularly from the P1092 promoter) and to the activation of genome replication.The analysis of HPV11 E1 expression plasmids showed that the E6/E7 region, together with the E1 coding region, is crucial for the production of functionally active E1 protein.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technology, University of Tartu, Tartu, Estonia. helen.isok-paas@ut.ee.

ABSTRACT

Background: Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is the clear unmet medical need in order to justify the development of drugs targeting human papillomavirus replication. The native host cells of HPVs are human primary keratinocytes which can be cultivated in raft cultures. However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains.

Methods: U2OS cells were transfected with HPV11 wt or E8- minicircle genomes and their gene expression was studied via 3' RACE, 5' RACE or via real time PCR methods. The DNA replication of these genomes was detected by Southern blot methods.

Results: The analysis of HPV11 transcripts in U2OS cells showed that the patterns of promoter use, splice sites and polyadenylation cleavage sites are identical to those previously characterized in human HPV-related lesions, human squamous carcinoma cell lines (e.g., SSC-4) and laryngeal papillomas. Transcriptional initiation from the three previously described HPV11 promoters in the E6 and E7 ORFs (P90, P264, and P674-714) were functional, and these promoters were used together with two promoter regions in the E1 ORF (P1092 and P1372). Mutating the E8 ORF ATG start codon to ACG eliminated the translation of fusion proteins from the E8 ORF coupled to E1 and E2 proteins C-terminal sequences, leading to the de-repression of gene expression (particularly from the P1092 promoter) and to the activation of genome replication. These data suggested that the expression of the functional E8^E2 protein is used to control viral gene expression and copy number of the HPV11 genome. The analysis of HPV11 E1 expression plasmids showed that the E6/E7 region, together with the E1 coding region, is crucial for the production of functionally active E1 protein.

Conclusions: The data presented in this paper suggest that in human osteosarcoma cell line U2OS the gene expression pattern of the HPV11 truly reflect the expression profile of the replicating HPV genome and therefore this cellular system is suitable for drug development program targeting HPV replication.

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Mapping of polyadenylation CSs in the HPV11 genome in U2OS cells via 3´ RACE. U2OS cells were transfected with the HPV11wt genome (A and B, lane 1) or together with the linearized pBabe-Neo construct (A and B, lanes 2 and 3). Mock transfection was used as a negative control (lane 4). PolyA+ mRNA was extracted at 4 or 10 days post-transfection. All distinct products were purified, cloned, sequenced and analyzed. (A) 3´ RACE analysis of the HPV11 early region CSs using the HPV11-specific primer pr3392. The indicated 3´ RACE products represent the potential CSs at nt 4384 and nt ~3248. (B) 3´ RACE analysis of the HPV11 late region CSs using the HPV11-specific primer pr7188. The indicated 3´ RACE products represent the potential late CSs at nt 7485, at nt ~7770 and at nt ~7587.
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Fig3: Mapping of polyadenylation CSs in the HPV11 genome in U2OS cells via 3´ RACE. U2OS cells were transfected with the HPV11wt genome (A and B, lane 1) or together with the linearized pBabe-Neo construct (A and B, lanes 2 and 3). Mock transfection was used as a negative control (lane 4). PolyA+ mRNA was extracted at 4 or 10 days post-transfection. All distinct products were purified, cloned, sequenced and analyzed. (A) 3´ RACE analysis of the HPV11 early region CSs using the HPV11-specific primer pr3392. The indicated 3´ RACE products represent the potential CSs at nt 4384 and nt ~3248. (B) 3´ RACE analysis of the HPV11 late region CSs using the HPV11-specific primer pr7188. The indicated 3´ RACE products represent the potential late CSs at nt 7485, at nt ~7770 and at nt ~7587.

Mentions: The putative HPV11 early polyadenylation CS has been mapped to the downstream region of the viral E5B ORF at nt 4384, and the late CS has been mapped to the downstream region of the L1 ORF at nt 7458 in the HPV11 genome [20,27]. The HPV11 polyadenylation CSs used in HPV11-transfected U2OS cells were mapped via 3´ RACE analysis of polyA+ mRNA samples extracted at 4 or 10 days (subconfluent and confluent samples) post-transfection. The HPV11-specific primers pr3392 and pr7188 were used, and the results are shown in Figure 3A and B, respectively. PolyA+ mRNA from mock-transfected cells was used as a negative control (Figure 3, lane 4). The sequencing of the clones revealed that the polyadenylation CSs previously described for HPV11 genome transcripts are also used in U2OS cells. We did not observe any major changes when different phases of replication were compared; however, the late polyadenylation CS was used more frequently (Figure 3B).Figure 3


The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome.

Isok-Paas H, Männik A, Ustav E, Ustav M - Virol. J. (2015)

Mapping of polyadenylation CSs in the HPV11 genome in U2OS cells via 3´ RACE. U2OS cells were transfected with the HPV11wt genome (A and B, lane 1) or together with the linearized pBabe-Neo construct (A and B, lanes 2 and 3). Mock transfection was used as a negative control (lane 4). PolyA+ mRNA was extracted at 4 or 10 days post-transfection. All distinct products were purified, cloned, sequenced and analyzed. (A) 3´ RACE analysis of the HPV11 early region CSs using the HPV11-specific primer pr3392. The indicated 3´ RACE products represent the potential CSs at nt 4384 and nt ~3248. (B) 3´ RACE analysis of the HPV11 late region CSs using the HPV11-specific primer pr7188. The indicated 3´ RACE products represent the potential late CSs at nt 7485, at nt ~7770 and at nt ~7587.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4414447&req=5

Fig3: Mapping of polyadenylation CSs in the HPV11 genome in U2OS cells via 3´ RACE. U2OS cells were transfected with the HPV11wt genome (A and B, lane 1) or together with the linearized pBabe-Neo construct (A and B, lanes 2 and 3). Mock transfection was used as a negative control (lane 4). PolyA+ mRNA was extracted at 4 or 10 days post-transfection. All distinct products were purified, cloned, sequenced and analyzed. (A) 3´ RACE analysis of the HPV11 early region CSs using the HPV11-specific primer pr3392. The indicated 3´ RACE products represent the potential CSs at nt 4384 and nt ~3248. (B) 3´ RACE analysis of the HPV11 late region CSs using the HPV11-specific primer pr7188. The indicated 3´ RACE products represent the potential late CSs at nt 7485, at nt ~7770 and at nt ~7587.
Mentions: The putative HPV11 early polyadenylation CS has been mapped to the downstream region of the viral E5B ORF at nt 4384, and the late CS has been mapped to the downstream region of the L1 ORF at nt 7458 in the HPV11 genome [20,27]. The HPV11 polyadenylation CSs used in HPV11-transfected U2OS cells were mapped via 3´ RACE analysis of polyA+ mRNA samples extracted at 4 or 10 days (subconfluent and confluent samples) post-transfection. The HPV11-specific primers pr3392 and pr7188 were used, and the results are shown in Figure 3A and B, respectively. PolyA+ mRNA from mock-transfected cells was used as a negative control (Figure 3, lane 4). The sequencing of the clones revealed that the polyadenylation CSs previously described for HPV11 genome transcripts are also used in U2OS cells. We did not observe any major changes when different phases of replication were compared; however, the late polyadenylation CS was used more frequently (Figure 3B).Figure 3

Bottom Line: However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains.Mutating the E8 ORF ATG start codon to ACG eliminated the translation of fusion proteins from the E8 ORF coupled to E1 and E2 proteins C-terminal sequences, leading to the de-repression of gene expression (particularly from the P1092 promoter) and to the activation of genome replication.The analysis of HPV11 E1 expression plasmids showed that the E6/E7 region, together with the E1 coding region, is crucial for the production of functionally active E1 protein.

View Article: PubMed Central - PubMed

Affiliation: Institute of Technology, University of Tartu, Tartu, Estonia. helen.isok-paas@ut.ee.

ABSTRACT

Background: Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is the clear unmet medical need in order to justify the development of drugs targeting human papillomavirus replication. The native host cells of HPVs are human primary keratinocytes which can be cultivated in raft cultures. However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains.

Methods: U2OS cells were transfected with HPV11 wt or E8- minicircle genomes and their gene expression was studied via 3' RACE, 5' RACE or via real time PCR methods. The DNA replication of these genomes was detected by Southern blot methods.

Results: The analysis of HPV11 transcripts in U2OS cells showed that the patterns of promoter use, splice sites and polyadenylation cleavage sites are identical to those previously characterized in human HPV-related lesions, human squamous carcinoma cell lines (e.g., SSC-4) and laryngeal papillomas. Transcriptional initiation from the three previously described HPV11 promoters in the E6 and E7 ORFs (P90, P264, and P674-714) were functional, and these promoters were used together with two promoter regions in the E1 ORF (P1092 and P1372). Mutating the E8 ORF ATG start codon to ACG eliminated the translation of fusion proteins from the E8 ORF coupled to E1 and E2 proteins C-terminal sequences, leading to the de-repression of gene expression (particularly from the P1092 promoter) and to the activation of genome replication. These data suggested that the expression of the functional E8^E2 protein is used to control viral gene expression and copy number of the HPV11 genome. The analysis of HPV11 E1 expression plasmids showed that the E6/E7 region, together with the E1 coding region, is crucial for the production of functionally active E1 protein.

Conclusions: The data presented in this paper suggest that in human osteosarcoma cell line U2OS the gene expression pattern of the HPV11 truly reflect the expression profile of the replicating HPV genome and therefore this cellular system is suitable for drug development program targeting HPV replication.

Show MeSH
Related in: MedlinePlus