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

HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B). (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B). (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.

Mentions: To test the efficiency of E1 expression from the designed vectors and to identify the effect of the expressed E1 protein on origin-containing plasmids, 100 ng of the HPV11URR plasmid was co-transfected with 100 ng of HPV11E2 and with various amounts of HPV11E1 expression plasmids (10, 100, 1000, or 5000 ng) into U2OS cells. E1 protein expression was evaluated via Western blotting using an HA-tag-specific antibody (Figure 6A). Such tagging of the E1 protein did not interfere with the functions of the protein during replication initiation (data not shown) and enabled us to evaluate E1 protein expression levels. E1 expression levels varied greatly depending on which of the E1 plasmids was used. The E1 protein expressed from the L+ plasmid could be detected when as little as 10 ng of the expression plasmid was transfected (Figure 6A, lane 1), whereas the L- plasmid E1 protein signal was detectable when at least a 100-times higher concentration was used (Figure 6A, lane 5). The plasmid containing one intron expressed E1 at the same level as the L+ plasmid (Figure 6A, compare lanes 1-3 and 7-9), indicating that the natural leader sequence of HPV11 can be substituted with the heterologous intron. The data suggested that sequences preceding the E1 ORF in polycistronic mRNA play an important role in determining E1 protein expression. The construct with a double intron had an even stronger effect, providing an extremely high level of E1 protein expression (Figure 6A, lanes 10-13).Figure 6


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)

HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B). (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B). (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.
Mentions: To test the efficiency of E1 expression from the designed vectors and to identify the effect of the expressed E1 protein on origin-containing plasmids, 100 ng of the HPV11URR plasmid was co-transfected with 100 ng of HPV11E2 and with various amounts of HPV11E1 expression plasmids (10, 100, 1000, or 5000 ng) into U2OS cells. E1 protein expression was evaluated via Western blotting using an HA-tag-specific antibody (Figure 6A). Such tagging of the E1 protein did not interfere with the functions of the protein during replication initiation (data not shown) and enabled us to evaluate E1 protein expression levels. E1 expression levels varied greatly depending on which of the E1 plasmids was used. The E1 protein expressed from the L+ plasmid could be detected when as little as 10 ng of the expression plasmid was transfected (Figure 6A, lane 1), whereas the L- plasmid E1 protein signal was detectable when at least a 100-times higher concentration was used (Figure 6A, lane 5). The plasmid containing one intron expressed E1 at the same level as the L+ plasmid (Figure 6A, compare lanes 1-3 and 7-9), indicating that the natural leader sequence of HPV11 can be substituted with the heterologous intron. The data suggested that sequences preceding the E1 ORF in polycistronic mRNA play an important role in determining E1 protein expression. The construct with a double intron had an even stronger effect, providing an extremely high level of E1 protein expression (Figure 6A, lanes 10-13).Figure 6

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