scholarly journals Cooperative role of c-MYC and E6/E7 from two molecular variants of human papillomavirus type 16 upon proliferation and in vitro transformation of primary human keratinocytes

2019 ◽  
Vol 98 (1) ◽  
pp. 52-58 ◽  
Author(s):  
Jimena Hochmann ◽  
Silvaneide Ferreira ◽  
João Sobrinho ◽  
Laura Sichero
Keyword(s):  
Human Papillomavirus ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Nuclear Antigen ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
In Vitro Transformation ◽  
Molecular Variants ◽  
E6 And E7

The roles of E6 and E7 oncoproteins of Human Papillomavirus type 16 (HPV-16) in the progression of immortalized epithelial cells to invasive tumors are not fully understood. Here, we establish a novel link between E6 and E7 of two molecular variants of HPV-16 (AA and E-350G), and c-MYC, regarding the cooperation in promoting malignant transformation of primary human foreskin keratinocytes (PHK). We aimed to study the synergistic effects of E6/E7 and c-MYC upon proliferation, and the in vitro transformation potential of PHK. We evaluated cellular proliferation through the expression of the Proliferating Cell Nuclear Antigen (PCNA) protein and colony formation abilities using soft agar and low attachment plates. We observed that E-350G-c-MYC PHKs exhibited discrete higher PCNA levels and formed significantly more colonies in both soft-agar and when growth in low-adhesion culture plates. Overall, we concluded that the E-350G variant co-transfected with c-MYC might promote malignant cellular transformation with a better efficiency than the AA-c-MYC counterpart. The enhanced oncogenic properties exhibited by the E-350G-c-MYC variant offer insights into mechanisms that may operate in human cervical neoplasia, given the higher frequency of its occurrence in the progression of high-grade precursor lesions to invasive carcinomas.

scholarly journals Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

2010 ◽  
Vol 84 (16) ◽  
pp. 8219-8230 ◽  
Author(s):  
Monika Somberg ◽  
Stefan Schwartz
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Splice Site ◽  
Binding Sites ◽  
Mrna Levels ◽  
Coding Region ◽  
Cervical Lesions ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

scholarly journals Adeno-Associated Virus Major Rep78 Protein Disrupts Binding of TATA-Binding Protein to the p97 Promoter of Human Papillomavirus Type 16

2000 ◽  
Vol 74 (5) ◽  
pp. 2459-2465 ◽  
Author(s):  
Pei-Fen Su ◽  
Shu-Yuan Chiang ◽  
Cheng-Wen Wu ◽  
Felicia Y.-H. Wu
Keyword(s):  
Human Papillomavirus ◽  
Binding Protein ◽  
Core Promoter ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Dependent Manner ◽  
Hpv 16 ◽  
Adeno Associated Virus ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7

ABSTRACT Adeno-associated virus type 2 (AAV) is known to inhibit the promoter activities of several oncogenes and viral genes, including the human papillomavirus type 16 (HPV-16) E6 and E7 transforming genes. However, the target elements of AAV on the long control region (LCR) upstream of E6 and E7 oncogenes are elusive. A chloramphenicol acetyltransferase assay was performed to study the effect of AAV on the transcription activity of the HPV-16 LCR in SiHa (HPV-positive) and C-33A (HPV-negative) cells. The results reveal that (i) AAV inhibited HPV-16 LCR activity in a dose-dependent manner, (ii) AAV-mediated inhibition did not require the HPV gene products, and (iii) the AAV replication gene product Rep78 was involved in the inhibition. Deletion mutation analyses of the HPV-16 LCR showed that regulatory elements outside the core promoter region of the LCR may not be direct targets of AAV-mediated inhibition. Further study with the electrophoretic mobility shift assay demonstrated that Rep78 interfered with the binding of TATA-binding protein (TBP) to the TATA box of the p97 core promoter more significantly than it disrupted the preformed TBP-TATA complex. These data thus suggest that Rep78 may inhibit transcription initiation of the HPV-16 LCR by disrupting the interaction between TBP and the TATA box of the p97 core promoter.

scholarly journals Transcriptional Activation of the Telomerase hTERT Gene by Human Papillomavirus Type 16 E6 Oncoprotein

2001 ◽  
Vol 75 (9) ◽  
pp. 4467-4472 ◽  
Author(s):  
Tim Veldman ◽  
Izumi Horikawa ◽  
J. Carl Barrett ◽  
Richard Schlegel
Keyword(s):  
Human Papillomavirus ◽  
Transcriptional Activation ◽  
Transcriptional Control ◽  
Regulatory Region ◽  
Rnase Protection ◽  
Hpv 16 ◽  
Htert Gene ◽  
Human Papillomavirus Type 16 ◽  
E6 Gene

ABSTRACT The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

scholarly journals Leaky Scanning Is the Predominant Mechanism for Translation of Human Papillomavirus Type 16 E7 Oncoprotein from E6/E7 Bicistronic mRNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7284-7297 ◽  
Author(s):  
Simon N. Stacey ◽  
Deborah Jordan ◽  
Andrew J. K. Williamson ◽  
Michael Brown ◽  
Joanna H. Coote ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Human Papillomaviruses ◽  
Start Codon ◽  
Leaky Scanning ◽  
Structural Element ◽  
Entry Site ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7 ◽  
Bicistronic Mrna

ABSTRACT Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5′ untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopusβ-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5′ end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5′ end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.

scholarly journals In Vitro Infection and Type-Restricted Antibody-Mediated Neutralization of Authentic Human Papillomavirus Type 16

1998 ◽  
Vol 72 (2) ◽  
pp. 959-964 ◽  
Author(s):  
Wendy I. White ◽  
Susan D. Wilson ◽  
William Bonnez ◽  
Robert C. Rose ◽  
Scott Koenig ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Neutralizing Antibodies ◽  
Cultured Cells ◽  
Severe Combined Immunodeficiency ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Model Systems ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16

ABSTRACT Human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Studies of model systems with animal papillomaviruses have demonstrated the importance of neutralizing antibodies in preventing papillomavirus-associated disease. The assessment of neutralizing antibody responses against HPV-16, previously hampered by the lack of a viral source, was enabled by the recent propagation of an HPV-16 stock in xenografted severe combined immunodeficiency (SCID) mice. HPV-16 infection of an immortalized human keratinocyte cell line was demonstrated by detection of an HPV-16-specific spliced mRNA amplified by reverse transcriptase PCR. Infection was blocked by preincubation of the virus with antiserum generated against HPV-16 virus-like particles (VLPs) composed of the major capsid protein, L1. To examine potential cross-neutralizing activity among the different genital HPV types, rabbit antisera to L1 VLPs corresponding to HPV-6, -11, -18, -31, -33, -35, -39, and -45 were assayed for the ability to block the HPV-16 infection of cultured cells. Antiserum raised against HPV-33 L1 VLPs was the only heterologous antiserum which inhibited HPV-16 infection. Thus, a neutralization assay for HPV-16 may help to characterize the components required to compose a broadly efficacious genital HPV vaccine.

scholarly journals Identification and functional analysis of sequence rearrangements in the long control region of human papillomavirus type 16 Af-1 variants isolated from Ugandan penile carcinomas

2000 ◽  
Vol 81 (12) ◽  
pp. 2969-2982 ◽  
Author(s):  
Maria Lina Tornesello ◽  
Franco M. Buonaguro ◽  
Luigi Buonaguro ◽  
Immacolata Salatiello ◽  
Elke Beth-Giraldo ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Control Region ◽  
Expression Vectors ◽  
Long Control Region ◽  
Hpv 16 ◽  
Expression Levels ◽  
Human Papillomavirus Type 16 ◽  
Transforming Activity ◽  
E6 And E7 ◽  
Cat Expression

Human papillomavirus type 16 (HPV-16) is the predominant HPV isolate found in malignancies of male and female lower genital tracts. However, only a small percentage of individuals infected with high-risk HPVs develop a genital neoplasia, suggesting that additional events at both the cellular and the virus level are necessary for the progression to cancer, including genetic mutations/rearrangements of viral sequences involved in the oncogenic process. In this study, the genetic stability of the long control region (LCR) (nt 7289–114), which regulates expression levels of oncoproteins E6 and E7, was analysed in HPV-16 isolates from penile carcinoma (PC) biopsies of patients recruited from Uganda, one of the countries with the highest incidence of genital cancers in both men and women. Nucleotide changes within the LCR region typical of the African-1 (Af-1) lineage were observed in all HPV-16 isolates. Two out of five samples showed further rearrangements of the enhancer region. The functional activity of LCR with Af-1 mutations and/or rearrangements was evaluated by cloning each LCR into CAT expression vectors, followed by transfection in several epithelial and non-epithelial cell lines. CAT expression levels driven by a rearranged LCR were significantly higher than those driven by Af-1 or European prototype LCRs. Furthermore, in the NIH3T3 focus formation assay, the transforming activity of E6 and E7 genes, driven by a mutated or rearranged LCR, was 1·4- to 3·0-fold higher, respectively. These results indicate that rearrangements within the LCR of HPV-16 isolated from African PCs are frequently found (2 out of 5, 40%). It is also shown that increased HPV LCR activity is associated with an increased E6/E7-mediated in vitro transforming activity, suggesting that natural variants can play a major role in the pathogenesis of genital carcinomas.

scholarly journals CD4-Positive and CD8-Positive Cytotoxic T Lymphocytes Contribute to Human Papillomavirus Type 16 E6 and E7 Responses

1999 ◽  
Vol 6 (4) ◽  
pp. 494-498 ◽  
Author(s):  
Mayumi Nakagawa ◽  
Daniel P. Stites ◽  
Joel M. Palefsky ◽  
Zachary Kneass ◽  
Anna-Barbara Moscicki
Keyword(s):  
Human Papillomavirus ◽  
T Lymphocytes ◽  
Natural Killer ◽  
Cell Population ◽  
Effector Cell ◽  
Target Cells ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7 ◽  
Ctl Responses

ABSTRACT Cytotoxic T-lymphocyte (CTL) responses to E6 and E7 were previously shown to be more commonly detectable in human papillomavirus type 16 (HPV-16)-positive women without squamous intraepithelial neoplasia (SIL) than in HPV-16-positive women with SIL (M. Nakagawa, D. P. Stites, S. Farhat, J. R. Sisler, B. Moss, F. Kong, A. B. Moscicki, and J. M. Palefsky, J. Infect. Dis. 175:927–931, 1997). The objective of this study was to characterize the phenotype(s) of the effector cell population responsible for HPV-16 E6- and E7-specific cytotoxic responses. Peripheral blood mononuclear cells were stimulated with HPV-16 E6 or E7 fusion protein. Cells from an autologous B-lymphoblastoid cell line, infected with vaccinia virus expressing E6 or E7, served as target cells. The effector cells were characterized by using natural-killer-cell removal, antibody blocking, and T-cell subset separation. Our results suggest that both CD4 and CD8 T lymphocytes contribute to HPV-16 E6- and E7-specific CTL responses although their relative contributions vary from individual to individual. On the other hand, natural killer cells in the effector cell population contribute to background activities but not to HPV-specific responses in this assay system.

scholarly journals Time Course of Humoral and Cell-Mediated Immune Responses to Human Papillomavirus Type 16 in Infected Women

2002 ◽  
Vol 9 (4) ◽  
pp. 877-882 ◽  
Author(s):  
Mayumi Nakagawa ◽  
Raphael Viscidi ◽  
Ian Deshmukh ◽  
Maria Da Costa ◽  
Joel M. Palefsky ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Immune Responses ◽  
Time Course ◽  
Cytotoxic T Lymphocyte ◽  
Dna Detection ◽  
Protective Role ◽  
Hpv 16 ◽  
Humoral Immune ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7

ABSTRACT The time course of cell-mediated and humoral immune responses was elucidated in eight women with human papillomavirus type 16 (HPV-16) infection by performing serial HPV-16 E6 and E7 cytotoxic T-lymphocyte (CTL) assays and HPV-16 virus-like particle (VLP) antibody analyses. Four subjects had a single incident of HPV-16 DNA detection, and four subjects had two periods of HPV-16 DNA detection. In two of the women in the latter group, the second episode of HPV-16 detection occurred in the presence of high titers of HPV-16 VLP antibody, bringing into question the protective role of humoral immunity in preventing repeated infection. However, all four subjects rapidly became HPV-16 DNA negative following the second detection of HPV-16 DNA, suggesting the presence of immunological memory. In addition, one subject rapidly became negative for HPV-16 DNA despite having no evidence of CTL or VLP antibody response prior to the second HPV-16 DNA detection, suggesting the presence of immunological responses at an undetectable level. Overall, seven of eight subjects (88%) had detectable HPV-16 E6 and/or E7 CTL responses and seven of eight women (88%) had detectable HPV-16 VLP antibody responses.

scholarly journals The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression

1999 ◽  
Vol 73 (3) ◽  
pp. 1918-1930 ◽  
Author(s):  
Walter Stünkel ◽  
Hans-Ulrich Bernard
Keyword(s):  
Human Papillomavirus ◽  
Control Region ◽  
Replication Origin ◽  
Chromatin Organization ◽  
Long Control Region ◽  
Promoter Elements ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Hpv 18

ABSTRACT The long control region (LCR) of human papillomavirus type 16 (HPV-16) has a size of 850 bp (about 12% of the viral genome) and regulates transcription and replication of the viral DNA. The 5′ segment of the LCR contains transcription termination signals and a nuclear matrix attachment region, the central segment contains an epithelial cell-specific enhancer, and the 3′ segment contains the replication origin and the E6 promoter. Here we report observations on the chromatin organization of this part of the HPV-16 genome. Treatment of the nuclei of CaSki cells, a cell line with 500 intrachromosomal copies of HPV-16, with methidiumpropyl-EDTA-Fe(II) reveals nucleosomes in specific positions on the LCR and the E6 and E7 genes. One of these nucleosomes, which we termed Ne, overlaps with the center of the viral enhancer, while a second nucleosome, Np16, overlaps with the replication origin and the E6 promoter. The two nucleosomes become positioned on exactly the same segments after in vitro assembly of chromatin on the cloned HPV-16 LCR. Primer extension mapping of DNase I-cleaved chromatin revealed Np16 to be positioned centrally over E6 promoter elements, extending into the replication origin. Ne covers the center of the enhancer but leaves an AP-1 site, one of the strongestcis-responsive elements of the enhancer, unprotected. Np16, or a combination of Np16 and Ne, represses the activity of the E6 promoter during in vitro transcription of HPV-16 chromatin. Repression is relieved by addition of Sp1 and AP-1 transcription factors. Sp1 alters the structure of Np16 in vitro, while no changes can be observed during the binding of AP-1. HPV-18, which has a similar arrangement ofcis-responsive elements despite its evolutionary divergence from HPV-16, shows specific assembly in vitro of a nucleosome, Np18, over the E1 binding site and E6 promoter elements but positioned about 90 bp 5′ of the position of Np16 on the homologous HPV-16 sequences. The chromatin organization of the HPV-16 and HPV-18 genomes suggests important regulatory roles of nucleosomes during the viral life cycle.

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