Papillomavirus: Viral vectors in the gene therapy and new therapeutic targets (original) (raw)
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Novel recombinant papillomavirus genomes expressing selectable genes
Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 " marker " genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle. Worldwide, human papillomavirus (HPV) infections are one of the most common sexually transmitted infections. While most infections are asymptomatic, persistent infection with certain HPV types has been shown to be the causative agent of an increasing number of human cancers 1. All known papillomaviruses have a small dsDNA circular genome of approximately 8 kb that encodes for six to eight genes. Because of this limited coding capacity, the HPV life cycle is strictly dependent on host factors and is tightly associated with keratinocyte differentiation. Following infection, the viral genome must amplify to a low copy number and become established as a stable, extra-chromosomal replicating element. This initial phase is followed by the maintenance phase, during which viral genomes are maintained at a constant copy number in actively dividing cells. Finally, terminal differentiation of the infected cell triggers high-level viral DNA amplification and capsid protein production resulting in the assembly of infectious virus. Because of this dependence on cellular differentiation, the viral life cycle cannot be studied using standard, monolayer cell culture systems. Early studies showed that viral particles isolated from warts were used to study the ability of the viral genome to be maintained 2. However, similar samples are hard to obtain and the observation that transfected human pap-illomavirus genomes could establish persistent replication in human primary foreskin keratinocytes was a major breakthrough 3. This transfection-based system has helped elucidate many events in the viral life cycle 3. Using this method, it has been shown that oncogenic HPV types will replicate in primary human keratinocytes and provide these cells with a growth advantage, eventually leading to their immortalization 4,5. A correlation has been observed between the oncogenic potential of HPV types and their ability to score in a quantitative colony forming assay 6. Traditionally, the viral DNA is co-transfected with a plasmid encoding a selectable marker. However, since the selectable gene is supplied in trans from a non-replicating plasmid, selection can only be maintained while the non-replicating plasmid is present in the cell (typically a few days). Furthermore, this can result in false positive (cells transiently resistant to the selectable marker but no HPV18) and false negative (cells containing HPV18 but not resistant to the selectable marker) results 6. More recently, HPV genomes packaged in viral particles (known as quasiviruses) 7 have been used to investigate early events in the viral life cycle 8–10. However, HPV-derived qua-siviruses can only contain a single plasmid or genome 7 .
HUMAN PAPILLOMAVIRUS BIOLOGY: REVIEW
Journal of Advanced Research
Sexually transmitted infections (STIs) are more dynamic than any other diseases in both developing and developed countries. Human Papillomaviruses (HPVs) are icosahedral, small, non-enveloped particle measuring ~55 nm in diameter. They are ~8000 base-pair (bp), double stranded circular DNA molecule which wrapped into a protein shell by two molecules namely, L1 and L2. The HPV genome has the coding capacity for two late proteins (L1 and L2) and for six early proteins (E1, E2, E4–E7) which are necessary for the replication of the viral DNA and for the assembly of newly produced virus particles within the infected cells. More than 100 HPV types have been characterized molecularly and about 40 types are able to infect the epithelial lining of the anogenital tract and other mucosal areas of the human body. Human papillomavirus (HPV) is a necessary cause of cervical, anogenital, upper aerodigestive tract and skin cancers. Other cofactors are necessary for progression from cervical HPV inf...
Cancer and Metastasis Review, 1987
Numerous studies over the past several years have demonstrated that human papillomaviruses (HPV) may play a significant role in the development of several types of human neoplasia. Although it has been accepted for some time that HPVs are responsible for benign epithelial tumors, data accumulated in more recent years have implicated this group of animal viruses in a number of premalignant lesions, as well as a variety of epithelially derived malignancies. Genital, oral, and some rare types of cutaneous cancers have all been found to contain varying degrees of HPV DNA. In several instances secondary tumors resulting from metastases to lymph nodes and lungs have also been demonstrated to contain HPV DNA. Although there is a strong correlation between the presence of the virus and the malignant phenotype in several of these cancers, the precise role of the virus in the development of malignant tumors has not yet been elucidated. A major difficulty in elucidating the role of papillomaviruses in oncogenesis has been the lack of an appropriate in vitro culture system that would permit the growth of the virus and allow an analysis of its transforming properties. Nevertheless. recent advances in molecular biology have permitted the molecular cloning and amplification of HPV viral DNA. thereby facilitating its use as a probe for the detection of miniscule amounts of H PV DNA and HPV RNA in tumor biopsies. Moreover. DNA transfections of cells in culture have been extremely useful in the study of viral DNA replication and transformation properties, providing information on the maintenance and oncogenicity of HPV DNA. These advances have implications for the improved detection of HPV infections, which will aid in patient diagnosis and prognosis. In addition, future treatment and prevention programs may come as a direct result of these basic studies on the mechanism of HPV-induced oncogenesis.
Journal of Virology, 2011
We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3- to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono- and oligomeric HPV genomes in these confluent cell cultur...
Biology and pathological associations of the human papillomaviruses: a review
The Malaysian journal of pathology, 1998
Historical cottontail rabbit papillomavirus studies raised early indications of a mammalian DNA oncogenic virus. Today, molecular cloning recognises numerous animal and human papillomaviruses (HPVs) and the development of in vitro transformation assays has escalated oncological research in HPVs. Currently, their detection and typing in tissues is usually by Southern blotting, in-situ hybridization and polymerase chain reaction methods. The complete papillomavirus virion constitutes a protein coat (capsid) surrounding a circular, double-stranded DNA organised into coding and non-coding regions. 8 early (E1-E8) open reading frames (ORFs) and 2 late (L1, L2) ORFs have been identified in the coding region of all papillomaviruses. The early ORFs encode proteins which interact with the host genome to produce new viral DNA while late ORFs are activated only after viral DNA replication and encode for viral capsid proteins. All papillomaviruses are obligatory intranuclear organisms with spec...
Current Perspectives in Human Papillomavirus [Working Title]
Current Perspectives in Human Papillomavirus, 2018
Cervical cancer is by far the most common HPV-related disease. About 99.7% of cervical cancer are caused by persistent genital high-risk human papillomavirus (HPV) infection. Worldwide, cervical cancer is one of the most common cancer in women with an estimated 528,000 new cases reported in 2012. Most HPV infections clear spontaneously but persistent infection with the oncogenic or high-risk types may cause cancer of the oropharynx and anogenital regions. The virus usually infects the mucocutaneous epithelium and produces viral particles in matured epithelial cells and then causes a disruption in normal cell-cycle control and the promotion of uncontrolled cell division leading to the accumulation of genetic damage. There are currently two effective prophylactic vaccines against HPV infection in many developed countries and these comprise of HPV types 16 and 18, and HPV types 6, 11, 16 and 18 virus-like particles. HPV testing in the secondary prevention of cervical cancer is clinically valuable in triaging low-grade cytological abnormalities and is also more sensitive than cytology as a primary screening. If these prevention strategies can be implemented in both in developed and developing countries, many thousands of lives could be saved.
Hypermethylation of the human papillomavirus transgenome in transgenic mice
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biospied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 61 02-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E l ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced EI-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E l ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells. ): Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa. Virology 159:389-398. Baker CC, Phelps WC, Lindgren V, Braun MJ, Gonda MA, Howley PM (1987): Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. Journal of Virology 61:962-971. Bernard BA, Bailly C, Lenoir MC, Darmon M, Thierry F, Moshe Y (1989): The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes. Journal of Virology 6343174324. Bream GL, Ohmstede CA, Phelps WC (1993): Characterization of human papillomavirus type ll E l and E2 expressed in insect cells. Journal of Virology 67:2655-2663. Chiang CM, Ustav M, Stenlund A, Ho TF, Broker TR, Chow LT (1992): Viral E l and E2 proteins support replication of homologous and heterologous papilloma-viral origins. Proceedings of the National Academy of Sciences, USA 89:5799-5803. Choo KB, Pan CC, Han SH (1987a): Integration of human papillomavirus E2 gene and invariable retention of the long cintrol region and the E6iE7 open reading frames. Virology 161:259-261. Choo KB, Pan CC, Liu MS, Ng HT, Chen CP, Lee YN, Chao CF, Meng CL, Yeh MY, Han SH (198713): Presence ofepisomal and integrated human papillomavirus DNA sequences in cervical carcinoma. Journal of Medical Virology 21:lOl-107. Choo KB, Lee HH, Pan CC, Wu SM, Liew LN, Cheung WF, Han SH (1988): Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma. Journal of Virology 62:1659-1666. Choo KB, Cheung WF, Liew LN, Lee HH, Han SH (1989): Presence of catenated human papillomavirus type 16 episomes in a cervical carcinoma cell line. Journal of Virology 63:782-789. Choo KB, Lee HH, Liew LN, Chong KY, Chou H F (1990): Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma. Virology 178:621-625. Clertant P, Seif I(1984): A common function for polyoma virus large-T papillomavirus E l proteins? Nature 311:276-279. Couturier J , Sastre-Garau X, Schneider-Maunoury S, Labib A, Orth G (1991): Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for proto-oncogene expression. Journal of Virology 65:45344538. Cullen AP, Reid R, Campion M, Lorincz AT (1991): Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. Journal of Virology 65: 606-612. Das B, Sharma JK, Gopalakrishna V, Luhra UK (1992): Analysis by polymerase chain reaction of physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions. Journal of Virology 73:2327-2336. Dean FB, Bullock P, Murakami Y, Wobbe R, Weissbach L, Hurwitz J (1987): Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.
International journal of oncology, 2018
Human papilloma viruses (HPV) are a small group of non‑enveloped viruses belonging to the Papillomaviridae family with strong similarities to polyoma viruses. The viral particles consist of a genome in the form of a circular double‑stranded DNA, encompassing eight open reading frames, as well as a non‑enveloped icosahedral capsid. HPV infection is considered the most common sexually transmitted disease in both sexes and is strongly implicated in the pathogenesis of different types of cancer. 'High‑risk' mucosal HPV types, predominantly types 16, 18, 31, 33 and 35, are associated with most cervical, penile, vulvar, vaginal, anal, oropharyngeal cancers and pre‑cancers. Screening for HPV is necessary for the prognosis and for determining treatment strategies for cancer. Novel HPV markers, including proteomic and genomic markers, as well as anti‑papillomavirus vaccines are currently available. The aim of this comprehensive review was to thoroughly present the updated information...