Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: Analysis by in situ hybridization

Virology ◽  
1992 ◽  
Vol 189 (1) ◽  
pp. 132-140 ◽  
Author(s):  
Matthias Dürst ◽  
Dagmar Glitz ◽  
Achim Schneider ◽  
Harald zur Hausen
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Human Papillomavirus ◽  
Dna Replication ◽  
Cervical Neoplasia ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16

scholarly journals Polypyrimidine Tract Binding Protein Induces Human Papillomavirus Type 16 Late Gene Expression by Interfering with Splicing Inhibitory Elements at the Major Late 5′ Splice Site, SD3632

2008 ◽  
Vol 82 (7) ◽  
pp. 3665-3678 ◽  
Author(s):  
Monika Somberg ◽  
Xiaomin Zhao ◽  
Monika Fröhlich ◽  
Magnus Evander ◽  
Stefan Schwartz
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Splice Site ◽  
Late Gene ◽  
Polypyrimidine Tract ◽  
Late Gene Expression ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Polypyrimidine Tract Binding Protein ◽  
Cellular Factors

ABSTRACT We have initiated a screen for cellular factors that can induce human papillomavirus type 16 (HPV-16) late gene expression in human cancer cells. We report that the overexpression of polypyrimidine tract binding protein (PTB), also known as heterologous nuclear ribonucleoprotein I (hnRNP I), induces HPV-16 late gene expression in cells transfected with subgenomic HPV-16 plasmids or with full-length HPV-16 genomes and in persistently HPV-16-infected cells. In contrast, other hnRNPs such as hnRNP B1/A2, hnRNP F, and hnRNP Q do not induce HPV-16 late gene expression. PTB activates SD3632, the only 5′ splice site on the HPV-16 genome that is used exclusively by late mRNAs. PTB interferes with splicing inhibitory sequences located immediately upstream and downstream of SD3632, thereby activating late gene expression. One AU-rich PTB-responsive element was mapped to a 198-nucleotide sequence located downstream of SD3632. The deletion of this element induced HPV-16 late gene expression in the absence of PTB. Our results suggest that the overexpression of PTB interferes with cellular factors that interact with the inhibitory sequences. One may speculate that an increase in PTB levels or a reduction in the concentration of a PTB antagonist is required for the activation of HPV-16 late gene expression during the viral life cycle.

scholarly journals The Human Papillomavirus Type 16 E7 Oncogene Is Required for the Productive Stage of the Viral Life Cycle

2000 ◽  
Vol 74 (14) ◽  
pp. 6622-6631 ◽  
Author(s):  
Elsa R. Flores ◽  
B. Lynn Allen-Hoffmann ◽  
Denis Lee ◽  
Paul F. Lambert
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Dna Replication ◽  
Epithelial Cells ◽  
Dna Synthesis ◽  
Copy Number ◽  
Keratinocyte Differentiation ◽  
Replication Machinery ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16

ABSTRACT The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.

scholarly journals A Downstream Polyadenylation Element in Human Papillomavirus Type 16 L2 Encodes Multiple GGG Motifs and Interacts with hnRNP H

2005 ◽  
Vol 79 (14) ◽  
pp. 9254-9269 ◽  
Author(s):  
Daniel Öberg ◽  
Joanna Fay ◽  
Helen Lambkin ◽  
Stefan Schwartz
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Polyadenylation Signal ◽  
Late Gene ◽  
Coding Region ◽  
Late Gene Expression ◽  
Hpv 16 ◽  
Virus Particles ◽  
Human Papillomavirus Type 16 ◽  
L1 And L2

ABSTRACT Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.

scholarly journals Influence of Codon Bias on Heterologous Production of Human Papillomavirus Type 16 Major Structural Protein L1 in Yeast

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Milda Norkiene ◽  
Alma Gedvilaite
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Codon Bias ◽  
Heterologous Gene Expression ◽  
Heterologous Production ◽  
Structural Protein ◽  
Hpv 16 ◽  
Major Structural Protein ◽  
Human Papillomavirus Type 16 ◽  
L1 Protein

Heterologous gene expression is dependent on multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and posttranslational modifications. Codon bias has a significant influence on protein yields. However, sometimes it is not clear which parameter causes observed differences in heterologous gene expression as codon adaptation typically optimizes many sequence properties at once. In the current study, we evaluated the influence of codon bias on heterologous production of human papillomavirus type 16 (HPV-16) major structural protein L1 in yeast by expressing five variants of codon-modified open reading frames (OFRs) encoding HPV-16 L1 protein. Our results showed that despite the high toleration of various codons used throughout the length of the sequence of heterologously expressed genes in transformed yeast, there was a significant positive correlation between the gene's expression level and the degree of its codon bias towards the favorable codon usage. The HPV-16 L1 protein expression in yeast can be optimized by adjusting codon composition towards the most preferred codon adaptation, and this effect most probably is dependent on the improved translational elongation.

scholarly journals A Splicing Enhancer in the E4 Coding Region of Human Papillomavirus Type 16 Is Required for Early mRNA Splicing and Polyadenylation as Well as Inhibition of Premature Late Gene Expression

2005 ◽  
Vol 79 (18) ◽  
pp. 12002-12015 ◽  
Author(s):  
Margaret Rush ◽  
Xiaomin Zhao ◽  
Stefan Schwartz
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Splice Site ◽  
Late Gene ◽  
Coding Region ◽  
Late Gene Expression ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Early Region ◽  
Splicing Enhancer

ABSTRACT Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3′ splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3′ splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3′ splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3′ splice site at position 5639 in the late region. Optimization of the position 3358 3′ splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3′ splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression.

scholarly journals Nuclear Matrix Attachment Regions of Human Papillomavirus Type 16 Repress or Activate the E6 Promoter, Depending on the Physical State of the Viral DNA

2000 ◽  
Vol 74 (6) ◽  
pp. 2489-2501 ◽  
Author(s):  
Walter Stünkel ◽  
Zhonghui Huang ◽  
Shyh-Han Tan ◽  
Mark J. O'Connor ◽  
Hans-Ulrich Bernard
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Reporter Gene ◽  
Hela Cells ◽  
Nuclear Matrix ◽  
Binding Sites ◽  
Matrix Attachment Regions ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 Gene

ABSTRACT Two nuclear matrix attachment regions (MARs) bracket a 550-bp segment of the long control region (LCR) containing the epithelial cell-specific enhancer and the E6 promoter of human papillomavirus type 16 (HPV-16). One of these MARs is located in the 5′ third of the LCR (5′-LCR-MAR); the other lies within the E6 gene (E6-MAR). To study their function, we linked these MARs in various natural or artificial permutations to a chimeric gene consisting of the HPV-16 enhancer-promoter segment and a reporter gene. In transient transfections of HeLa cells, the presence of either of these two MARs strongly represses reporter gene expression. In contrast to this, but similar to the published behavior of cellular MARs, reporter gene expression is stimulated strongly by the E6-MAR and moderately by the 5′-LCR-MAR in stable transfectants of HeLa or C33A cells. To search for binding sites of soluble nuclear proteins which may be responsible for repression during transient transfections, we performed electrophoretic mobility shift assays (EMSAs) of overlapping oligonucleotides that represented all sequences of these two MARs. Both MARs contain multiple sites for two strongly binding proteins and weak binding sites for additional factors. The strongest complex, with at least five binding sites in each MAR, is generated by the CCAAT displacement factor (CDP)/Cut, as judged by biochemical purification, by EMSAs with competing oligonucleotides and with anti-CDP/Cut oligonucleotides, and by mutations. CDP/Cut, a repressor that is down-regulated during differentiation, apparently represses HPV-16 transcription in undifferentiated epithelials cells and in HeLa cells, which are rich in CDP/Cut. In analogy to poorly understood mechanisms acting on cellular MARs, activation after physical linkage to chromosomal DNA may result from competition between the nuclear matrix and CDP/Cut. Our observations show that cis-responsive elements that regulate the HPV-16 E6 promoter are tightly clustered over at least 1.3 kb and occur throughout the E6 gene. HPV-16 MARs are context dependent transcriptional enhancers, and activated expression of HPV-16 oncogenes dependent on chromosomal integration may positively select tumorigenic cells during the multistep etiology of cervical cancer.

scholarly journals Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry

Cytometry ◽  
1994 ◽  
Vol 15 (3) ◽  
pp. 245-257 ◽  
Author(s):  
Majid Siadat-Pajouh ◽  
Ammasi Periasamy ◽  
Andrea H. Ayscue ◽  
Anna B. Moscicki ◽  
Joel M. Palefsky ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Human Papillomavirus ◽  
Image Cytometry ◽  
Human Papillomavirus Type 16

scholarly journals Productive Replication of Adeno-Associated Virus Can Occur in Human Papillomavirus Type 16 (HPV-16) Episome-Containing Keratinocytes and Is Augmented by the HPV-16 E2 Protein

2000 ◽  
Vol 74 (8) ◽  
pp. 3494-3504 ◽  
Author(s):  
Phyllis Ogston ◽  
Kenneth Raj ◽  
Peter Beard
Keyword(s):  
Cell Cycle ◽  
Human Papillomavirus ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Hpv 16 ◽  
Adeno Associated Virus ◽  
E2 Protein ◽  
Human Papillomavirus Type 16 ◽  
Productive Replication

ABSTRACT We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.

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