Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
1.
Front Microbiol ; 14: 1259510, 2023.
Article in English | MEDLINE | ID: mdl-37795297

ABSTRACT

Objective: In past decades, the role of high-risk HPV (HR-HPV) infection in cancer pathogenesis has been extensively studied. The viral E7 protein expressed in pre-malignant cells has been identified as an ideal target for immunological intervention. However, the cultivation of HPV in vitro remains a significant challenge, as well as the lack of methods for expressing the HPV E7 protein and generating replication-competent recombinant viral particles, which posed a major obstacle to further exploration of the function and carcinogenic mechanisms of the E7 oncoprotein. Therefore, it is imperative to investigate novel methodologies to construct replication-competent recombinant viral particles that express the HPV E7 protein to facilitate the study of its function. Methods: We initiated the construction of recombinant viral particles by utilizing the ccdB-Kan forward/reverse screening system in conjunction with the Red/ExoCET recombinant system. We followed the infection of C33A cells with the obtained recombinant virus to enable the continuous expression of HPV16 E7. Afterwards, the total RNA was extracted and performed transcriptome sequencing using RNA-Seq technology to identify differentially expressed genes associated with HPV-induced oncogenicity. Results: We successfully established replicative recombinant viral particles expressing HPV16 E7 stably and continuously. The C33A cells were infected with recombinant viral particles to achieve overexpression of the E7 protein. Subsequently, RNA-Seq analysis was conducted to assess the changes in host cell gene expression. The results revealed an upregulation of the CD36 gene, which is associated with the HPV-induced oncogenic pathways, including PI3K-Akt and p53 signaling pathway. qRT-PCR analysis further identified that the upregulation of the CD36 gene due to the expression of HPV16 E7. Conclusion: The successful expression of HPV16 E7 in cells demonstrates that the replicated recombinant virus retains the replication and infection abilities of Ad4, while also upregulating the CD36 gene involved in the PI3K-Akt signaling and p53 pathways, thereby promoting cell proliferation. The outcome of this study provides a novel perspective and serves as a solid foundation for further exploration of HPV-related carcinogenesis and the development of replicative HPV recombinant vaccines capable of inducing protective immunity against HPV.

2.
Front Cell Infect Microbiol ; 12: 929666, 2022.
Article in English | MEDLINE | ID: mdl-35832386

ABSTRACT

High-risk human papillomavirus infection may develop into a persistent infection that is highly related to the progression of various cancers, including cervical cancer and head and neck squamous cell carcinomas. The most common high-risk subtypes are HPV16 and HPV18. The oncogenic viral proteins expressed by high-risk HPVs E6/E7 are tightly involved in cell proliferation, differentiation, and cancerous transformation since E6/E7 mRNAs are derived from the same pre-mRNA. Hence, the alternative splicing in the E6/E7-coding region affects the balance of the E6/E7 expression level. Interrupting the balance of E6 and E7 levels results in cell apoptosis. Therefore, it is crucial to understand the regulation of E6/E7 splice site selection and the interaction of splicing enhancers and silencers with cellular splicing factors. In this review, we concluded the relationship of different E6/E7 transcripts with cancer progression, the known splicing sites, and the identified cis-regulatory elements within high-risk HPV E6/E7-coding region. Finally, we also reviewed the role of various splicing factors in the regulation of high-risk HPV oncogenic E6/E7 mRNA splicing.


Subject(s)
Oncogene Proteins, Viral , Papillomavirus Infections , Alternative Splicing , Human papillomavirus 16/genetics , Humans , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/genetics , RNA Splicing Factors/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism
3.
Genes (Basel) ; 13(4)2022 04 07.
Article in English | MEDLINE | ID: mdl-35456460

ABSTRACT

Breast cancer is one of the most common malignant tumors in women worldwide. Early diagnosis, treatment, and prognosis of breast cancer are global challenges. Identification of valid predictive diagnosis and prognosis biomarkers and drug targets are crucial for breast cancer prevention. This study characterizes differentially expressed genes (DEGs) based on the TCGA database by using DESeq2, edgeR, and limma. A total of 2032 DEGs, including 1026 up-regulated genes and 1006 down-regulated genes were screened. Followed with WGCNA, PPI analysis, GEPIA 2, and HPA database verification, thirteen hub genes including CDK1, BUB1, BUB1B, CDC20, CCNB2, CCNB1, KIF2C, NDC80, CDCA8, CENPF, BIRC5, AURKB, PLK1, MAD2L1, and CENPE were obtained, and they may serve as potential therapeutic targets of breast cancer. Especially, overexpression of CCNB1 and PLK1 are strongly associated with the low survival rate of breast cancer patients, demonstrating their potentiality as prognostic markers. Moreover, CCNB1 and PLK1 are highly expressed in all breast cancer stages, suggesting that they could be further studied as potential drug targets. Taken together, our study highlights CCNB1 and PLK1 as potential anti-breast cancer drug targets and prognostic markers.


Subject(s)
Breast Neoplasms , Computational Biology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Cycle Proteins/genetics , Cyclin B1/genetics , Female , Gene Expression Regulation, Neoplastic , Genes, cdc , Humans , Prognosis , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , Polo-Like Kinase 1
4.
Nucleic Acids Res ; 50(7): 3867-3891, 2022 04 22.
Article in English | MEDLINE | ID: mdl-35357488

ABSTRACT

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three 'AC(A/G)AGG'-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236-286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.


Subject(s)
DNA-Binding Proteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins , Human papillomavirus 16 , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins/genetics , DNA-Binding Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Human papillomavirus 16/genetics , Humans , Oncogene Proteins, Viral/metabolism , Oncogenes , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism
5.
Arch Virol ; 167(2): 563-570, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34860285

ABSTRACT

We have determined the effect of seven serine- and arginine-rich (SR) proteins and 15 heterogenous nuclear ribonucleoproteins (hnRNPs) on human papillomavirus type 16 (HPV16) late gene expression. Of the seven SR proteins analyzed here, SRSF1, SRSF3, and SRSF9 induced HPV16 late gene expression, and five of the SR proteins affected HPV16 L1 mRNA splicing. Of the 15 hnRNP proteins analyzed here, hnRNP A2, hnRNP F, and hnRNP H efficiently induced HPV16 late gene expression, and all of the hnRNPs affected HPV16 L1 mRNA levels or mRNA splicing. Thus, the majority of SR proteins and hnRNPs have the potential to regulate HPV16 L1 mRNA splicing. Strict control of the expression of the immunogenic L1 and L2 capsid proteins may contribute to the ability of HPV16 to cause persistence.


Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins , Serine , Arginine , Gene Expression , Gene Expression Regulation, Viral , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Humans , RNA, Messenger/genetics , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors/genetics
6.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: mdl-32759322

ABSTRACT

Human papillomavirus 16 (HPV16) 5'-splice site SD226 and 3'-splice site SA409 are required for production of the HPV16 E7 mRNAs, whereas unspliced mRNAs produce E6 mRNAs. The E6 and E7 proteins are essential in the HPV16 replication cycle but are also the major HPV16 proteins required for induction and maintenance of malignancy caused by HPV16 infection. Thus, a balanced expression of unspliced and spliced mRNAs is required for production of sufficient quantities of E6 and E7 proteins under physiological and pathophysiological conditions. If splicing becomes too efficient, the levels of unspliced E6 mRNAs will decrease below a threshold level that is no longer able to produce E6 protein quantities high enough to significantly reduce p53 protein levels. Similarly, if splicing becomes too inefficient, the levels of spliced E7 mRNAs will decrease below a threshold level that is no longer able to produce E7 protein quantities high enough to significantly reduce pRb protein levels. To determine how splicing between SD226 and SA409 is regulated, we have investigated how SA409 is controlled by the cellular proteins hnRNP A1 and hnRNP A2, two proteins that have been shown previously to control HPV16 gene expression. We found that hnRNP A1 and A2 interacted directly and specifically with a C-less RNA element located between HPV16 nucleotide positions 594 and 604 downstream of SA409. Overexpression of hnRNP A1 inhibited SA409 and promoted production of unspliced E6 mRNAs at the expense of the E7 mRNAs, whereas overexpression of hnRNP A2 inhibited SA409 to redirect splicing to SA742, a downstream 3'-splice site that is used for generation of HPV16 E6̂E7, E1, and E4 mRNAs. Thus, high levels of either hnRNP A1 or hnRNP A2 inhibited production of the promitotic HPV16 E7 protein. We show that the hnRNP A1 and A2 proteins control the relative levels of the HPV16 unspliced and spliced HPV16 E6 and E7 mRNAs and function as inhibitors of HPV16 E7 expression.IMPORTANCE Human papillomavirus type 16 (HPV16) belongs to the high-risk-group of HPVs and is causing a variety of anogenital cancers and head and neck cancer. The two HPV16 oncoproteins E6 and E7 prevent apoptosis and promote mitosis and are essential for completion of the HPV16 life cycle and for transformation of the infected cell and maintenance of malignancy. E6 and E7 are produced from two mRNAs that are generated in a mutually exclusive manner by alternative splicing. While E6 protein is made from the unspliced mRNA, E7 is made from the spliced version of the same pre-mRNA. Since sufficient quantities of both E6 and E7 are required for malignant transformation, this intricate arrangement of gene expression renders E6 and E7 expression vulnerable to external interference. Since antiviral drugs to HPV16 are not available, a detailed knowledge of the regulation of HPV16 E6 and E7 mRNA splicing may uncover novel targets for therapy.


Subject(s)
Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Human papillomavirus 16/metabolism , Papillomavirus E7 Proteins/metabolism , RNA Splicing , RNA, Viral/metabolism , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Human papillomavirus 16/genetics , Humans , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/genetics , RNA, Viral/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism
7.
Virus Res ; 285: 198004, 2020 08.
Article in English | MEDLINE | ID: mdl-32380211

ABSTRACT

Human papillomaviruses (HPVs) produce a large number of alternatively spliced mRNAs, including a number of differently spliced mRNAs with the potential to produce E2 protein. To identify the alternatively spliced HPV16 mRNA with the highest ability to produce E2 protein, we have generated E2 cDNA expression plasmids representing the most common, alternatively spliced E2 mRNAs, and assessed their translational potential. Our results revealed that an mRNA initiated at the HPV16 late promoter p670 and spliced from the HPV16 5'-splice site SD880 to the HPV16 3'-splice site SA2709, located immediately upstream of the E2 ATG, produced higher levels of E2 than any of the other alternatively spliced, E2-encoding mRNAs. Utilization of a known, alternative 3'-splice site located upstream of the E2 ATG named SA2582, generated mRNAs with lower ability to produce E2 than mRNAs spliced to SA2709. Finally, analysis of HPV16 mRNA splicing demonstrated that SA2709 was more efficiently spliced to the upstream 5'-splice site SD880 than to the upstream 5'-splice site SD226. In conclusion, the HPV16 mRNA with the greatest ability to produce E2 protein is generated from the HPV16 late promoter and is spliced between HPV16 5'-splice site SD880 and HPV16 3'-splice site SA2709.


Subject(s)
DNA-Binding Proteins/biosynthesis , Human papillomavirus 16/metabolism , Oncogene Proteins, Viral/biosynthesis , Alternative Splicing , RNA, Messenger , RNA, Viral
8.
Int J Cancer ; 144(2): 297-310, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30303514

ABSTRACT

Here we show that treatment of the HPV16-positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan-induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down-regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1-negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half-life of the HPV16 E6 and E7 mRNAs renders HPV16-driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Apoptosis/drug effects , Melphalan/pharmacology , Squamous Cell Carcinoma of Head and Neck/virology , Tonsillar Neoplasms/virology , Animals , Cell Line, Tumor , Half-Life , Human papillomavirus 16 , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Oncogene Proteins, Viral/biosynthesis , Oncogene Proteins, Viral/drug effects , Papillomavirus E7 Proteins/biosynthesis , Papillomavirus E7 Proteins/drug effects , Papillomavirus Infections/complications , RNA Stability/drug effects , Repressor Proteins/biosynthesis , Repressor Proteins/drug effects , Xenograft Model Antitumor Assays
9.
J Clin Invest ; 126(7): 2425-36, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27214553

ABSTRACT

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor-containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1ß by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1ß secretion was minimal in CFT073-infected macrophages; however, IL-1ß release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1ß secretion by CFT073 and tcpC-deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1ß levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.


Subject(s)
Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli , Animals , Bone Marrow Cells/metabolism , Caspase 1/metabolism , Cytosol/metabolism , Escherichia coli Proteins/metabolism , Female , HEK293 Cells , Humans , Immunity, Innate , Interleukin-1beta/metabolism , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Protein Domains , Virulence Factors/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...