Limits...
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.

Show MeSH

Related in: MedlinePlus

Mapping of HPV11 promoter activity from E6/E7-containing E1 expression plasmids. (A) U2OS cells were transfected with 1 μg of different HPV11 expression plasmids (indicated at top of panel A and schematically introduced in panel B). PolyA+ mRNA was extracted at 24 h post-transfection, and 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. The products from a-e were sequenced, and their structures are shown in panel B. Mock-transfected U2OS cells were used as a negative control (lane 5). (B) Schematic map of the 5′ RACE products from the E1 expression plasmids, together with HPV11 E1 L+, L-, Int1 and Int2 expression vector maps. All of the E1 protein-coding transcripts (indicated with letters from a-e, shown at left) are shown, indicating their exons (solid boxes), introns (lines) and potential TSSs in E6 and E7 ORFs (nt numbers).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Mapping of HPV11 promoter activity from E6/E7-containing E1 expression plasmids. (A) U2OS cells were transfected with 1 μg of different HPV11 expression plasmids (indicated at top of panel A and schematically introduced in panel B). PolyA+ mRNA was extracted at 24 h post-transfection, and 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. The products from a-e were sequenced, and their structures are shown in panel B. Mock-transfected U2OS cells were used as a negative control (lane 5). (B) Schematic map of the 5′ RACE products from the E1 expression plasmids, together with HPV11 E1 L+, L-, Int1 and Int2 expression vector maps. All of the E1 protein-coding transcripts (indicated with letters from a-e, shown at left) are shown, indicating their exons (solid boxes), introns (lines) and potential TSSs in E6 and E7 ORFs (nt numbers).

Mentions: All of the previously described HPV11 promoters have been mapped to the E6/E7 protein coding region at nt 90, nt 264 and nt 674-714. In our L+ E1 expression system, the first viral promoter P90 is disrupted, and the E6 ORF starts at nt 105, whereas the remaining promoters and potential splice donor sites at leader sequence are intact in the E6/E7 ORF region. The CMV promoter is an extremely strong promoter; however, viral promoters may also function and initiate transcription on their own or splicing may still contribute to the generation of translatable E1 mRNA. Therefore, we searched for evidence of functional viral promoters in the L+ E1 expression vector or for evidence of a splicing event occurring in the E6/E7 ORF, as in the case of the high-risk HPVs. One microgram of different E1 expression vectors was transfected into U2OS cells, and polyA+ mRNA was extracted at 24 h post-transfection. A 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. All of the observed 5′ RACE products shown in Figure 7A were gel purified, cloned and sequenced, and the results are shown in Figure 7B. As expected, strong signals could be detected from transcripts starting from the CMV promoter region. The E1 transcript from the L+ expression vector was unspliced, and the CMV promoter mimicked the early promoter at P90 (Figure 7A and B, species a). The L- mRNA was also unspliced, and the CMV promoter served as the late promoter at P674-714 (Figure 7A and B, species c). The insertion of the intron between the CMV promoter and the E1 coding region (Int1) showed that the intron was spliced out in all of the sequenced clones (Figure 7A and B, species d). The E1 expression vector Int2, which contained two introns between the CMV promoter and the E1 coding region, provided the following double-spliced products: a) both of the introns were spliced out or b) a single intron was spliced, leaving the first intron intact (75% of clones) (Figure 7A and B, species e). A weak signal could be detected for the L+ sample at the level of the 750 bp size marker (Figure 7A and B, species b). The analysis of this product showed that the viral promoters were functional in the presence of the CMV promoter (Figure 7A and B, species b) because we were able to detect the following two transcripts: one starting at P264 and the other at the P674-714 promoter region. However, we did not detect any splicing event occurring in the E6/E7 region.Figure 7


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 HPV11 promoter activity from E6/E7-containing E1 expression plasmids. (A) U2OS cells were transfected with 1 μg of different HPV11 expression plasmids (indicated at top of panel A and schematically introduced in panel B). PolyA+ mRNA was extracted at 24 h post-transfection, and 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. The products from a-e were sequenced, and their structures are shown in panel B. Mock-transfected U2OS cells were used as a negative control (lane 5). (B) Schematic map of the 5′ RACE products from the E1 expression plasmids, together with HPV11 E1 L+, L-, Int1 and Int2 expression vector maps. All of the E1 protein-coding transcripts (indicated with letters from a-e, shown at left) are shown, indicating their exons (solid boxes), introns (lines) and potential TSSs in E6 and E7 ORFs (nt numbers).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Mapping of HPV11 promoter activity from E6/E7-containing E1 expression plasmids. (A) U2OS cells were transfected with 1 μg of different HPV11 expression plasmids (indicated at top of panel A and schematically introduced in panel B). PolyA+ mRNA was extracted at 24 h post-transfection, and 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. The products from a-e were sequenced, and their structures are shown in panel B. Mock-transfected U2OS cells were used as a negative control (lane 5). (B) Schematic map of the 5′ RACE products from the E1 expression plasmids, together with HPV11 E1 L+, L-, Int1 and Int2 expression vector maps. All of the E1 protein-coding transcripts (indicated with letters from a-e, shown at left) are shown, indicating their exons (solid boxes), introns (lines) and potential TSSs in E6 and E7 ORFs (nt numbers).
Mentions: All of the previously described HPV11 promoters have been mapped to the E6/E7 protein coding region at nt 90, nt 264 and nt 674-714. In our L+ E1 expression system, the first viral promoter P90 is disrupted, and the E6 ORF starts at nt 105, whereas the remaining promoters and potential splice donor sites at leader sequence are intact in the E6/E7 ORF region. The CMV promoter is an extremely strong promoter; however, viral promoters may also function and initiate transcription on their own or splicing may still contribute to the generation of translatable E1 mRNA. Therefore, we searched for evidence of functional viral promoters in the L+ E1 expression vector or for evidence of a splicing event occurring in the E6/E7 ORF, as in the case of the high-risk HPVs. One microgram of different E1 expression vectors was transfected into U2OS cells, and polyA+ mRNA was extracted at 24 h post-transfection. A 5′ RACE analysis was performed with the HPV11-specific primer pr1265c. All of the observed 5′ RACE products shown in Figure 7A were gel purified, cloned and sequenced, and the results are shown in Figure 7B. As expected, strong signals could be detected from transcripts starting from the CMV promoter region. The E1 transcript from the L+ expression vector was unspliced, and the CMV promoter mimicked the early promoter at P90 (Figure 7A and B, species a). The L- mRNA was also unspliced, and the CMV promoter served as the late promoter at P674-714 (Figure 7A and B, species c). The insertion of the intron between the CMV promoter and the E1 coding region (Int1) showed that the intron was spliced out in all of the sequenced clones (Figure 7A and B, species d). The E1 expression vector Int2, which contained two introns between the CMV promoter and the E1 coding region, provided the following double-spliced products: a) both of the introns were spliced out or b) a single intron was spliced, leaving the first intron intact (75% of clones) (Figure 7A and B, species e). A weak signal could be detected for the L+ sample at the level of the 750 bp size marker (Figure 7A and B, species b). The analysis of this product showed that the viral promoters were functional in the presence of the CMV promoter (Figure 7A and B, species b) because we were able to detect the following two transcripts: one starting at P264 and the other at the P674-714 promoter region. However, we did not detect any splicing event occurring in the E6/E7 region.Figure 7

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