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The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

ABSTRACT: The human osteosarcoma cell line U2OS is useful for studying genome replication of human papillomavirus (HPVs) subtypes that belong to different phylogenetic genera. In this study, we defined the HPV18 transcription map in U2OS cells during transient replication, stable maintenance and vegetative amplification by identifying viral promoter regions, transcription polyadenylation and splicing sites during HPV18 genome replication. Mapping of the HPV18 transcription start sites in U2OS cells revealed five distinct promoter regions (P102, P520, P811, P1193 and P3000). With the exception of P3000, all of these regions have been previously identified during productive HPV18 infection. Collectively, the data suggest that U2OS cells are suitable for studying the replication and transcription properties of HPVs and to serve as a platform for conducting high-throughput drug screens to identify HPV replication inhibitors. In addition, we have identified mRNA species that are initiated from the promoter region P3000, which can encode two E2C regulator proteins that contain only the C-terminal hinge and DNA-binding and dimerization domains of E2. We show that these proteins regulate the initial amplification of HPV18 by modulating viral transcription. Moreover, we show that one of these proteins can act as a transcriptional activator of promoter P102.

SUBMITTER: Toots M

PROVIDER: S-EPMC4280167 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

Toots Mart M Männik Andres A Kivi Gaily G Ustav Mart M Ustav Ene E Ustav Mart M

PloS one 20141230 12

The human osteosarcoma cell line U2OS is useful for studying genome replication of human papillomavirus (HPVs) subtypes that belong to different phylogenetic genera. In this study, we defined the HPV18 transcription map in U2OS cells during transient replication, stable maintenance and vegetative amplification by identifying viral promoter regions, transcription polyadenylation and splicing sites during HPV18 genome replication. Mapping of the HPV18 transcription start sites in U2OS cells reveal ...[more]

PMID: 25548925

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Project description:BackgroundAlthough 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.MethodsU2OS 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.ResultsThe 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.ConclusionsThe 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.

Project description:Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5' RACE, 3' RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as "early" promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as "late" promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.

Project description:Human papillomaviruses (HPVs) replicate in the cutaneous and mucosal epithelia, and the infectious cycle is synchronous with the differentiation program of the host keratinocytes. The virus initially infects dividing cells in the lower layers of the epithelium, where it establishes a persistent infection. The viral genome is maintained as a low-copy-number, extrachromosomal element in these proliferating cells but switches to the late stage of the life cycle in differentiated cells. The cellular chromatin adaptor protein Brd4 is involved in several stages and processes of the viral life cycle. In concert with the viral transcriptional regulator E2, Brd4 can repress transcription from the early viral promoter. Brd4 and E2 form a complex with the viral genome that associates with host chromosomes to partition the viral genome in dividing cells; Brd4 also localizes to active sites of productive HPV DNA replication. However, because of the difficulties in producing HPV viral particles, the role of Brd4 in modulating viral transcription and replication at the initial stage of infection is unclear. In this study, we have used an HPV18 quasivirus-based genome delivery system to assess the role of Brd4 in the initial infectivity of primary human keratinocytes. We show that, upon infection of primary human keratinocytes with HPV18 quasivirus, Brd4 activates viral transcription and replication. Furthermore, this activation is independent of the functional interaction between Brd4 and the HPV18 E2 protein.ImportanceHPVs lack encapsidated proteins and so rely exquisitely on host cellular factors to initiate their gene expression programs in newly infected cells. Brd4 is an important cellular chromatin adaptor molecule that normally activates host transcription initiation and elongation. In this study, we further optimize and utilize a quasivirus infection system to show that Brd4 activates HPV18 transcription at early stages of infection. HPVs are important human pathogens causing a wide range of cutaneous and tumorigenic morbidities. Therefore, specifically targeting this protein could provide a new target of therapeutic prevention of establishment of HPV infections.

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The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

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The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

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