Isolation of Fidelity Variants of RNA Viruses and

Characterization of Virus Mutation Frequency (2011)

Stéphanie Beaucourt, Antonio V. Bordería, Lark L. Coffey, Nina F. Gnädig, Marta Sanz-Ramos, Yasnee Beeharry, and
Marco Vignuzzi

Ovo je onaj 2011. Rad u kojem se objašnjava metodologija. Nisu koristili 5-FU za dobivanje polimeraze,
samo za provjeru rezistencije na druge mutagene. Puna crta je wild type, isprekidana je Coxsackie A372V.
Rađeno na HeLa stanicama. Radili su prvo provjeru citotoksičnosti stanica, ali ne piše koje koncentracije
su ok za stanice, ali očito do 200 µM, supernatant skupljen 48h p.i.)
Fidelity Variants of RNA Dependent RNA Polymerases
Uncover an Indirect, Mutagenic Activity of Amiloride
Compounds (2010)
Laura I. Levi, Nina F. Gnädig, Stéphanie Beaucourt, Malia J. McPherson, Bruno Baron, Jamie J. Arnold,
and Marco Vignuzzi

RNA mutagen, amiloride compound and cation assays

HeLa cell monolayers in 6-well plates were pretreated for 2 hours (ribavirin, AZC, FU, NaCl, CaCl, MgCl2,
or MnCl2) or 10 hours (amiloride compounds) with different concentrations of compound as indicated.
We chose and verified concentrations of compounds that were not toxic to cells over a 72 hours period.
For amiloride compounds, we chose and confirmed concentrations corresponding to virus inhibitory
concentration (IC50) values that were not toxic to cells, as determined by Harrison et al.. Cells were then
infected at an MOI = 0.01 with passage 2 virus. 48 hours post-infection, virus was harvested by one
freeze-thaw cycle and virus titers (TCID50 or plaque assay) were determined.
Isolation of RNA mutagen resistant CVB3. CVB3 was passaged 20 times in 50 µM ribavirin, 50 µM AZC or
mock treated HeLa cells. Every 5 passages the virus population was sequenced. The emergence of a
single point mutation resulting in a A372V change in the RdRp is indicated by a solid circle. (C) A372V is
resistant to three different base analog RNA mutagens. HeLa cells treated with 300 µM of ribavirin or
AZC, or 150 µM FU, were infected with either wild type CVB3 (black bars) or A372V variant (gray bars) at
an MOI of 0.01. Control infections (no drug) were also performed. At 48 hours after infection, progeny
virus was titered by TCID50 assay.

The role of A372V in resistance was

confirmed by introducing the
mutation back into the CVB3 wild
type cDNA infectious clone and
comparing the growth of A372V to
wild type virus treated with 3 RNA
mutagens with different nucleotide
structures: ribavirin (300 µM), AZC
(300 µM) and 5-fluorouracil (FU, 150
µM).
Effective Lethal Mutagenesis of Influenza Virus by Three
Nucleoside Analogs (2015)
Matthew D. Pauly and Adam S. Lauring

• RBV nucleoside analog, 5-FU base analog

• The base analog 5-fluorouracil is processed intracellularly into a nucleoside analog and has
demonstrated activity as a lethal mutagen against LCMV, exonuclease-deficient coronaviruses,
and FMDV in vitro
• 5-Fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) (Sigma-Aldrich F6627) was dissolved in DMSO
at 384 mM. Aliquots of all stocks were stored at −20°C.

The viability of MDCK cells after drug treatment was measured using MTT assay. Briefly, 24-well plates were seeded
with 20,000 MDCK cells in 500 μl of medium. The following day the medium was replaced with mutagen medium.
Viability was measured 24 h later.

Cytotoxicity was measured using the CytoTox-Glo cytotoxicity assay (Promega G9290), according the manufacturer's
protocol. Briefly, 3,200 MDCK cells per well were seeded in a white, flat-bottom, 96-well plate (Fisher 353296). Drugs
were diluted in viral infection medium and added to the cells as described above. Luminescence was measured after
24 h using a Synergy HT microplate reader both before and after digitonin permeabilization of the cell membrane.
 Nucleoside analogs have structural similarity to cellular nucleosides and may reduce viral titer through pleiotropic
effects on cellular polymerases and metabolic pathways. We investigated this possibility by quantifying both direct
cytotoxicity and the effect of each drug on cellular viability. Cellular viability is distinct from direct cytotoxicity, as it
reflects both cell proliferation and cell death over the assay period. We assayed relative cell viability using the MTT
assay, which measures mitochondrial succinate dehydrogenase activity. The CytoTox-Glo assay, which quantifies the
release of cellular proteases, was used to assay drug-induced cytotoxicity.

Using the MTT assay, we found a modest decrease in cellular viability in both 120 μM ribavirin and 480 μM 5-
fluorouracil and a 50% decrease in cellular viability in 20 μM 5-azacytidine.
Using the protease release assay, we determined that direct cytotoxicity was minimal for both ribavirin and 5-
fluorouracil up to the maximal concentrations tested (60 μM and 200 μM, respectively).
Given the relatively small reductions in cellular viability at the doses used, the large decreases in viral titer observed
with each of the three nucleosides are unlikely to be due to drug-associated cytotoxicity.

• When virus was treated with 20 μM ribavirin, there was a greater-than-5-fold reduction in
specific infectivity, which persisted at higher concentrations. We found that treatment with 12.5
μM 5-azacytidine was sufficient to cause a greater-than-10-fold decrease in specific infectivity,
with larger reductions at higher drug concentrations. Similar reductions in specific infectivity
were achieved at 100 μM 5-fluorouracil. These reductions in specific infectivity are consistent
with a mutagenic mode of action for each of the nucleoside analogs:
Arbovirus high fidelity variant loses fitness in mosquitoes
and mice (2011)
Lark L. Coffey, Yasnee Beeharry, Antonio V. Bordería, Hervé Blanc, and Marco Vignuzzi

Ništa ne pišu o citotoksičnosti na stanicama.

By serial passage of CHIKV in ribavirin or fluorouracil (5-FU), we selected a mutagen-resistant variant

with a single amino acid change (C483Y) in the nsP4 RdRp gene that increases replication fidelity. In a
mosquito host, Aedes aegypti, high fidelity virus presents lower infection and dissemination titers than
wild type. Higher fidelity CHIKV also produces truncated viremias and lower organ titers compared with
wild type in newborn mice. These results suggest that increased replication fidelity and reduced genetic
diversity impose a fitness cost in invertebrate and vertebrate arbovirus hosts.

black – untreated

white – RBV (100 μM)

gray – 5-FU (77 μM)

Wild-type (WT) CHIKV rescued from an infectious clone was serially passaged 20 times in the presence or absence of 100 μM
ribavirin or 10 μg/mL 5-FU (77 μM). After one passage (p1), mean ribavirin- and 5-FU–treated CHIKV titers were reduced by
≈2.0 logs (P < 0.0 01) compared with CHIKV titers for untreated replicates. By p5, the mutagen-treated CHIKV resisted
treatment with both compounds: No significant reduction in titer was observed for ribavirin-treated CHIKV, and 5-FU–treated
CHIKV achieved significantly higher titers than untreated CHIKV (P < 0.007). This phenotype was observed through the series to
p20, indicating that mutagen resistance had been generated.
Resistance of CHIKV WT and C483Y to mutagens. Matched titers of WT (black line) and C483Y (gray line) were inoculated in
triplicate in HeLa cells with increasing concentrations 5-FU
Lethal mutagenesis of the prototypic arenavirus
lymphocytic choriomeningitis virus (LCMV) (2003)
Carmen M. Ruiz-Jarabo,a Calvin Ly,b Esteban Domingo,a and Juan Carlos de la Torre

FU (Sigma) and Rib (gift from R. Robins) were dissolved in DMEM to yield stock solutions of 2.5 mg/ml
and 10 mM, respectively. These solutions were sterilized by filtration (0.22 m membrane filter, Costar).
Stock solutions were aliquoted, stored at -20°C and diluted as needed.

We first examined the effect of increasing amounts of FU and Rib on the viability of BHK21 cells (Fig. 1).
For this, we treated BHK-21 cells with increasing concentrations of FU or Rib, and at the indicated post-
exposure times cell viability was determined by staining with the supravital dye neutral red and
exclusion of the dye trypan blue. Cells treated with FU concentrations of 50 or 200 g/ml exhibited more
than 90 and 80%, respectively, viability after 48 h treatment. Increasing the concentration of FU to 800
g/l resulted in significant levels (more than 30%) of cell death. Consistent with a previously reported low
cell toxicity of Rib (Huggins et al., 1984), all the Rib concentrations we tested had only minimal effects
on cell survival after 48 h treatment (Fig. 1).

FU (Sigma) and Rib (gift from

R. Robins) were dissolved in
DMEM to yield stock solutions
of 2.5 mg/ml and 10 mM,
respectively. These solutions
were sterilized by filtration
(0.22 μm membrane filter,
Costar). Stock solutions were
aliquoted, stored at
20°C and diluted as needed.

BHK-21 cell viability in the presence of FU and Rib. BHK-21 cells were seeded (1 x 105 cells/well) on M6-well plates. 12h later,
cells were treated with the indicated concentrations of FU or Rib. At the indicated times after drug exposure, numbers of viable
cells were determined as described in Material and Methods. Percent cell viabilities were normalized with respect to the
corresponding untreated control cells. Values represent the average (+/-SD) of two independent experiments, with each time
point done in triplicate.

50 μg/ml = 385 μM 200 μg/ml = 1538 μM 800 μg/ml = 6154 μM

At the concentrations tested, Rib had a more severe effect than FU on viral RNA synthesis.

For these studies we used FU and Rib at concentrations of 1538 μM (200 μg/ml) and 400 μM,
respectively. These drug concentrations were selected based on their effects on cell survival and LCMV
multiplication during one-step growth.
An increased replication fidelity mutant od foot-and-mouth
disease virus retains fitness in vitro and virulence in vivo
(2013)
Jianxiong Zeng, Haiwei Wang, Xiaochun Xie, Decheng Yang, Guohui Zhou, Li Yu
Coronaviruses Lacking Exoribonuclease Activity Are
Susceptible to Lethal Mutagenesis: Evidence for
Proofreading and Potential Therapeutics (2013)
Everett Clinton Smith, Hervé Blanc, Marco Vignuzzi and Mark R. Denison

Murine astrocytoma delayed brain tumor cells (DBT cells)

Vero

Compounds and cell viability studies

5-fluorouracil (5-FU) was made as 200 mM stock solutions in DMSO. Low concentration (µM) working
stocks were prepared as needed in sterile water prior to dilution in DMEM. Viability of DBT and Vero cells
was assessed using CellTiter-Glo (Promega) in 96-well plate format according to manufacturer's
instructions. DBT and Vero cells were seeded into opaque tissue culture grade 96-well plates, and DMEM
containing 5-FU was added to each well to achieve the concentrations indicated. Water or DMSO vehicle
controls were performed, in addition to a 20% ethanol control for cell death. The cells were then
incubated at 37°C for either 12 or 24 h, and cell viability was determined using a Veritas Microplate
Luminometer (Promega). The resultant values were then normalized to untreated cells.

Drug sensitivity studies and plaque assays

Subconfluent monolayers of DBT cells in 6-well plates were pretreated for 30 min at 37°C with 1 mL of
DMEM containing vehicle or the indicated concentration of RBV, 5-FU, MPA, or GUA. The drug was then
removed and cells were infected with MHV-ExoN+ or ExoN− viruses at an MOI of 1 plaque forming units
(PFU)/cell (single-cycle) or 0.01 (multi-cycle) for 30 min at 37°C. Virus was then removed and 1 mL of
DMEM containing vehicle, RBV, 5-FU, MPA, or GUA was added to each well. Cells were then incubated
at 37°C for either 12 (single-cycle) or 24 (multi-cycle) h. The supernatant was harvested and virus titer
was determined by plaque assay on DBT cells. For SARS-CoV studies, subconfluent monolayers of Vero
cells in T25 flasks were pretreated for 30 min at 37°C with DMEM containing vehicle, RBV, or 5-FU. The
drug was removed and cells were infected with either SARS-ExoN+ or ExoN− viruses at an MOI of 0.1
PFU/cell (single-cycle) for 30 min. The virus was removed and DMEM containing vehicle, RBV, or 5-FU
was added back. Cells were then incubated for 24 h, at which point the supernatant was harvested and
virus titer was determined by plaque assay on Vero cells. All treated samples were normalized to the
untreated vehicle control, and values were expressed as fold change from untreated virus titers.
5-FU na DBT stanicama 12h p.i.

SC - MOI 0.1 MC - MOI 0.01

5-FU na Vero 24h p.i.

24h p.i. skupljen supernatant

Effects of 5-Fluorouracil on Morphology, Cell Cycle,
Proliferation, Apoptosis, Autophagy and ROS Production
in Endothelial Cells and Cardiomyocytes (2015)
Chiara Focaccetti, Antonino Bruno, Elena Magnani, Desirée Bartolini, Elisa Principi, Katiuscia Dallaglio,
Eraldo O. Bucci, Giovanna Finzi, Fausto Sessa, Douglas M. Noonan, and Adriana Albini

Human umbilical vein endothelial cells (HUVECs)

Human cardiac myocytes (HCMs)

Human colorectal cancer (CRC) cell lines HCT-116 and HT29

Murine CT26 CRC cells

Cell proliferation assay

HUVECs were seeded on 1% gelatin coated tissue culture plates at 2x103 cell/well in 96-well plate. HCMs
were plated at the same density in their specific growth medium. HCT-116 and CT26 were seeded at
1x103 cells/well, HT29 at 3x103 cells/well, in 96-well plates. After a 24 hour incubation in their respective
media to allow for cell attachment, the medium was changed and the cells were treated with serial 10-
fold dilutions (10 nM to 1 mM) of 5-FU dissolved in DMSO. As negative controls, growth medium with
DMSO alone were used, 0.1% saponin was the positive control. All conditions were incubated for up to
96 hours and 6–8 replicates were done for each time-point. The MTT reagent was added to a final
concentration of 0.5 mg/ml to each well. After 5 hours incubation at 37°C, medium was removed,
formazan crystals were dissolved with 100 μl of DMSO and absorbance read at 570 nm in a micro-plate
reader. Non-viable cells are unable to reduce the MTT dye, giving an indirect measure of 5-FU effects on
cell number. The concentration of drug that reduced cell proliferation by 50% (EC50) was calculated by
non-linear regression fit using GraphPad Prism.
Cytostatic/cytotoxic effects of 5-FU on cardiomyocytes and endothelial cells.

The cytostatic effect of 5-FU on HCMs (A) was not marked but statistically significant starting at 48 hours and from 10 μM 5-FU.

The inhibitory effect of 5-FU was significant in endothelial cells (B) already after 24 hours at the intermediate concentration of
10 μM.

The inhibition of cell viability was maintained up to 96 hours. Drug efficacy was also tested on the HT29 and HCT116 colorectal
cancer cell lines (C, D). Mean ± S.E.M. of four different experiments for each cell type is shown.
Cytotoxic and Cytokinetic Effects of Thymidine, 5-
Fluorouracil, and Deoxycytidine on HeLa Cells in Culture
(1981)
Jerrold Fried,2 Amaury G. Perez, Jeffrey M. Doblin, and Bayard D. Clarkson

Chart shows the survival of cells exposed to FUra at 10-5, 5 x 10-5, and 10-4 M for up to 3 days