Patent Application: US-53632406-A

Abstract:
the invention discloses a method of maintaining hepatitis c virus growing indefinitely in cell culture . the method includes providing a culture of cells susceptible to infection by hcv ; introducing to the cell culture an inoculum containing an infective dose of hcv ; contacting the inoculated cell culture with a growth medium containing an excess of uridine and cytidine ; and changing spent growth medium with fresh growth medium containing the excess of uridine and cytidine on a predetermined schedule .

Description:
compounds . interferon - a ( ifn - α ), aphidicolin , uridine , and cytidine were purchased from sigma . cyclosporin a ( cs a ) was purchased from alexis corporation ( san diego , calif .). 2 ′- c - methyl - adenosine ( 2cma ) was a gift from dr . steve carroll ( merck inc .). sirna . hairpin sirnas directed against the ns5b sequence of hcv 1b and the firefly luciferase gene were expressed in the hairpin format from a lentiviral vector as previously described ( 25 ). viral vectors were produced in 293 - t cells and viral transductions were done according to standard procedures . cell lines . clone b and huh . 8 cells were obtained from apath llc through the nih aids reagents program . the i / 5a - gfp cell line was provided by dr . charles rice . replicon cells were routinely maintained in dulbecco modified medium supplemented with antibiotics , 10 % fetal bovine serum , and 500 μg / ml g418 . huh - 7 cells were maintained in a similar medium but without the g418 . when replicon cells were treated with various hcv inhibitors , the g418 was also omitted from the medium . flow cytometry and cell cycle analysis . cells were routinely fixed in 2 % paraformaldehyde before flow cytometry analysis , which was performed with a facscanto flow cytometer ( bd biosciences ). live cell sorting was performed under sterile conditions with a facsaria flow cytometer ( bd biosciences ). the approximately 5 % of cells ( 2 × 10 7 cells total ) with the strongest gfp signal were sorted back for expansion at each round of live - cell sorting . for cell - cycle analysis , 1 × 10 6 cells were stained with 50 μg / ml propidium iodide in the presence of 200 μg / ml rnase a and 0 . 1 % triton x - 100 for 30 minutes before being subjected to facs analysis . microscopy . for confocal microscopy , the cells were seeded onto glass coverslips deposited in six - well plates . after treatment , cells were fixed in 4 % paraformaldehyde on the coverslip , which was then mounted on a glass slide for microscopic observations . fluorescence and dic images of the cells were examined with a zeiss lsm 510 laser scanning confocal microscope equipped with multi - photon laser and recorded with the accompanying lsmib - 3 . 2 software . antibodies and western blots . an anti - ns5a monoclonal antibody was purchased from virogen ( boston , mass .) and an anti - ku86 monoclonal antibody from sigma . a monoclonal antibody against gfp was obtained from clontech / bd biosciences . to detect membrane - associated hcv proteins , we directly lysed the replicon cells in sds page sample buffer . we used 2 × 10 5 cells per 160 μl of sds page sample buffer for all our western blotting experiments . blotting and detection were all performed according to standard procedures . for detection of ku86 signal , the membrane was first stripped of the ns5a signal by incubation in stripping buffer ( 100 mm 2mercaptoethanol , 2 % sds , 62 . 5 mm tris - hcl , ph 6 . 7 ) for 30 minutes at 50 ° c . rna extraction and northern blots . total rna was extracted with trizol reagent ( invitrogen , san diego , calif .). equal amounts of rna ( 10 μg per sample ) were loaded onto formaldehyde - containing agarose gels for electrophoresis . the transfer of rna onto a nitrocellulose membrane , random labeling of a radioactive probe , and hybridization were all performed according to standard molecular - biology protocols . the probe for detecting hcv rna is a cdna fragment that corresponds to nucleotides 8024 to 9563 of hcv 1 b . the exposure and detection of the radioactive signal was done with a storm 860 phosphor - imaging system ( amersham / ge healthcare ). a novel gfp - based flow cytometry assay for hcv replication . recently , a modified hcv replicon with the egfp gene inserted into the coding region of ns5a has been generated for study of the subcellular localization of the hcv replication complex ( 16 ). in this replicon cell line ( i / 5a - gfp ), the ns5a - gfp fusion protein exhibits properties of both the fluorescent protein and the hcv ns5a as it supports hcv replication and renders the cells harboring the hybrid replicon fluorescent under a fluorescence microscope . when we subjected i / 5a - gfp cells to flow - cytometry analysis , we found the gfp signal of the majority (˜ 70 %) of the cells to be indistinguishable from that of clone b , a replicon cell line without gfp but containing the same s2204i mutation in the ns5a gene ( 1 ) ( fig1 ). to obtain a cell line with minimal facs - profile overlap with the gfp - negative replicon cells , we then performed consecutive rounds of cell sorting on i / 5a - gfp - 6 cells in which the cells with the strongest gfp were recovered . a population with no greater than 15 % gfp - negative cells was obtained after four rounds of sorting and was used for the present study . we designated these cells gs4 ( fig1 ). immunoblotting analysis using both anti - ns5a and anti - gfp antibodies revealed only one species of ns5a - gfp hybrid protein in these cells , indicating that no green fluorescence was contributed by unfused or truncated gfp proteins ( fig2 ). when examined under a fluorescence microscope , the green fluorescence of the gs4 cells exhibited the characteristic punctuate subcellular localization of hcv nonstructural proteins ( fig3 ). the gfp signal of these cells was then analyzed by flow cytometry 4 days after treatment with increasing amounts of inhibitors , including interferon - α ( ifn - α ), 2 ′- cmethyl - adenosine ( 2cma ), and cyclosporin a ( cs a ). the inhibitors effectively reduced the percentage of gfp positive cells as well as the mean gfp intensity of the treated population ( fig4 ). results for both parameters showed good dosage - dependent inhibition by the inhibitors ( fig5 ). the 1050 ( 0 . 25 μm for 2cma , 0 . 5 μg / ml for csa , and 10 - 25 units / ml for ifn - α ) obtained with this assay closely resemble results from previous studies where hcv rna level was measured ( 2 , 6 , 19 , 26 ). we next determined whether a sirna whose direct target is the hcv genome could suppress ns5a - gfp expression in gs4 cells by degrading viral rna . we constructed lentiviral vectors that expressed the hairpin forms of sirnas whose targets were either luciferase or the ns5b gene as previously described ( 25 ). the ns5b sirna had been shown to inhibit rna replication by targeting the hcv replicon rna for degradation ( 27 ); the luciferase sirna served as a negative control . when expressed in the gs4 cells , the ns5b sirna , but not the luciferase sirna , specifically inhibited ns5a - gfp expression ( fig6 ). we consistently observed 50 - 60 % inhibition with the sirna , corresponding to the transduction efficiency of the replicon cells with our lentiviral vectors ( fig7 and data not shown ). taken together , these results show that the gs4 cell line is a suitable indicator cell line for hcv replication and that the flow cytometry - based assay can be used to study replicon inhibition in vitro . serum starvation does not inhibit hcv expression as cell confluency does . we applied this assay to test a number of non - drug - based treatments that had been reported to inhibit hcv replication in vitro . these included cell confluency , 39 ° c . incubation , and serum starvation . gs4 cells were treated for 4 days before facs analysis . to our surprise , even though cell confluency and 39 ° c . treatment efficiently inhibited replicon as previously reported ( 7 , 20 , 22 , 31 ), serum starvation had no effect even when cells were maintained in culture medium with 0 % serum for 96 hours , after which the replicon cells started to die off ( fig8 ). lack of inhibition of ns5a - gfp expression by serum starvation was also confirmed by fluorescence microscopy ( fig9 ). to rule out the possibility that the failure of serum starvation to suppress replicon expression is a result of our multiple rounds of cell sorting and specific to this gfp - containing replicon , we repeated the experiment using a different replicon cell line that does not contain gfp and had not been subjected to cell sorting . cell line 1b is a replicon clone derived from a hcv - n based replicon ( 6 , 9 ). we subjected these cells to serum starvation for 4 days and then detected ns5a expression using western blot . decreasing amount of serum had no effect on the ns5a level , whereas confluency had a dramatic inhibitory effect ( fig1 ). on the other hand , serum starvation had a significant effect on cell growth ( fig1 ). note that 1 % or no serum had a greater effect on cell growth than did confluency yet had no effect on ns5a expression . cell cycle perturbation failed to suppress hcv replication and expression . failure of growth arrest to inhibit replicon expression prompted us to examine the effect of cell - cycle perturbation on replicon expression . propidium iodide ( pi ) staining of the cells permitted acquisition of dual fluorescence facs profiles showing the cell - cycle distribution of gfp - positive and - negative cells . we found no correlation between positive gfp signal and any of the cell - cycle stages in the cycling cells ( fig1 , no treatment ). incubation at 39 ° c . inhibited ns5a - gfp expression without any effect on cell - cycle profile . more importantly , although cell confluency inhibited hcv expression , aphidicolin failed to do so even though it effectively arrested cell cycle at the g1 / s transition as expected ( fig1 ). even though confluency also affected cell cycle to a certain degree , its effect on cell - cycle progress was far less dramatic than that of aphidicolin . to eliminate the possibility that these growth - arrest treatments somehow stabilize the gfp and maintain it throughout the treatment , we took advantage of our finding that 39 ° c . treatment results in reversible inhibition of hcv replication in our assay . we first eliminated the gfp signal from gs4 cells by incubating them at 39 ° c . for 4 days and then transferred the cells back to 37 ° c . so that hcv replication could recover . as shown in fig1 , over a recovery period of 6 days at 37 ° c ., more than 40 % of the cells regained the gfp signal , indicating hcv replication and viral protein expression had resumed . we tested aphidicolin and serum starvation using this assay along with ifn - α . although ifn - a efficiently blocked the recovery of the ns5a - gfp expression in this assay , aphidicolin and serum starvation again did not prevent the resumption of hcv replication , which led to the recovery of ns5a - gfp expression ( fig1 ). measurement of cell number in these samples during the course of the recovery indicated that aphidicolin and serum starvation effectively blocked cell growth as expected ( fig1 ). taken together , these results show that neither serum starvation nor aphidicolin - induced cell - cycle arrest can produce the same inhibitory effect exerted by cell confluency . reversal of confluency - mediated replicon inhibition by exogenous nucleosides . inhibitors of de novo synthesis of nucleosides can suppress hcv replication ( 23 ), suggesting that lowered nucleoside level in confluent cells accounts for the reduction in viral rna level . we tested this hypothesis by adding exogenous nucleosides to the culture media of confluent gs4 cells and then comparing the ns5agfp expression with that of confluent cells without added nucleosides . different nucleosides were tested , and the combination of uridine and cytidine ( uc , 50 - 200 μm each ) was found to rescue hcv expression to various degrees . a nearly complete reversal of the inhibitory effect was observed when the cells were kept in the confluency state for up to 4 days in the presence of 200 μm of uc , as demonstrated by both flow cytometry and fluorescence microscopy ( fig1 and 17 ). the reversal is evident throughout the duration of the experiment ( 6 days after confluency ) although longer incubation time in the confluent state caused the percentage of gfp - positive cells to decrease even in the cells to which uc had been added . to validate our results at the viral rna level , we repeated the inhibition and rescue experiments and directly examined hcv rna by northern blotting . gs4 cells were treated with various inhibitors / agents for 3 days before the total rnas were subjected to northern analysis for hcv rna . 2cma and cell confluency effectively eliminated hcv rna signal ( fig1 , top panel , lanes 2 and 6 ), whereas serum starvation had no effect ( lane 4 ). aphidicolin also only had a minimal effect , not enough to explain the confluency - based inhibition ( lane 3 ). importantly , exogenously added uc partially rescued rna replication from confluency - based inhibition ( compare lane 7 to lane 6 ). these results confirm that previous results obtained at the gfp expression level reflect hcv rna replication . additional experiments with a replicon cell line containing a wild type hcv sequence . we carried out additional experiments in a replicon cell line that does not contain the two modifications of gs4 cells : the s2204i mutation and the gfp insertion in ns5a . huh . 8 is replicon cell line that contains the wild - type sequence of ns5a ( 1 ). huh . 8 cells were cultured under various conditions for 4 days before being analyzed by western blot for ns5a expression . as a positive control , 1 μm of 2cma effectively eliminated any ns5a expression ; aphidicolin and serum starvation again did not inhibit hcv expression ( fig1 ). serum starvation actually appeared to have increased ns5a expression , as it did with the gs4 cells ( fig8 and data not shown ). in contrast , confluency inhibited ns5a expression , and more importantly , the exogenously added uc again counteracted this inhibition ( fig1 , compare lanes 6 and 7 ). these data indicate that the lack of hcv inhibition by serum starvation and aphidicolin and the mechanism of confluency - based viral inhibition are not results of the modifications found in gs4 cells . we have previously used a gfp - based cell - sorting assay to identify cellular proteins involved in hiv replication ( 25 ) and wished to develop a similar assay for hcv replication in vitro . taking advantage of a modified hcv replicon with the egfp gene inserted into the coding region of ns5a ( 16 ), we obtained a derivative cell line that we named gs4 by live cell sorting and expansion . the gs4 cells we have developed have a much higher level of hcv expression than that of the original i / 5a - gfp cells yet still respond very well to inhibitors . even though the stronger gfp signal also makes it much easier to visualize the fluorescence under a microscope , the real strength of the assay based on the gs4 cells lies in its amenability to flow - cytometry analysis . the assay is simple and yet quantitative . no cell lysis or addition of exogenous substrates is needed . it is also rapid , and the results are visual . the speed of flow - cytometry analysis makes the test very fast , and many samples can be processed in parallel . it measures and displays results from individual cells , eliminating the need to normalize the results to cell numbers in different samples . this feature is especially important in situations where antiviral effects must be distinguished from those of cytostasis , for example in the case of cell confluency . most importantly , the more favorable flowcytometry profile ( minimized overlapping between the positive and the negative populations ) makes it more suitable for screening projects using a reverse genetics approach , where distinct populations with desired fluorescence intensities can be recovered by cell sorting . in the study reported here , we applied the assay to examine the effect of cell growth on hcv replication and the mechanism of cell confluency based inhibition of hcv rna . the most straightforward mechanism for confluency - mediated inhibition is that exponential cell growth is required for efficient hcv replication . indeed , other means of arresting cell growth have also been shown to inhibit hcv replication to various degrees ( 17 , 22 ). in one study , serum starvation reduced the rna abundance in a replicon cell harboring a selectable full - length hcv rna based on the genotype 1b hcv - n , whereas aphidicolin treatment , which arrested the cell cycle as expected , had no effect on the abundance of hcv rna or protein in these cells ( 22 ). in contrast , murata et al . ( 17 ) observed an inhibitory effect by aphidicolin on hcv replicon in their report of tgf - β as an inhibitor of hcv replication in vitro . during the course of our study , we found that serum starvation or aphidicolin failed to inhibit hcv rna replication or protein expression without killing the replicon cells , whereas cell confluency did so very effectively . tgf - β had only a marginal effect in our system ( data not shown ). the reason for the difference in the sensitivity of different replicons to these different treatments is not clear . properties of specific replicons and sensitivities of different assays were some of the possibilities . we performed these experiments in three different replicon lines and used various assays including flow cytometry , fluorescence microscopy , western blotting , and northern blotting , and all gave similar results . in any event , because the replicon cells that we used here did respond as expected to cell confluency , we concluded that growth arrest was not the general mechanism of confluency - based viral inhibition . note also that , because all these treatments significantly inhibit cell growth , the cell numbers at the time of the assay are dramatically lower in the treated group , making accurate normalization , for distinguishing antiviral from cytostatic effects , difficult . because our flow cytometry - based assay looks at the hcv protein expression in individual cells , it does not need the normalization . a second hypothesis , not mutually exclusive with that of growth arrest , is that the reduced intracellular pools of nucleosides account for the decrease of hcv rna in confluent cells because these cells depend on salvage nucleoside biosynthesis pathways and thus have a lower level of intracellular ntps . we reasoned that , if this were the case , adding back exogenous nucleoside should alleviate the block on hcv replication and rescue viral expression . our results indicate that this was indeed at least partly the case , as exogenously added uridine and cytidine rescued hcv rna replication and protein expression in confluent cells of several different replicon lines . these exogenous nucleosides probably rescue hcv replication by increasing the level of building blocks for the synthesis of viral genome rna . the failure of the nucleosides to reverse completely the inhibitory effect posed by confluency beyond four days suggests that additional mechanisms exist for confluency - based hcv inhibition . the finding that serum starvation and cell confluency have drastically different effects on steady - state hcv replicon rna level is somewhat surprising , as they both induce a cytostatic state of the cells that is believed to be detrimental to hcv replication . our results nevertheless suggest that serum starvation and confluency cause distinct changes in the physiological state of the cells that is relevant for hcv replication . the results with exogenously added nucleosides imply that serum starvation does not result in the reduction of intracellular ntp pools as confluency does , at least not to the extent that it limits hcv replication . cell growth / cycle arrest induced by serum starvation or aphidicolin has been reported to increase replication / infection of other viruses ( 5 , 15 ). the transcription of the borna disease virus ( bdv ), a negative - stranded rna virus , was enhanced by serum starvation . interestingly , the rna species that was up - regulated the most was the 1 . 9 - kb rna without the 5 ′ cap and 3 ′ poly ( a ) tail , similar to the hcv genomic rna ( 15 ). the finding that serum starvation can increase hcv expression under certain culture conditions is intriguing . the dynamics of hcv replication in response to cellular environment may be relevant in vivo , as detection of hcv in pathologic samples has been variable , possibly because replication levels fluctuate . the exquisite sensitivity of hcv replication to intracellular nucleoside level may provide an explanation for the difficulty of detecting and recovering large quantities of viruses in vivo . hence , the present invention includes a method of maintaining hepatitis c virus ( hcv ) replicating indefinitely in cell culture , the method comprising providing a culture of cells susceptible to infection by hcv , introducing to the cell culture an inoculum containing an infective dose of hcv , contacting the inoculated cell culture with a growth medium containing uridine and cytidine , and adding exogenous uridine and cytidine to avoid lowered levels of nucleosides in the cell culture . accordingly , in the drawings and specification there have been disclosed typical preferred embodiments of the invention and although specific terms may have been employed , the terms are used in a descriptive sense only and not for purposes of limitation . the invention has been described in considerable detail with specific reference to these illustrated embodiments . it will be apparent , however , that various modifications and changes can be made within the spirit and scope of the invention as described in the foregoing specification and as defined in the appended claims . 1 . blight , k . j ., a . a . kolykhalov ., and c . m . rice . 2000 . efficient initiation of hcv rna replication in cell culture . science 290 : 1972 - 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