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

Schematic map of the HPV11 genome and primers used in RACE and RT-PCR analysis. (A) Linear depiction of the HPV11 genome, showing the open reading frames (ORFs), long control region (LCR), promoters (P90, P264 and P674-714) and polyadenylation CSs (Cs4384 and Cs7485). The number before each ORF indicates the first nucleotide of the start codon. The primers used in transcriptome analysis are indicated at the top of the figure with dashed arrows. The number given with each primer represents its 5´ end binding position in the HPV11 genome. (B) Sequences of primers used for RACE and RT-PCR analysis. The position of each primer in the HPV11 genome and its orientation and sequence are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Schematic map of the HPV11 genome and primers used in RACE and RT-PCR analysis. (A) Linear depiction of the HPV11 genome, showing the open reading frames (ORFs), long control region (LCR), promoters (P90, P264 and P674-714) and polyadenylation CSs (Cs4384 and Cs7485). The number before each ORF indicates the first nucleotide of the start codon. The primers used in transcriptome analysis are indicated at the top of the figure with dashed arrows. The number given with each primer represents its 5´ end binding position in the HPV11 genome. (B) Sequences of primers used for RACE and RT-PCR analysis. The position of each primer in the HPV11 genome and its orientation and sequence are indicated.

Mentions: Previously, HPV11 gene transcription has been studied in human clinical samples [20,21,24,27] and in a human squamous carcinoma cell line [26] employing various detection mechanisms as PCR [21,23,26], cDNA cloning [27], nuclease mapping [24,25], retrovirus mediated gene transfer [22,23] and RACE analysis [26]. To compare viral transcription in U2OS cells with these previous findings, we decided to map all of the transcripts of the HPV11 genome generated during its replication in these cells. Thus, we extracted polyA+ mRNA from U2OS cells transfected with the HPV11wt or HPV11E8- genome and analyzed the samples by 3′ RACE, 5′ RACE, and RT-PCR analyses. The primer sequences used in this study and their positions in the HPV11 genome are shown in Figure 2.Figure 2


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)

Schematic map of the HPV11 genome and primers used in RACE and RT-PCR analysis. (A) Linear depiction of the HPV11 genome, showing the open reading frames (ORFs), long control region (LCR), promoters (P90, P264 and P674-714) and polyadenylation CSs (Cs4384 and Cs7485). The number before each ORF indicates the first nucleotide of the start codon. The primers used in transcriptome analysis are indicated at the top of the figure with dashed arrows. The number given with each primer represents its 5´ end binding position in the HPV11 genome. (B) Sequences of primers used for RACE and RT-PCR analysis. The position of each primer in the HPV11 genome and its orientation and sequence are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Schematic map of the HPV11 genome and primers used in RACE and RT-PCR analysis. (A) Linear depiction of the HPV11 genome, showing the open reading frames (ORFs), long control region (LCR), promoters (P90, P264 and P674-714) and polyadenylation CSs (Cs4384 and Cs7485). The number before each ORF indicates the first nucleotide of the start codon. The primers used in transcriptome analysis are indicated at the top of the figure with dashed arrows. The number given with each primer represents its 5´ end binding position in the HPV11 genome. (B) Sequences of primers used for RACE and RT-PCR analysis. The position of each primer in the HPV11 genome and its orientation and sequence are indicated.
Mentions: Previously, HPV11 gene transcription has been studied in human clinical samples [20,21,24,27] and in a human squamous carcinoma cell line [26] employing various detection mechanisms as PCR [21,23,26], cDNA cloning [27], nuclease mapping [24,25], retrovirus mediated gene transfer [22,23] and RACE analysis [26]. To compare viral transcription in U2OS cells with these previous findings, we decided to map all of the transcripts of the HPV11 genome generated during its replication in these cells. Thus, we extracted polyA+ mRNA from U2OS cells transfected with the HPV11wt or HPV11E8- genome and analyzed the samples by 3′ RACE, 5′ RACE, and RT-PCR analyses. The primer sequences used in this study and their positions in the HPV11 genome are shown in Figure 2.Figure 2

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