Patent Application
20130150262
This application contains sequence listing.
The present invention relates to the fields of virology, cell biology, cell culturing, and drug development. More particularly the invention provides a method for screening for anti-HPV substances and a kit for screening for anti-HPV substances. The invention also provides plasmids for transfecting cell lines and cell lines capable of supporting all replication phases of Human Papilloma Virus.
The continuous interest to study the human papillomaviruses (HPV) has been generated from their association with specific human cancers. HPV infects basal proliferating cells of the epithelium and induces the formation of benign tumors. In some cases this infection may lead to progression and formation of malignant carcinomas. The complete papillomavirus virion constitutes a protein coat (capsid) surrounding a circular, double-stranded DNA organized into coding and non-coding regions. Eight early (E1-E8) open reading frames (ORFs) and two late (L1, L2) ORFs have been identified in the coding region of papillomaviruses. The early ORFs encode proteins involved in viral DNA replication during the establishment, in continuous maintenance state and late amplification (E1 and E2), in regulation of viral gene expression and chromosome tethering (E2), virus assembly (E4), immortalisation and transformation (E6 and E7/high-risk HPVs only). Late ORFs are activated only after cell differentiation and encode viral capsid proteins (L1 and L2). In the noncoding Upstream Regulatory Region (URR) the promoters, enhancer and other regulatory elements in addition to the replication origin are located.
The current view divides the papillomaviral life cycle into three stages. First, following initial entry into the cell nucleus in the basal layer of the epithelium, where the apparatus necessary for replication exists, the PV genome is amplified, viral DNA is synthesized faster than chromosomal DNA, the copy number raises (up to 50-300 copies per cell) (for review, see Kadaja M, Silla T, Ustav E, Ustav M. Papillomavirus DNA replication—from initiation to genomic instability. Virology. 2009 Feb. 20; 384(2):360-8.). The second stage represents stable replication of HPV DNA in S-phase synchronized with chromosomal replication and maintenance of viral DNA as extrachromosomal multicopy nuclear episomes as a result of segregation/partitioning of the viral genome into the daughter cells.
At this stage only early genes are expressed and neither the synthesis of capsid proteins L1 and L2 nor virion assembly occurs. Early gene products provide transforming proteins that ensure clonal expansion of infected cells. If infected cells detach from the basal membrane and reach upper layers of the skin or mucosa, they stop dividing and start differentiation (keratinisation). It triggers onset of the third step, vegetative viral DNA replication during which a) viral DNA amplification is initiated again, and then b) late proteins are synthesized and viral particles assembled (for review, see Kadaja M, Silla T, Ustav E, Ustav M. Papillomavirus DNA replication—from initiation to genomic instability. Virology. 2009 Feb. 20; 384(2):360-8.).
Modelling of these replication stages in cells has been problematic in the case of human papillomaviruses. Most of the tissue culture cells do not support any mode of HPV genomic replication. Attempts to get viral genomic DNA replication going from transfected plasmids of β-papillomavirus types has completely failed in any keratinocyte cell lines or primary keratinocytes. Also, it has been difficult to generate reproducible human cell lines that carry stable HPV replicating genomes, especially that of the “low risk”-HPV types. The stable replication of HPV episomes has been accomplished just by a handful of laboratories. The episomal state has been shown to be allowed only in the presence of feeders or in conditions of raft cultures. W12, a frequently used HPV-16 cell line, has originated from a patient sample, but while cultivating W12 cells in monolayer, integration events have been shown to take place instead of maintenance of the episomal state of the viral genome.
Nevertheless, the replication of HPV replication origin containing plasmids can be demonstrated in many different cell lines of different species in case the production of E1 and E2 proteins is provided from heterologous expression vectors. The main factor which restricts the replication to certain epithelial cells is therefore the availability of coordinated expression of cellular transcription factors for the transcription of the mRNAs for viral proteins.
The vaccines targeting HPV-16 and HPV-18 or HPV-6b, HPV-11, HPV-16 and HPV-18 have been developed and are becoming increasingly available in many countries. It should be considered as a great achievement in fighting against cervical cancers. However, it is not sufficient, because the vaccines target at best only for four subtypes of hundreds of papillomaviruses, including “high risk”-type of mucosal or cutaneous skin papillomaviruses. Additionally, it has been shown convincingly that HPV-16 and HPV-18 are prevalent viruses found in cervical carcinomas in developed countries. According to the molecular epidemiological analysis of the spread of the virus in developing countries, like in Sub-Saharan regions of Africa, other virus isolates like HPV-52 and HPV-35 are prevalent.
There is an urgent need for the small-molecule drugs, which can be used to block effectively the replication of the papillomavirus genome, therefore lowering the viral load per cell and avoiding the generation of viral particles and therefore the spread of the virus. Furthermore, there is a need for small-molecule drugs, which could be used in various stages of virus infection to stop the viral replication at that specific stage. However, this objective has been difficult to achieve due to the lack of an effective cellular system for screening for drug candidates. This cellular system should be compatible with the high-throughput and high-content format of the screening of the drug candidates and allow identifying the active substances in reproducible and cost-effective format. Furthermore such cellular system should allow detection of compounds inhibiting any of the replications phases of all types of HPV-viruses. Animal xenograft models have been described previously by J. Duan, WO0040082 (A reproducible xenograft animal model for hosting and propagating human papillomavirus (HPV)), as well as primary keratinocytes are applied for hosting the viral genome by Kreider et al. 1993 and 1998, (U.S. Pat. No. 5,541,058, In vitro assay system for testing the effectiveness of anti-papilloma viral agents; U.S. Pat. No. 6,200,745, Vitro assay system using a human cell line for testing the effectiveness of anti-papilloma viral agents). However, these methods do not allow high-throughput screening for drug candidates, and a simpler and more convenient method is necessarily required. Our group has previously discovered the ability of human osteosarcoma cell line U2OS to support the in vitro cultivation of HPV (K. Salk, 2009 Studies on the mechanisms of the DNA replication of high- and low-risk human papillomavirus in different cell lines. MSc thesis /in Estonian/; University of Tartu Press). However, maintenance of episomal HPV by itself is not sufficient for a high-throughput screening assay to identify possible HPV replication inhibitors.
This invention provides solutions to the above described shortcomings of current technology and others.
Accordingly it is an object of this invention to provide a cellular system supporting all phases of HPV DNA replication to allow determination of inhibitory effects of drug candidates on various phases of HPV DNA replication.
It is another object of this invention to provide a method to screen for factors inhibiting the HPV DNA replication at all different replication phases of HPV life cycle by detecting a product of a reporter gene or a reaction product of a protein encoded by a reporter gene enabling.
Another object of this invention is to provide a method to screen for factors inhibiting DNA replication of all types of human papilloma viruses, including high-risk, low-risk and cutaneous HPV on various phases of HPV DNA replication.
Another object of this invention is to provide extrachromosomally maintainable plasmids carrying HPV DNA sequences for transfection of cell lines.
Yet another object of this invention is to provide cell lines supporting all phases of HPV DNA replication for use in high-throughput screening of HPV replication inhibitors.
Another object of this invention is to provide an in vitro system to screen compounds capable of inhibiting initial replication of HPV DNA for use as vaccines.
Yet another object of this invention is to provide an in vitro system to screen compounds capable of inhibiting stable maintenance of HPV DNA replication for use as vaccines and cure.
An even further object of this invention is to provide an in vitro system to screen compounds capable of inhibiting vegetative amplificational replication phase of HPV DNA to prevent or cure viral infections in nondividing cells in upper layers of skin.
A yet another object of this invention is to identify compounds capable of inhibiting HPV DNA replication either in initial replication, stable maintenance, or vegetative amplification phase of all types of HPV.
Another object of this invention is candidate compounds for treating and curing infections and conditions caused by any type of HPV where the compound is indentified by the method of this invention.
Initial replication or transient replication refers to HPV DNA replication at establishment of the infection.
Stable maintenance or latent maintenance refers to the latent stage of viral replication cycle where viral DNA is stably maintained at an almost constant copy number in dividing host cells.
Vegetative amplificational replication or late amplificational replication refers to exponential viral DNA amplification when epithelial cells detach the basement membrane.
The present invention provides a method for identifying compounds capable of inhibiting Human Papillomavirus (HPV) DNA replication as well as plasmids for transfecting cells, cell lines capable of supporting all phases of HPV DNA replication and a kit for identifying the compounds capable of inhibiting HPV DNA replication.
The present invention provides a method and a system, wherein HPV genomic or subgenomic DNA is inserted into a cell line, and wherein all the phases of HPV DNA replication are supported, and further the influence of a compound on the HPV DNA replication is determined. The U2OS cell line was identified as a feasible host cell line to support HPV DNA replication. Now, according to the present invention, U2OS cells are identified as a suitable host for the propagation of genomes of all types of mucosal and cutaneous tissue specific HPVs and for the HPV genome-related constructs. It is also demonstrated that amplificational replication of the HPV genome, resembling amplification in the vegetative phase of the viral life-cycle occurs, when HPV positive U2OScell clones are maintained in high density for extended periods with regular media for at least 4 to 12 days.
Thus, a method is provided, wherein the quantitative detection of replicated HPV DNA or, more preferably, detection of a product of a reporter gene, a fusion protein including a reporter gene, or a reaction product of a protein encoded by a reporter gene enables screening for factors inhibiting the HPV DNA replication at all different replication phases of HPV life cycle: a) the initial amplificational replication demonstrated by the transient replication assay; b) the stable HPV DNA replication, synchronous with cellular DNA replication, demonstrated by the analysis of low to high HPV-content subclones; and c) the amplificational replication resembling vegetative phase of the viral DNA replication. This kind of novel system and method can be widely used in pharmacological research and high through-put screening for new potential drug candidates for prevention or therapy of infections by various subtypes of HPVs.
A preferred embodiment of this invention is a method for identifying compounds capable of inhibiting HPV DNA of all types of HPV at intial replication, stable maintenance or vegetative amplification phase of replication comprising the following steps:
The presence or absence of the inhibitory effect is detected as is described below.
According to one preferred embodiment, the invention provides a method for identifying compounds capable of inhibiting HPV DNA latent replication, which comprises the following steps:
In an another preferred embodiment, the invention provides a method for identifying compounds capable of inhibiting induced HPV DNA vegetative amplificational replication, which comprises the following steps:
According to the present invention launch of vegetative amplification of step d above is achieved with high risk HPV, with low risk HPV and even with cutaneous beta-papilloma viruses.
The inhibitory effect can be determined by any methods known in the art, which enables quantitative detection of the extrachromosomal (plasmid) DNA. However, most preferable methods comprise, but are not limited to, inserting nucleic acid sequences, which encode a reporter gene, to the episomally replicating construct. These reporter genes may encode any directly detectable and measurable proteins known in the art, or proteins catalyzing a reaction, product of which can be measured quantitatively or semiquantitatively, e.g. by visual observation with a microscope. The measurable product may remain inside the cell or may be excreted into the media. Examples of such reporter genes comprise, but are not limited to, dGFP, luciferase, secreted alkaline phosphatase, Gaussia luciferase, Renilla luciferase, dGFP-Luciferase fusion gene. Preferably, the nucleic acid sequence of the reporter gene is inserted to the region of HPV genome, which encodes for the L genes.
Most preferably the nucleic acid sequence of the reporter gene substitutes the L1 or L2 genes or both of them in the HPV genome. According to one preferred embodiment the reporter gene sequences are inserted in E2 ORF after E1 coding sequence. The subclones provided for selection from the generated cell banks are chosen from the ones carrying the variety of copy numbers ranging from low to high copy numbers of HPV plasmid per cell.
The subtypes of HPV provided in the present invention comprise, but are not limited to, HPV-18, HPV-16, HPV-6b, HPV-11, HPV-5 and HPV-8. These subtypes belong to mucosal high-risk, low-risk and cutaneous type of HPV subgroups, thus providing previously undescribed means for detecting substances capable for inhibiting the DNA replication of low-risk and skin-type of HPVs. The latent phase of HPV DNA replication provided in the invention, models the viral DNA replication process occurring in the dividing cells at the basal and suprabasal layer of the skin, infected by HPV. The vegetative amplificational replication phase of HPV replication provided in the invention models the viral DNA replication process occurring in nature in nondividing cells in the upper layers of the skin.
Moreover, the present invention provides a kit for identifying compounds capable of inhibiting HPV DNA initial, stable and amplificational replication. This kit comprises at least: human osteosarcoma cell line U2OS, or another cell line enabling the stable replication of HPV DNA; an episomally maintainable construct with complete or partial sequences of HPV DNA with L1 or L2 genes or both substituted with the reporter genes, or alternatively the reporter genes being inserted in E2 ORF before E1 coding sequence, for introduction into the cell line; a compound or a library of compounds to be screened for anti-HPV activity; and a means for assessing transcriptional activity of HPV DNA in the cells.
Hereby, experimental data is provided to illustrate the ability of U2OS cell line to support HPV DNA replication at establishment, at latent maintenance phase as well as the unexpected phenomena of the induction of exponential viral DNA amplification mimicking the vegetative phase of the infection. The data is provided by way of examples and the scope of the invention is presented in the claims.
U2OS cells were transfected with HPV-16 genome (
Prior to transfection, the HPV DNAs were cleaved out from the vector backbone: HPV-18 genome from pBR322 vector with EcoRI; HPV-6b from pBR322 with BamHI; HPV-16 and HPV-11 genomes from pUC19 with BamHI; HPV-8 DNA from pUC9 vector with BamHI; HPV-5 from pBR322 with Sad. Linear HPV fragments (ca 8 kb) were religated at low DNA concentrations (5 μg/ml) for 16 hrs at 4° C.
A sample from U18 #1.13 cell line was taken from the cell bank, cells were grown as regular monolayers, and 106 cells were seeded into each of the six 100 mm culture dishes for additional cultivation. 2 ml of fresh culture medium (IMDM) was added every two days, but no splitting of the cells was performed. Time points for analysis were taken the next day after adding the medium during 12 day growth period with 2-days interval. Time dependent growth series to obtain dense cell cultures are presented.
Time dependent growth series to obtain dense cell cultures are presented. The cells were counted with Invitrogen Countess cell counter before analysis.
Total DNA was isolated by standard procedures, and DNA concentrations were measured by NanoDrop spectrophotometer ND-1000.
The replication signal intensities of U18 #1.13 cell line were measured using Phosphor-Imager and ImageQuant software. The HPV-18 genome copy number was estimated by standard curves of marker lanes. Three different series are summarized.
Most of the late region (L1 and L2 ORFs) of the HPV-18 genome was removed by cleavage with ApaI and BpiI. The removed region was replaced with the fragment containing the sequences needed for propagation of the plasmid in E. coli cells (pMB1 origin of replication and beta-lactamase resistance markergene (bla) amplified from pUC18 cloning vector). The inserted bacterial sequences can be removed by HindIII digestion.
Expression cassette that includes synthetic 5′ intron element, codon optimised sequence encoding Gaussia luciferase marker gene, as well as bovine growth hormone polyadenylation signal, were inserted into the pUCHPV-18E so that the early region of the HPV-18 genome remained intact. The bacterial sequences can be removed by HindIII digestion.
Human papillomaviruses show strong tropism for epithelial cells. It was discovered that human osteosarcoma cell line U2OS, exhibiting epithelial adherent morphology, although derived from a moderately differentiated osteosarcoma, supported very effectively the HPV E1 and E2 protein dependent viral DNA replication, when the expression-vectors for viral replication proteins were used together with reporter plasmids containing viral origin. U2OS cells encode wild-type pRb and p53.
Hereafter it was investigated, whether the viral trans factors (E1 and E2) could act in their native configurations supporting the replication of the viral genomes in U2OS monolayer cultures. A set of four different cutaneous type of papillomaviruses were included, two of them belonging to high-risk type (HR/HPV-18 and HR/HPV-16) and two to low-risk type (LR/HPV-11 and LR/HPV-6b) according to their prognosis for cancer development. Additionally, two subtypes, the HPV-5 and HPV-8 as skin infecting β-papillomaviruses, were included. The U2OS cells were transfected with HPV-16 genome (
As seen in
The fact that the diverse groups of HPV circular genomes of HPV-6b, HPV-11, HPV-16, HPV-18, HPV-5 and HPV-8, respectively, are capable of establishing viral DNA replication in U2OS cells, suggests that the viral regulatory elements are adequately functional for supporting DNA replication of these virus types and that viral and cellular transcription and replication factors are adequately expressed. Thus, a compound capable for the inhibition of the first amplificational step of viral DNA replication in U2OS cell culture may be considered as a potential candidate for HPV treatment/prevention of HPV infection. The observation is valid at least for high-risk and low-risk mucosal HPVs as well as cutaneous HPVs.
Quite strong HPV genomic DNA replication signal in U2OS cells in transient assays suggested further evaluation of the capacity of HR- and LR-HPV plasmids for stable episomal replication. For this purpose we co-transfected into U2OS cells 5 μg of HPV-6b, or HPV-11, HPV-16, HPV-18, HPV-5, HPV-8 circular plasmid together with 5 μg AraD carrier DNA and with 2 μg of Eco01091-linearized of pNeo-EGFP or EcoRI-linearized pBabeNeo plasmid, encoding antibiotic resistance marker, which would allow the selection for the transfected cells. 48 hrs after the transfection G418 selection was performed. After two to three weeks of cultivation with G418 selection, the low-molecular weight (LMW) Hirt extracts from whole cell population (“pool” DNA) were analyzed by Southern blotting with radioactively labelled probes against the appropriate HPV types. The analysis shows that all tested samples contained HPV genomes at quite comparable levels, which indicates that the selected cells contained the HPV replicon (
For the detection of cloned human cell lines that carry extrachromosomal replicating HPV episomes, dilutions of 5000, 10 000 and 50 000 cells per 100 mm dish were transferred from selected cell population and the single cell colonies were picked, expanded, and grown up under the G418 selection. Total genomic DNA was extracted from these clones and Southern blot analysis was performed with 10 μg of EcoRI-linearized (
For isolated HPV-positive subclones long term follow up was performed by Southern blot analysis to determine the stability of the episomal maintenance replication continuing into later passages. The majority of the tested cell lines were stable in monolayer cultures with regular cultivation conditions in monolayer cultures during at least two months of inspection (example with HPV-18 subclone #1.13 on
HPV-18 #1.13 subclone was cultivated in regular monolayer cell culture conditions during 11 weeks starting from the detection of positive HPV-18 signal. The stability of extrachromosomal HPV-18 DNA over the time course was determined by Southern blot analysis of linearized (EcoRI) low-molecular weight DNA samples from Hirt lysates, extracted every time from one 100 mm culture dish. In parallel series equal amount (2 μg) of linearized cellular DNA (total DNA) was loaded and compared during the same time course. The HPV-18 full length genome specific probe was used.
The fact that the diverse group of HPV circular genomes of HPV-6b, HPV-11, HPV-16, HPV-18, HPV-5 and HPV-8, respectively, are capable of maintaining viral DNA replication in U2OS cells in monolayer cultures, further suggests that the viral regulatory elements are adequately functional for supporting at least stable or latent viral DNA replication step of these virus types and that viral and cellular transcription and replication factors are adequately expressed. Thus, a compound capable for the inhibition of the latent step of DNA replication in U2OS cell culture may be considered a potential candidate for HPV treatment/prevention in the latent phase of HPV infection. The observation is valid at least for high-risk and low-risk mucosal HPVs as well as cutaneous HPVs. The establishment of subclones with the HPV plasmid copy numbers varying from low to high confirms the usefulness of created tools, desired in the search for anti-HPV drugs.
In the productive stage of PV life cycle, amplification of the viral genome occurs in differentiated cells within the upper layer of epidermis. To study the productive stage of viral life cycle in tissue culture, the three-dimensional architecture of the epithelium has been usually tried to be reproduced with organotypic or raft cultures, suspension in methylcellulose, feeder cells, by using regulated culture and growth conditions.
We used an alternative method, only dense cell cultures to imitate differentiation-dependent viral amplification. For this purpose equal number of cells (for example 1×106 cells per 10 cm culture dish) of appropriate HPV-positive cell clone were split on several dishes (for example 6) and maintained as regular confluent monolayers grown up to high densities. The total DNA or low molecular weight (Hirt) DNA samples were collected at day 2, 4, 6, 8, 10, 12, ( . . . ), isolated and analyzed.
Using the HPV-18 positive cell line H18 #1.13 as an example, the induction of HPV DNA amplification is shown in
To characterize the appearance of intracellular HPV DNA episome formation supplementary to Southern blot analysis, the interphase and metaphase fluorescence in situ hybridization (FISH) was performed for studied subclones (Invitrogen Corporation, TSA™ Kit #22). Examples for interphase FISH for HPV-18 subclone #1.13 are shown in
As seen from these examples, HPV plasmid goes through an amplificational replication stage in confluent U2OS cells, bringing its copy number up to tens of thousands per cell, and therefore it is applicable for a person skilled in the art to use it in a high-throughput system for screening for agents exhibiting anti-HPV properties. Thus, a compound capable for the inhibition of amplificational DNA replication in U2OS cell culture may be considered a potential candidate for HPV treatment/prevention in the amplificational phase of HPV infection. The observation is valid at least for high-risk and low-risk mucosal HPVs as well as cutaneous HPVs.
Most of the late region (L1 and L2 ORFs) of the HPV-18 genome was removed by cleavage with ApaI and BpiI. The removed region was replaced with the fragment containing the sequences needed for the propagation of the plasmid in E. coli cells (pMB1 origin of replication and beta-lactamase resistance marker gene (bla) amplified from pUC18 cloning vector). The inserted bacterial sequences were removed by HindIII digestion. As a result, a plasmid construct with HPV-18 early region was obtained. The map of the plasmid is presented in
Expression cassette that includes synthetic 5′ intron element, codon optimised sequence encoding Gaussia luciferase marker gene, as well as bovine growth hormone polyadenylation signal, were inserted into the pUCHPV-18E so that the early region of the HPV-18 genome remained intact. The bacterial sequences were removed by HindIII digestion. As a result, a plasmid with HPV-18 early region was constructed, which carries a reporter gene enabling quantitative or semi-quantitative detection of extrachromosomal high-risk mucosal HPV-18 DNA. The map of the plasmid is presented in
The plasmid was made from the pUCHPV-18E-Gluc by insertion of the Herpes Simplex virus 1 (HSV 1) derived thymidine kinase (TK) promoter region in front of the Int-Gluc-bgh expression cassette. The bacterial sequences were removed by HindIII digestion. As a result, a plasmid with HPV-18 early region was constructed, which carries a TK promoter-regulated reporter gene enabling quantitative or semi-quantitative detection of extrachromosomal high-risk mucosal HPV-18 DNA. The map of the plasmid is presented in
Examples 4-6 present a HPV-based construct, where L1 and L2 genes have been removed and replaced with a reporter gene. Accordingly, a useful instrument for quantitative or semi-quantitative assessment of the amount of replicated extrachromosomal DNA is provided.
Constructs pMC-18L2-Rluc (SEQ ID NO:4) and pMC-18L2-Rluc-pA(SEQ ID NO:5) were cloned as parental plasmids for preparation of HPV18 markergenomes 18L2-Rluc (SEQ ID NO:6) and 18L2-Rluc-pA (SEQ ID NO:7), respectively (
The markergenomes were constructed by inserting markergene (Renilla luciferase (Rluc) in this particular example into the late region (L1 and L2 ORFs) of HPV18 genome downstream from the sequences needed for polyadenylation of the viral early transcripts. As cellular transcription factor binding sites containing heterologous promoter sequences could interfere the HPV gene we did not include any promoter into the markergene expression cassette. Instead of this, the Rluc cDNA was linked with human VCIP mRNA 5′UTR for promotion of the markergene expression. It has been demonstrated that VCIP mRNA 5′UTR contains internal ribosome entry site (IRES) functional in U2OS cells (Blais et al., 2006).
pMC18L2-Rluc
First, the VCIP mRNA 5′UTR product was amplified from genomic DNA of U2OS cells using primers VCIP_F_PpuMI (SEQ ID NO: 8) and VCIP_R_MCS (SEQ ID NO:9). The VCIP mRNA 5′UTR and Rluc cDNA (derived from Rluc expression vector as NcoI-NotI fragment) was joined in cloning vector pTZ57R/T (Fermentas, Lithuania) resulting pTZ-VCIP-Rluc. For generation of the pMC18L2-Rluc (SEQ ID NO:4), the VCIP-Rluc fragment (cut out with PpuMI and Esp3I from pTZ-VCIP-Rluc) was inserted into the parental plasmid pMC-HPV18 for wt HPV18 opened with restriction enzymes PpuMI and BbsI.
pMC18L2-Rluc-pA
Rluc cDNA with 3′-linked bovine growth hormone gene polyadenylation region (pA) (derived from Rluc expression vector as NcoI-PacI fragment) was joined with VCIP mRNA 5′UTR product in the cloning vector pTZ57R/T (Fermentas, Lithuania) resulting the plasmid TZ-VCIP-Rluc-pA. For generation of the pMC18L2-Rluc-pA (SEQ ID NO:5), the VCIP-Rluc-pA fragment (cut out with PpuMI and Esp3I) was inserted into the plasmid pMC-HPV18 opened with restriction enzymes PpuMI and BbsI.
We also constructed the parental plasmid pMC18-E1-Rluc-E2 (SEQ ID NO:10) of another type of the markergenome, 18-E1-Rluc-E2 (SEQ ID NO: 11) (
The constructions were made as follows: Rluc cDNA fused with 5′ nucleotides of E2 ORF (including the Psp1406I site in E2 ORF) and 3′ part of the 2A peptide coding sequence was amplified from Rluc expression vector using primers E2-Rluc_F_Psp1406 and Rluc2A_R_Eam (SEQ ID NO:12 and 13, respectively). Also, the 5′ end of the E2 ORF (including the AatII site in the E2 ORF) fused with 5′ part of the 2A peptide coding sequence was amplified from HPV18 genomic DNA using primers 2AE2—F_Eam and E2—R_AatII (SEQ ID NO: 14 and 15, respectively). The amplified fragments were joined in pUC57-kana cloning vector using the Eam1105I site present in 2A peptide coding sequence. Finally, the pMC18-E1-Rluc-E2 (SEQ ID NO: 10) was generated by insertion of the E2′-Rluc-2A-E2′ construction from pUC57 into the pMC-HPV18 using the Psp1406I and AatII cloning sites present in the E2 ORF.
Transient replication assay was performed in U2OS cells in order to test the replication capability of the constructed markergenomes in comparison with wt HPV18 genome. First, the wt HPV18 genome and 18L2-Rluc, 18L2-Rluc-pA, 18-E1-Rluc-E2 markergenomes were prepared from their parental plasmids (pMC-HPV18, pMC-18L2-Rluc, pMC-18L2-Rluc-pA and pMC-18-E1-Rluc-E2, respectively) by removing almost completely the bacterial backbone sequences using the method described in Kay et al., 2010. Then the U2OS-EGFP-Fluc cells (U2OS derived cell line expressing EGFP and firefly luciferase) were transfected with 1 μg of HPV18 genome or 1 μg of each markergenome or mock transfected (neg. control). Forty-eight and 72 hours after transfection the low molecular weight DNA was isolated from the cells, digested with the restriction endonuclease linearizing the HPV18 genome or markergenomes and with DpnI (destroys the unreplicated input DNA). The digested DNA samples were analyzed by Southern blotting using early region of the HPV18 genome as the probe. The results shown on the
Similarly to replication assay described in Example 9 above, the markergene expression assay was performed in U2OS-EGFP-Fluc cells in order to test the markergene expression capability of the 18L2-Rluc-pA and 18-E1-Rluc-E2 markergenomes. The U2OS-EGFP-Fluc cells were transfected with 1 μg of HPV18 genome negative control (contains no markergene) or 1 μg of each markergenome. Forty-eight and 72 hours after transfection the cells were lysed and activities of firefly luciferase (expressed by U2OS-EGFP-Fluc cells) and Renilla luciferase (expressed by markergenomes) were measured in lysates using Dual-Luciferase® Reporter Assay System kit (Promega, US). The results shown in
U2OS cell lines transfected with plasmids of Examples 7 and 8 were tested for induction of vegetative amplification of HPV DNA similarly as described in Example 3. The results (not shown) prove that the cell lines are supporting all replications phases of HPV DNA including the vegetative amplification phase, and therefore are useful in establishing an in vitro system for high throughput screening for drugs inhbiting HPV DNA replication during any one of the replication phases.
A kit was completed by combining human osteosarcoma cell line U2OS, extrachromosomally maintainable HPV DNA plasmid pUCHPV-18E-TKGluc wherein the L1 and L2 genes are substituted with Gaussia luciferase marker gene. This construct was transfected into the U2OS cell line, the stable cell lines identified and cultivated to confluency. Any library of chemical compounds available or generated by a person skilled in the art can be applied to the preconfluent and/or confluent cell culture to screen the provided compounds from the library for their anti-HPV activity at stable maintenance and/or amplificational stage of viral DNA replication. The Gaussia luciferase reporter gene works as a means for quantitative or semi-quantitative assessment of replicated extrachromosomal DNA, as the amount of the fluorescent product of the inserted gene is readily detectable for a person skilled in the art either quantitatively by measuring the fluorescence or semi-quantitatively by visual observation with fluorescence microscope. Similarly a kit was completed by using U2OS cell line and extrachromosomally maintainable plasmids pMC-18L2Rluc, pMC-18L2Rluc-pA and pMC18-E1-Rluc-E2 The skilled artisan will recognise that instead of HPV 18-genome, genome from another type of human papilloma virus may be used.
Complete or partial sequence of HPV DNA carrying all necessary viral cis-sequences and trans-factors necessary for all steps of viral replication cycles was introduced into human osteosarcoma cell line U2OS using electroporation or chemical transfection methods know in the art. The clones of U2OS cell lines that carry extrachromosomally replicating HPV plasmids was isolated using selection marker providing resistance to G418. The identified cell clones carrying different HPV copy numbers per cell were characterized, grown and cell banks of these cell clones were generated. The cells of the subclone chosen for the identification of HPV latent replication inhibitors were seeded at low density into 96 well plates, drug candidates at different concentrations were added to the growth media, and cells were grown until confluent. Alternatively cells may be seeded into 384 well plates to increase the throughput. As another, preferred option, the cell culture was maintained for at least 5 to 7 days on the plates to become confluent, the potential drug candidates were added to the growth medium after cells had become confluent. The copy-number of HPV extrachromosomal copies was determined in the cells by direct differential measurement of the viral DNA in the cells or using reporters. Subsequently, the compound under investigation was applied to the cultivation vessel of the U2OS cell clone monolayers; the presence or absence of the inhibitory effect of the compound on viral DNA stable or amplificational replication in the cells was assessed by measuring the amount of the product of the reporter gene or the amount of extrachromosomal DNA; finally the compound was identified as a candidate for HPV DNA replication inhibitor, if inhibitory effect on HPV DNA replication of a certain concentration of the compound at certain copy number level at certain growth phase is observed at certain growth conditions.
One skilled in the art will recognize that the examples above are illustrative and do not limit the scope of the invention. There are various ways of modifications that would fall under the spirit of this invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EE2010/000010 | May 2010 | EE | national |
This application is a continuation in part application of International Application Number PCT/EE2010/000010 filed on May 19, 2010 which is incorporated herein by reference in its entirety.
