Remédio
para hepatite C é a aposta da Fiocruz para tratar zika
Os
vírus da zika e da hepatite C têm enzima chamada RNA polimerase, que ajuda a
multiplicação. Agente antiviral sofosbuvir age sobre essa enzima.
Um
remédio contra a Hepatite C é a mais nova aposta dos cientistas pro tratamento
da zika. O estudo é coordenado pela Fundação Oswaldo Cruz.
Foram
apenas oito meses de pesquisas e um resultado que pode salvar vidas.
A
descoberta é de pesquisadores da Fiocruz, do Instituto D'Or, da UFRJ, do Instituto
Nacional de Doenças Negligenciadas, em parceria com indústrias farmacêuticas.
Thiago
Moreno Lopes e Souza, especialista em antivirais, coordena o estudo na
CTDS/Fiocruz, partiu da semelhança entre os vírus da zika e da hepatite C. Os
dois têm uma enzima chamada RNA polimerase, que ajuda a multiplicar o vírus.
Sofosbuvir - já usado no tratamento da hepatite C - age exatamente sobre essa
enzima.
“Trabalhando
com remédios que já são aprovados, a gente consegue realizar essa pesquisa de
uma maneira mais rápida, mais acelerada. E a gente tem no sofosbuvir um
potencial novo, um potencial remédio que poderia ser adaptado para a utilização
em zika”,
O
próximo passo foi testar esse medicamento em laboratório contra o vírus da
zika. E o resultado foi mesmo da hepatite C. O teste foi feito em seis tipos de
células. Em cinco, o remédio bloqueou a multiplicação e eliminou o vírus da
doença, além de ajudar a recuperar células que foram infectadas e preservar as
que estavam saudáveis
A
pesquisa foi publicada nesta quarta-feira (18) na revista científica inglesa,
“Scientific reports” (disponível abaixo). E entre todos os medicamentos já
testados contra zika, esse tem a grande vantagem de atuar diretamente no vírus,
sem afetar o funcionamento da célula.
O
estudo mostra que o remédio provoca tantas mutações no vírus que ele não
consegue mais se multiplicar.
Mais
uma boa notícia nessa luta do mundo contra uma doença que deixou uma geração
com sequelas da microcefalia.
“A
gente imagina até o segundo semestre terminar os ensaios em animais de
laboratório. É um primeiro passo de uma esperança no combate à zika”, diz o
coordenador do estudo.
Os
pesquisadores esperam começar os testes com pacientes no fim do ano ou no
início de 2018.
The clinically approved
antiviral drug sofosbuvir inhibits Zika virus replication
· […]
· Scientific
Reports 7, Article number: 40920 (2017)
· doi:10.1038/srep40920
Received:
18
October 2016
Accepted:
13
December 2016
Published
online:
18
January 2017
Abstract
Zika virus (ZIKV) is a
member of the Flaviviridae family, along with other agents of
clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses.
Since ZIKV causes neurological disorders during fetal development and in
adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for
use against HCV and targets the protein that is most conserved among the
members of the Flaviviridae family, the viral RNA polymerase.
Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting
conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in
different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma
(SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to
the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir
also induced an increase in A-to-G mutations in the viral genome. Together, our
data highlight a potential secondary use of sofosbuvir, an anti-HCV drug,
against ZIKV.
Introduction
Zika virus (ZIKV) is a
member of the Flaviviridae family, which includes several
agents of clinical significance, such as dengue (DENV), hepatitis C (HCV), West
Nile (WNV) and Japanese encephalitis (JEV) viruses. This emerging pathogen is
an enveloped positive-sense single-stranded RNA virus. Although ZIKV is an
arthropod-borne virus (arbovirus) transmitted by mosquitos of the genus Aedes1, transmission through
sexual contact has been described2. The perception that ZIKV
causes a mild and self-limited infection3,4 has been jeopardized
in recent years, with outbreaks in the Pacific Islands and the Americas1,5. For instance, ZIKV spread
explosively across the Brazilian territory and to neighboring countries in
2015, infecting more than 4 million people6. As the ZIKV epidemic has
scaled up, the virus has been associated with congenital malformations,
including microcephaly, and a broad range of neurological disorders in adults,
including Guillain-Barré syndrome (GBS)7,8. These morbidities
associated with ZIKV infection led the World Health Organization (WHO) to
declare the Zika outbreak to be a public health emergency of international
concern.
Antiviral treatments against
ZIKV are therefore necessary not only to mitigate ZIKV-associated morbidities
but also to impair the chain of transmission. Some broad-spectrum antivirals,
such as interferons (IFNs), ribavirin and favipiravir, are not suitable for use
against ZIKV because they can be harmful to pregnant women9. Alternatively, others
have studied the use of Food and Drug Administration (FDA)-approved small
molecule drugs against ZIKV10,11,12,13,14,15. Overall, these drugs
might exert their anti-ZIKV activity at least in part by interfering with the
cellular pathways important for ZIKV replication10,11,12,13,14,15.
The gene encoding the viral
RNA polymerase shows the highest degree of conservation among the members of
the Flaviviridae family16, and the clinically
approved anti-HCV drug sofosbuvir targets this protein. Sofosbuvir is an
uridine nucleotide prodrug, which is triphosphorylated within cells to target
the viral RNA polymerase17. Sofosbuvir is a class B
FDA-approved drug. Moreover, Australia’s regulatory agency on drug
administration, the Therapeutic and Goods Administration (TGA), categorizes
sofosbuvir as class B1: “Drugs which have been taken by only a limited number
of pregnant women and women of childbearing age, without an increase in the
frequency of malformation or other direct or indirect harmful effects on the
human fetus having been observed.” Altogether, this information motivated us to
investigate whether the chemical structure of sofosbuvir possesses anti-ZIKV
activity. In the interest of disseminating public health information, we
disclosed a preprint of our data showing the anti-ZIKV activity of sofosbuvir18. In the present
investigation, we further studied the pharmacology of sofosbuvir in neuronal
and non-neuronal cell types. We observed a direct inhibition of the viral RNA
polymerase and an increase in A-to-G mutations in the viral genome due to
sofosbuvir treatment, highlighting a potential secondary use of sofosbuvir.
Materials and Methods
Reagents
The antiviral sofosbuvir (β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine) was donated by the BMK
Consortium: Blanver Farmoquímica Ltda; Microbiológica Química e Farmacêutica
Ltda; Karin Bruning & Cia. Ltda,
(Taboão da Serra, São Paulo, Brazil). Ribavirin was received as a donation from
the Instituto de Tecnologia de Farmacos (Farmanguinhos, Fiocruz). Sofosbuvir
triphosphate (STP) (β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine
triphosphate), ribavirin triphosphate (RTP) and AZT triphosphate (AZT-TP) were
purchased (Codontech.org, CA and Sierra Bioresearch, AZ). Interferon-alpha was
purchased from R&D Bioscience. All small molecule inhibitors were dissolved
in 100% dimethylsulfoxide (DMSO) and subsequently diluted at least 104-fold
in culture or reaction medium before each assay. The final DMSO concentrations
showed no cytotoxicity. The materials for cell culture were purchased from
Thermo Scientific Life Sciences (Grand Island, NY), unless otherwise mentioned.
Cells
Human neuroblastoma
(SH-Sy5y; ATCC) and baby hamster kidney (BHK-21) cells were cultured in
MEM:F-12 (1:1) and MEM, respectively. African green monkey kidney (Vero) and
human hepatoma (Huh-7) cells were cultured in DMEM. Aedes albopictus cells
(C6/36) were grown in L-15 medium supplemented with 0.3% tryptose phosphate
broth, 0.75 g/L sodium bicarbonate, 1.4 mM glutamine, and nonessential amino
acids. The culture medium of each cell type was supplemented with 10% fetal
bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin, and 100 μg/mL streptomycin19,20. The mammalian cells were
incubated at 37 °C in 5% CO2, whereas the mosquito cells were
maintained at 26 °C. Passages of the SH-sy5y cells included both adherent and
non-adherent cells.
Virus
ZIKV was isolated from a
serum sample of a confirmed case from Rio de Janeiro, Brazil. This sample was
received and diagnosed by the Reference Laboratory for Flavivirus, Fiocruz,
Brazilian Ministry of Health, as part of the surveillance system against
arboviruses3. Brazilian ZIKV was
originally isolated in C6/36 cells, titered by plaque-forming assays and
further passaged at a multiplicity of infection (MOI) of 0.01. The virus was
passaged by inoculating C6/36 cells for 1 h at 26 °C. Next, the residual virus
particles were removed by washing with phosphate-buffered saline (PBS), and the
cells were cultured for an additional 9 days. After each period, the cells were
lysed by freezing and thawing and centrifuged at 1,500 × g at 4 °C
for 20 min to remove cellular debris.
ZIKV was purified between
fractions of 50% and 20% sucrose. The sucrose gradients were made in 40 mL
ultracentrifuge tubes (Ultra-clear; Beckman, Fullerton, CA) in PBS without Ca++ and
Mg++ (pH 7.4). The tubes were allowed to stand for 2 h at room
temperature. Up to 20 mL of virus was added to each tube and centrifuged in an
SW 28 rotor (Beckman) at 10,000 rpm for 4 h at 4 °C. The fractions were
collected and assayed for total protein and for virus-induced hemagglutination
(HA) analysis using turkey red blood cells (Fitzgerald Industries International,
North Acton, MA). The fractions displaying HA activity (≥16 UHA/50 μL) were pooled and dialyzed against PBS without Ca++ and
Mg++ (pH 7.4) and 10% sucrose overnight at 4 °C. The virus
pools were filtered through 0.22-μm membranes (Chemicon, Millipore,
Bedford, NY). The infectious virus titers were determined by plaque assays in
BHK-21 cells, and the virus was stored at −70 °C for further studies.
Cytotoxicity assay
Monolayers of 104 BHK-21,
5 × 104 SH-Sy5y, 1.5 × 104 Vero or 1.5 × 104Huh-7
cells in 96-well plates were treated for 5 days with various concentrations of
sofosbuvir or ribavirin as a control. Then, 5 mg/ml
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) in DMEM was added to the cells in the presence of 0.01% of N-methyl
dibenzopyrazine methyl sulfate (PMS). After incubating for 4 h at 37 °C, the
plates were read in a spectrophotometer at 492 nm and 620 nm21. The 50% cytotoxic
concentration (CC50) was calculated by a non-linear regression
analysis of the dose–response curves.
Plaque-forming assay
Monolayers of BHK-21 cells
in 6-well plates were exposed to different dilutions of the supernatant from
the yield-reduction assays for 1 h at 37 °C. Whenever virus yields were
extremely low, viral particles from the supernatants were concentrated through
80-kDa centrifugal exclusion filters (Merk Millipore, Darmstadt, Germany) prior
to plaque-forming assay. Next, the cells were washed with PBS, and culture
medium containing 1% FBS and 3% carboxymethylcellulose (Fluka) (overlay medium)
was added to cells. After 5 days at 37 °C, the monolayers were fixed with 10%
formaldehyde in PBS and stained with a 0.1% solution of crystal violet in 70%
methanol, and the virus titers were calculated by scoring the plaque-forming
units (PFU).
Yield-reduction assay
Monolayers of 104 BHK-21,
5 × 104 SH-Sy5y, 1.5 × 104 Vero or 1.5 × 104Huh-7
cells in 96-well plates were infected with ZIKV at the indicated MOIs for 1 h
at 37 °C. The cells were washed with PBS to remove residual viruses, and
various concentrations of sofosbuvir, or interferon-alpha as a positive control,
in culture medium with 1% FBS were added. After 24 h, the cells were lysed, the
cellular debris was cleared by centrifugation, and the virus titers in the
supernatant were determined as PFU/mL. A non-linear regression analysis of the
dose-response curves was performed to calculate the concentration at which each
drug inhibited the plaque-forming activity of ZIKV by 50% (EC50).
Preparation of ZIKV RNA
polymerase
ZIKV RNA polymerase (ZVRP)
was obtained from ZIKV-infected BHK-21 cells. The cells were infected with ZIKV
at an MOI of 10 for 24 h, lysed with buffer A [containing 0.25 M potassium
phosphate (pH 7.5), 10 mM 2-mercaptoethanol (2-ME), 1 mM EDTA, 0.5% Triton
X-100, 0.5 mM phenylmethane sulfonyl fluoride (PMSF) and 20% glycerol],
sonicated and centrifuged at 10,000 × g for 10 min at 4 °C. The
resulting supernatant was further centrifuged at 100,000 × g for
90 min at 4 °C and passed through two ion-exchange columns, DEAE- and
phospho-cellulose19. Alternatively, the ZIKV
NS5 region encoding the nucleotides responsible for the RNA-dependent RNA
polymerase (RDRP) activity were cloned into the pET-41b+ vector (Novagen)
between the BamHI and SacI sites. ZVRP expression was induced by adding
isopropyl β-D-1-thiogalactopyranoside
(IPTG) to the E. coli strain BL21. The cells were lysed in
buffer A, and the N-terminal GST-tag was used to purify the protein using a GST
spin purification kit (ThermoFisher Scientific) according to the manufacturer’s
instructions.
RNA polymerase inhibition
assay
ZVRP inhibition assays were
adapted from a previous publication22. The reaction mixture for
ZVRP activity measurements was composed of 50 mM HEPES (pH 7.3), 0.4 mM of each
ribonucleotide (ATP, GTP, CTP and labelled UTP), 0.4 mM dithiothreitol, 3 mM
MgCl2, and 500 ng of ZIKV genomic RNA or cell extracts. The ZIKV RNA
was obtained using a QIAmp viral RNA mini kit (Qiagen, Dusseldorf, Germany)
according to the manufacturer’s instructions, except for the use of the RNA
carrier. The reaction mixtures were incubated for 1 h at 30 °C in the presence
or absence of the drugs. The reactions were stopped with addition of EDTA at a
final concentration of 10 mM.
The labeled UTP mentioned
above represents an equimolar ratio between biotinylated-UTP and
digoxigenin-UTP (DIG-UTP) (both from Roche Life Sciences, Basel, Switzerland).
The detection of incorporated labeled UTP nucleotides was performed by an
amplified luminescent proximity homogeneous assay (ALPHA; PerkinElmer, Waltham,
MA). In brief, streptavidin-donor and anti-DIG-acceptor beads were incubated
with the stopped reaction mixture for 2 h at room temperature. Then, the plates
containing the mixtures were read in an EnSpire® multimode
plate reader (PerkinElmer). Different types of blank controls were used, such
as reaction mixtures without cellular extracts and a control reaction mixture
without inhibitor and beads. In addition, the extract from mock-infected cells
was also assayed to evaluate the presence of RNA-dependent RNA polymerase
activity unrelated to ZIKV. Non-linear regression curves were generated to
calculate the IC50 values for the dose-response effects of the
compounds.
Antiviral activity in human
induced pluripotent stem (iPS) cell-derived neural stem cells (NSCs) and brain
organoids
NSCs and brain organoids
derived from human iPS cells were prepared as previously described23. The NSCs (20 × 103 cells/well
in a 96-well plate) were infected at MOIs of either 1.0 or 10 for 2 h at 37 °C.
Next, the cells were washed, and fresh medium containing sofosbuvir was added.
The cells were treated daily with sofosbuvir at the indicated concentrations.
The NSCs were observed daily for 8 days after infection. Virus titers were
determined from the culture supernatant using a plaque-forming assay. The cell
supernatant was also used for ZIKV genome analysis. Cell death was measured by
adding 2 μM CellEvent caspase-3/7
reagent and the fluorescent dye ethidium homodimer23 at days 4 and 8, when
the culture supernatants were collected. Images were acquired with an Operetta
high-content imaging system with a 20x objective and high numerical apertures
(NA) (PerkinElmer, USA). The data were analyzed using the high-content image
analysis software Harmony 5.1 (PerkinElmer, USA). Seven independent fields were
evaluated from triplicate wells per experimental condition.
Brain organoids were
infected with ZIKV at 3 × 105 PFU/mL for 2 h, and the medium
containing virus particles was then replaced with fresh medium. Sofosbuvir was
added to the fresh medium daily for one week. The culture supernatant was
collected to monitor virus infectivity and for RNA sequencing.
Comparative molecular
modeling
The amino acid sequence
encoding ZVRP (UniProtKB ID: B3U3M3) was obtained from the EXPASY proteomic
portal24 (http://ca.expasy.org/). The template search was
performed using the Blast server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the Protein Data
Bank25(PDB; http://www.pdb.org/pdb/home/home.do) as the database and the
default options. The T-COFFEE algorithm was used to generate a multiple
alignment between the amino acid sequences of the template proteins and ZVRP.
Subsequently, the construction of the SFV-ZVRP complex was performed using
MODELLER 9.16 software26, which employs spatial
restriction techniques based on the 3D-template structure. The preliminary
model was refined in the same software, using three cycles of the default
optimization protocol. The structural evaluation of the model was then
performed using two independent algorithms in the SAVES server (http://nihserver.mbi.ucla.edu/SAVES_3/): PROCHECK software27(stereochemical quality
analysis) and VERIFY 3D28 (compatibility
analysis between the 3D model and its own amino acid sequence by assigning a
structural class based on its location and environment and by comparing the
results with those of crystal structures).
Genome assembly
A 0.3-mL aliquot of supernatant
containing ZIKV (at least 2 × 105 PFU) was filtered through
0.22-μm filters to remove residual cells. The viral RNA
was extracted using a QIAamp Viral RNA Mini Kit (Qiagen®) with
RNase-free DNase (Qiagen®) treatment, omitting carrier RNA. Double-stranded
cDNA libraries were constructed using a TruSeq Stranded Total RNA LT kit
(Illumina®) with Ribo-zero treatment according to the manufacturer’s
instructions. The library size distribution was assessed using a 2100
Bioanalyzer (Agilent®) with a High Sensitivity DNA kit (Agilent®),
and the quantification was performed using a 7500 Real-time PCR System (Applied
Biosystems®) with a KAPA Library Quantification Kit (Kapa
Biosystems). Paired-end sequencing (2 × 300 bp) was performed with a MiSeq
Reagent kit v3 (Illumina®). The sequences obtained were preprocessed
using the PRINSEQ software to remove reads smaller than 50 bp and sequences
with scores of lower quality than a Phred quality score of 20. Paired-End reAd
merger (PEAR) software was used to merge and extend the paired-end Illumina
reads using the default parameters29,30. The extended reads were
analyzed against the Human Genome Database using the DeconSeq program, with an
identity and coverage cutoff of 70%, to remove human RNA sequences31. Non-human reads were
analyzed against all GenBank viral genomes (65 052 sequences) using the BLAST
software with a 1e-5 e-value cutoff. The sequences rendering a genome were
assembled with SPAdes 3.7.1 software32 followed by a
reassembly with the CAP3 program33.
Sequence comparisons
The sequences encoding the
C-terminal portion of the RNA polymerase from members of the Flaviviridae family
were acquired from the complete sequences deposited in GenBank. An alignment
was performed using the ClustalW algorithm in the Mega 6.0 software. The
sequences were analyzed using the neighbor-joining method with pairwise
deletion and a bootstrap of 1,000 replicates, and the P distances
were registered. The sequences were also analyzed for the mean evolutionary
rate.
Statistical analysis
All assays were performed
and codified by one professional. Subsequently, a different professional
analyzed the results before the identification of the experimental groups. This
approach was used to keep the pharmacological assays blind. All experiments
were carried out at least three independent times, including technical
replicates in each assay. The dose-response curves used to calculate the EC50 and
CC50values were generated by Excel for Windows. The dose-response
curves used to calculate the IC50 values were produced by Prism
GraphPad software 5.0. The equations to fit the best curve were generated based
on R2 values ≥ 0.9. ANOVA tests were also used, with P values < 0.05
considered statistically significant. The statistical analyses specific to each
software program used in the bioinformatics analysis are described above.
Results
Sofosbuvir fits into the
ZVRP predicted structure
The RNA polymerase
structures from WNV (PDB #2HFZ)34, JEV (PDB #4K6M)35, DENV (PDB #5DTO)36 and HCV (PDB #4WTG)37 share 72, 70, 68, and
25% sequence identity, respectively, with the orthologous ZIKV enzyme. Despite
its relatively low sequence identity to ZIKV, the HCV enzyme structure is
complexed with sofosbuvir, and the amino acids residues that interact with the
drug are highly conserved (approximately 80%) among the members of the Flaviviridae family37. The region encoding the
C-terminal portion of the Flaviviridae RNA polymerase contains
around 800 amino acid residues. Of these, we have highlighted in yellow those
that are identical among members of the Flaviviridae family
(see Supplementary
Material 1). The residues critical for RDRP activity are
conserved among different viral species and strains, including an African ZIKV
strain from the 1950 s and those circulating currently, DENV and different
genotypes of HCV (see Supplementary
Material 1)38.
Based on the HCV
RNA-dependent RNA polymerase domains, we constructed a 3D model of the
orthologous ZIKV enzyme (Fig. 1). Sofosbuvir was predicted
to be located among the palm and fingers region of ZIKV RNA polymerase (Fig. 1A), an area important for
coordinating the incorporation of incoming nucleotides into the new strand of
RNA37. Consequently, the amino
acid residues relevant to the sofosbuvir interaction are some of those critical
for natural nucleotide incorporation and thus RDRP activity (Fig. 1B)37.
Figure 1: Putative ZIKV RNA
polymerase in complex with sofosbuvir.
Based on the crystal structure
of the HCV RNA polymerase in complex with sofosbuvir diphosphate (PDB accession
#4WTG), the putative structure of the ZVRP was constructed. Using the T-COFFEE
server, the amino acid sequence of the ZVRP (UniProtKB ID: B3U3M3) was aligned
with orthologous RNA polymerases from other members of the Flaviviridae family,
specifically hepatitis C virus (HCV; PDB #4WTG, West Nile virus (WNIV; PDB
#2HFZ), Japanese encephalitis virus (JEV; PDB #4K6M), and dengue virus (DENV;
PDB #5DTO). The MODELLER 9.16 software was used to build a 3D model of ZIKV RNA
polymerase, with subsequent refinement performed using three cycles of the
default optimization protocol. The structural evaluation of the model was
performed using two independent algorithms, PROCHECK software and VERIFY 3D. (A)
The 3D model of ZIKV RNA polymerase is presented. (B) The residues
presumably required for the interaction of ZVRP with sofosbuvir and Mg++ions.
Sofosbuvir inhibits ZVRP in
a dose-dependent fashion
Next, we evaluated whether
sofosbuvir triphosphate (STP), the bioactive compound, could inhibit ZIKV RDRP
activity. Fractions containing the ZIKV RDRP activity were partially purified
from infected cells19. STP inhibited ZIKV RDRP
activity with an IC50 value of 0.38 ± 0.03 μM (Fig. 2A). Ribavirin-triphosphate
(RTP) and AZT-TP were used as positive and negative controls, respectively (Fig. 2A). RTP and AZT-TP exhibited
IC50values of 0.21 ± 0.06 and >10 μM, respectively (Fig. 2A). Moreover, the
recombinant expression of the C-terminal portion of ZVRP confirmed the STP
antiviral activity, with an IC50 value of 0.61 ± 0.08 μM (Fig. 2B). The AZT-TP and RTP IC50 values
for recombinant ZVRP were >10 and 0.62 ± 0.05 μM, respectively (Fig. 2B). Of note, the small
discrepancy observed in the STP IC50 values against the
partially purified and recombinant ZVRP could be because the total protein
content was used to normalize the assay conditions; therefore, the purified
recombinant preparation possessed a higher specific activity. Altogether, the
data from Fig. 2 confirmed the
molecular modeling prediction that sofosbuvir docks onto the ZVRP structure,
revealing that the chemical structure of sofosbuvir inhibits ZIKV RDRP
activity.
Figure 2: Sofosbuvir
inhibits ZIKV RDRP activity.
Cell extracts from
ZIKV-infected cells (A) or recombinant ZVRP (B) were assayed for
RDRP activity using viral RNA as the template and labeled UTP as the tracer.
Biotinylated-UTP and digoxigenin-UTP were detected by ALPHA technology using an
EnSpire® multimode plate reader (PerkinElmer). The molecules
assayed were sofosbuvir triphosphate (STP), ribavirin triphosphate (RTP) and
AZT triphosphate (AZT-TP). As a control, the RDRP activity was measured in
extracts from mock-infected cells (mock). The data represent means ± SEM of
five independent experiments.
Sofosbuvir inhibits ZIKV
replication in a cell lineage-, MOI- and dose-dependent manner
Before investigating ZIKV
susceptibility to sofosbuvir using cell-based assays, a Brazilian ZIKV isolate
was characterized for further use as a reference strain for the experimental virology.
The full-length viral genome was sequenced (GenBank accession #KX197205), and
the characteristic plaque-forming units (PFU) and cytopathic effects (CPE) were
determined in BHK-21 cells (see Supplementary
Fig. S1). Another concern was to establish whether another plaque-forming
viral agent was co-isolated with ZIKV, which could result in a misleading
interpretation of the antiviral activity. A metagenomic analysis revealed that
ZIKV was the only full-length genome of a plaque-forming virus detected in
BHK-21 cells (see Supplementary
Material 2).
The sofosbuvir
phosphoramidate prodrug must be converted to its triphosphate analog in the
cellular environment to become active. Despite a general perception that this
process is an exclusive feature of hepatocytes17, sofosbuvir may also
become active within neuroepithelial stem cells39. Indeed we investigated
whether sofosbuvir inhibits ZIKV replication in different cellular systems.
BHK-21, SH-Sy5y, Huh-7 or Vero cells were inoculated at different MOIs and
treated with various concentrations of sofosbuvir. The supernatants from these
cells were collected, and the infectious virus progeny were titered. Sofosbuvir
induced an MOI- and dose-dependent inhibition of ZIKV replication (Fig. 3A, Tables 1 and 2, and see Supplementary
Fig. S2A–D). The potency and efficiency to inhibit ZIKV
replication were higher in Huh-7 and SH-Sy5y cellsthan in BHK-21 cells (Fig. 3A and Table 1 and 2, and see Supplementary
Fig. S2A–C). Of note, even high concentrations of sofosbuvir
did not inhibit ZIKV replication in Vero cells, indicating a cell-dependent
inhibition of ZIKV replication (Fig. 3A and Tables 1 and 2, and see Supplementary
Fig. S2D). IFN-alpha and ribavirin were used as positive controls to inhibit
ZIKV replication (Fig. 3B and Tables 1 and 2, and see Supplementary
Fig. S2A–D).
Figure 3: The antiviral
activity of sofosbuvir against ZIKV.
BHK-21, SH-sy5y, Huh-7 or
Vero cells were infected with ZIKV at the indicated MOIs and exposed to various
concentrations of sofosbuvir (A) or IFN-alpha (B), and the viral
replication was measured by plaque-forming assays after 24 h of infection. The
data represent means ± SEM of three independent experiments.
Table 1: Antiviral activity
and cytotoxicity of sofosbuvir at an MOI of 1.0.
Table 2: Antiviral activity
and cytotoxicity of sofosbuvir at an MOI of 0.5.
The sofosbuvir cytotoxicity
was also cell type-dependent (Tables 1 and 2). Our results indicate
that the selectivity index (SI; which represents the ratio between the CC50 and
EC50 values) for sofosbuvir varied from 184 to 1191 (Tables 1 and 2) – being safer at an MOI
of 0.5 in the hepatoma and neuroblastoma cell lines. For comparison, the SI
values for sofosbuvir were almost 30 times higher than for ribavirin (Table 1). Our data indicate that
the sofosbuvir chemical structure possesses anti-ZIKV activity.
Sofosbuvir inhibits ZIKV
replication in human primary NSCs and brain organoids
Since the results regarding
the pharmacologic activity of sofosbuvir against ZIKV replication in lineage
cells were promising, we next investigated whether sofosbuvir could be
neuroprotective in a cellular model that corresponds to the early stages of
brain development23. Human iPS cell-derived
neural stem cells (NSCs) were infected with ZIKV and treated with sofosbuvir.
Sofosbuvir produced a pronounced inhibition of ZIKV replication in NSCs
challenged with MOIs of 1.0 or 10 after 4 to 8 days of infection (Fig. 4A), as only marginal virus
titers were detected in sofosbuvir-treated cells. This drastic reduction in
viral replication in sofosbuvir-treated NSCs impaired ZIKV-mediated
neuropathogenesis by inducing cell death. Whereas ZIKV-infected NSCs exhibited
considerable levels of caspase-3/7 activation and plasma membrane permeability
at 8 days post infection, sofosbuvir significantly protected these cells from
death (Fig. 5, and see Supplementary
Figs S3 and S4).
Figure 4: Sofosbuvir
inhibits ZIKV replication in human iPS cell-derived NSCs and brain organoids.
NSCs (A) were
infected at the indicated MOIs and brain organoids (B) were infected
with 5 × 107 PFU/mL of ZIKV. The ZIKV-infected cells were
treated with the indicated concentrations of sofosbuvir for different periods
of time post infection. At the indicated time points, the culture supernatants
were collected, and the virus was titered by plaque-forming assays. The data
represent means ± SEM of five independent experiments. The virus production in
the presence of the treatments was significantly reduced when compared to
untreated cells (P < 0.01).
Figure 5: Sofosbuvir
protects human iPS cell-derived NSCs from ZIKV-induced cell death.
NSCs were labeled for
activated caspase-3/7 (A,C) and cell permeability (B,D) 4 or 8
days after infection. The data represent means ± SEM of five independent experiments.
***indicates P < 0.001 for the comparison between the mock- and
ZIKV-infected cells. ###Indicates P < 0.001
and ##Indicates P < 0.01 for the comparison between the
ZIKV-infected cells treated with or without sofosbuvir.
Subsequently, we used
ZIKV-infected brain organoids as a three-dimensional model to assess the
antiviral activity of sofosbuvir. Brain organoids have been used as a
sophisticated cellular model to evaluate the impact of ZIKV on early brain
development and as a translational system for in-depth investigations of the
cellular and molecular events related to microcephaly23. We again observed a
pronounced reduction of ZIKV production due to sofosbuvir treatment (Fig. 4B). Altogether, these data
indicate that sofosbuvir is effective against ZIKV in neuronal cell systems
relevant to the physiopathology of the virus.
Observational analysis of
mutations in the ZIKV genome associated with sofosbuvir treatment
Systematically, we observed
that sofosbuvir was more effective in reducing ZIKV infectivity than viral RNA
levels in the supernatant of the cultures (Fig. 6). Independent of whether
sofosbuvir activity was measured in ZIKV-infected Huh-7 cells, NSCs or BHK-21
cells, a concentration of 2 μM of this drug was in
general almost two times more potent in inhibiting ZIKV-induced cell death and
viral infectivity than viral RNA production (Fig. 6). These data suggest that
virus particles containing genomic RNA may be produced under sofosbuvir
treatment but that these particles are unable to efficiently infect new cells
and produce cytopathogenicity.
Figure 6: Inhibition of
ZIKV-related infectivity and RNA production by sofosbuvir.
ZIKV-infected Huh-7 cells,
NSCs and BHK-21 cells were treated with or without sofosbuvir. The culture
supernatants were collected 24 h after infection to determine the virus
infectivity by plaque-forming assays and the viral RNA loads by real time
RT-PCR analysis. The data are presented as the percentage over the control
(untreated cells). *Indicates a significant difference (P < 0.05)
between the black and white bars. **Indicates a significant difference (P < 0.01)
between the black and white bars.
We thus sequenced the ZIKV genome
from infected Huh-7 cells, NSCs and brain organoids treated with sofosbuvir at
2 μM, a concentration that does not allow for viral
RNA extinction. The frequencies of the overall transition mutations in
sofosbuvir-treated sequences was significantly increased when compared to
control sequences (P < 0.05) for all infected host cell models (Table 3). More specifically, the
increased mutation frequency was greater for A-to-G changes, which represented
80 ± 5% of all transition mutations in the sofosbuvir-treated sequences (Table 3). In addition, A-to-G
mutations were approximately five times more abundant in sofosbuvir-treated
sequences than in control sequences (Table 3). Of note, there were no
significant differences in the frequencies of other transitions and
transversions between sofosbuvir-treated and control sequences. Our
observations indicate that in addition to its direct inhibition of the ZIKV RNA
polymerase, sofosbuvir may also increase mutations in the viral genome.
Table 3: Mutations in the
ZIKV sequence from viruses propagated in cells treated with 2 μM sofosbuvir.
Discussion
ZIKV is a member of
the Flaviviridae family, along with other clinically relevant
viruses such as DENV, WNV, JEV and HCV. In this family, ZIKV was considered to
be a virus causing only mild and self-limited infections4. However, based on
clinical evidence and experimental data, ZIKV infection has been associated
with neurological-related morbidities, with impacts on the development of the
human nervous system and neurological complications in adults7,8,23,40,41,42,43. Antiviral treatment
options are thus required to block viral replication. Here, we show that
uridine nucleotide analog anti-HCV drug sofosbuvir possesses anti-ZIKV
activity.
Several ongoing studies
have demonstrated the anti-ZIKV activity of clinically approved drugs10,11,12,13,14,15,18. Here, we show that
sofosbuvir-triphosphate inhibits ZVRP in a dose-dependent fashion. The
predicted ZVRP structure suggests that sofosbuvir binds to amino acid residues that
are critical for ribonucleotide incorporation, such as Arg473, Gly538, Trp539,
and Lys691. The fluoride radical in the sofosbuvir ribosyl moiety is
coordinated by Asn612, an interaction that is involved with the drug
selectivity to the viral RDRP, which may avoid nonspecific effects towards the
cellular DNA-dependent RNA polymerase. Lys458 seems to be the docking residue
for the uridine analog.
Next, using cell-based
assays, we demonstrated that sofosbuvir inhibits ZIKV replication in BHK-21,
SH-Sy5y and Huh-7 cells. Although ZIKV replication was susceptible to
sofosbuvir in these cells, the magnitude of antiviral potency and efficiency
varied among these cells. Taking as a reference an MOI of 1.0, the potency of
antiviral activity observed in hepatoma cells was five times higher, and in
neuroblastoma cells was two times higher, than that observed in BHK-21 cells.
This is consistent with the demonstration that sofosbuvir converts to its
bioactive form in liver cells17. Most recently, another
group also demonstrated that sofosbuvir may become active in human
neuroepithelial stem cells using functional assays against ZIKV39. Indeed, we further
confirmed that human iPS cell-derived NSCs and brain organoids can be protected
by sofosbuvir, even when these cells are challenged with exceedingly high MOIs.
Brain organoids represent a translational three-dimensional cell culture model
for the study of ZIKV-associated microcephaly23. Therefore, the antiviral
activity observed by sofosbuvir using human neural progenitors and brain
organoids represents a pragmatic demonstration of the potential secondary use
of this clinically approved anti-HCV drug, because it impairs ZIKV
neuropathogenesis, as shown here and by others39.
On the other hand, we observed
no inhibition of viral replication with even 50 μM of sofosbuvir in Vero cells. Similarly, in a recent study from
Eyer et al.12, African ZIKV
susceptibility to sofosbuvir was screened in Vero cells, and this compound did
not emerge as a potential hit. Although Eyer et al.12 and our group used
different viral strains, we obtained similar results in Vero cells.
Interestingly, sofosbuvir is a substrate for glycoprotein-P44. Proteomic data indicate
that Vero cells express this multi-drug resistance ABC-transporter, which may
cause the efflux of sofosbuvir from the cell45,46,47.
Using different read outs
to monitor sofosbuvir anti-ZIKV activity, we observed that this drug reduced
virus infectivity more than it inhibited the production of viral RNA. Unlike
most antiviral nucleoside analogs48and despite the presence of
the 3′-OH radical in its chemical structure37, sofosbuvir acts as a
chain terminator. To impair the incorporation of the incoming nucleotides, the
2′-F radical disrupts the hydrogen bonding network37. Similarly, the 2′-F in
sofosbuvir forms a hydrogen bond with Asn612, which may disrupt the hydrogen
bonding of incoming nucleotides to ZIKV RNA polymerase. Indeed, our results
indicate that sofosbuvir directly inhibits the viral RNA polymerase. In
addition to this mechanism, ZIKV genome sequences from sofosbuvir-treated cells
had an increased rate of A-to-G mutations when compared to untreated cells. It
is plausible to interpret that both the direct inhibition of ZIKV RNA
polymerase and induction of mutations in the viral genome may be triggered by
sofosbuvir. For instance, ribavirin, another ribonucleoside analog, may inhibit
viral replication by direct targeting the viral RNA polymerase and by the
induction of error-prone replication49,50.
ZIKV-associated
microcephaly and GBS highlight that antiviral interventions are urgently
needed. Our data reveal that a clinically approved drug possesses antiviral
activity against ZIKV and is active in cells derived from peripheral organs and
the CNS. Sofosbuvir may induce error-prone ZIKV replication. Together, our data
highlight the potential secondary use of sofosbuvir, an anti-HCV drug, against
ZIKV.
Additional Information
How to cite this article: Sacramento, C. Q. et
al. The clinically approved antiviral drug sofosbuvir inhibits Zika virus
replication. Sci. Rep. 7, 40920; doi: 10.1038/srep40920
(2017).
Publisher's note: Springer Nature
remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
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Acknowledgements
Thanks are due to Drs
Carlos M. Morel, Marcio L. Rodrigues, Renata Curi and Fabrícia Pimenta for
their strong advisement and support regarding the technological development
underlying this project. This work was
supported by Conselho Nacional de Desenvolvimento e Pesquisa (CNPq), Fundação
de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
Author
information
Author
notes
1.
o Carolina
Q. Sacramento
o , Gabrielle
R. de Melo
o , Caroline
S. de Freitas
o & Natasha
Rocha
These authors contributed
equally to this work.
Affiliations
1. Laboratório
de Imunofarmacologia, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz
(Fiocruz), Rio de Janeiro, RJ, Brazil
o Carolina
Q. Sacramento
o , Gabrielle
R. de Melo
o , Caroline
S. de Freitas
o , Natasha
Rocha
o , Natalia
Fintelman-Rodrigues
o , Andressa
Marttorelli
o , André
C. Ferreira
o , Giselle
Barbosa-Lima
o , Juliana
L. Abrantes
o , Yasmine
Rangel Vieira
o , Fernando
A. Bozza
o , Patrícia
T. Bozza
o & Thiago
Moreno L. Souza
2. Instituto
Nacional de Infectologia (INI), Fiocruz, Rio de Janeiro, RJ, Brazil
o Carolina
Q. Sacramento
o , Gabrielle
R. de Melo
o , Caroline
S. de Freitas
o , Natasha
Rocha
o , Natalia
Fintelman-Rodrigues
o , Andressa
Marttorelli
o , André
C. Ferreira
o , Giselle
Barbosa-Lima
o , Juliana
L. Abrantes
o , Yasmine
Rangel Vieira
o , Estevão
Portela Nunes
o , Fernando
A. Bozza
o & Thiago
Moreno L. Souza
3. National
Institute for Science and Technology on Innovation on Neglected Diseases
(INCT/IDN), Center for Technological Development in Health (CDTS), Fiocruz, Rio
de Janeiro, RJ, Brazil
o Carolina
Q. Sacramento
o , Gabrielle
R. de Melo
o , Caroline
S. de Freitas
o , Natasha
Rocha
o , Natalia
Fintelman-Rodrigues
o , Andressa
Marttorelli
o , André
C. Ferreira
o , Giselle
Barbosa-Lima
o , Yasmine
Rangel Vieira
o & Thiago
Moreno L. Souza
4. Instituto de
Tecnologia de Fármacos (Farmanguinhos), Fiocruz, Rio de Janeiro, RJ, Brazil
o Lucas
Villas Bôas Hoelz
o , Mônica
M. Bastos
o & Nubia
Boechat
5. Laboratório
de Vírus Respiratório e do Sarampo, IOC, Fiocruz, Rio de Janeiro, RJ, Brazil
o Milene
Miranda
6. Instituto de
Biologia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ,
Brazil
o Milene
Miranda
o , Diogo
A. Tschoeke
o , Luciana
Leomil
o & Fabiano
L. Thompson
7. Instituto de
Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
RJ, Brazil
o Juliana
L. Abrantes
o & Stevens
K. Rehen
8. Laboratório
de Virologia Comparada e Ambiental, IOC, Fiocruz, Rio de Janeiro, RJ, Brazil
o Eduardo
de Mello Volotão
9. SAGE –COPPE,
UFRJ, Rio de Janeiro, RJ, Brazil
o Diogo
A. Tschoeke
o , Luciana
Leomil
o & Fabiano
L. Thompson
10. Núcleo em Ecologia e Desenvolvimento
Sócio-Ambiental de Macaé (NUPEM), Universidade Federal do Rio de Janeiro,
Macaé, Rio de Janeiro, Brazil
o Diogo
A. Tschoeke
11. D’Or Institute for Research and
Education (IDOR), Rio de Janeiro, RJ, Brazil
o Erick
Correia Loiola
o , Pablo
Trindade
o & Stevens
K. Rehen
12. Laboratório de Flavivírus, IOC,
Fiocruz, Rio de Janeiro, RJ, Brazil
o Ana
M. B. de Filippis
13. BMK Consortium: Blanver Farmoquímica
Ltda; Microbiológica Química e FarmacêuticaLtda; Karin Bruning & Cia, Ltda,
Brazil
o Karin
Brüning
Contributions
C.Q.S.,
G.R.deM., N.R., L.V.B.H., M.M., C.S.deF., N.F.-R., A.M., A.C.F., G.B.-L.,
J.L.A., Y.R.V., E.deM.V., E.N.P., D.A.T., L.L., E.C.L and P.T. – experimental
execution and analysis S.K.R., M.M.B., F.A.B., P.T.B., N.B., F.L.T.,
A.M.B.deF., K.B. and T.M.L.S. – data analysis, manuscript preparation and
revision. K.B. and T.M.L.S. – conceptualized the study All authors revised and
approved the manuscript.
Competing interests
The authors declare no
competing financial interests.
Corresponding author
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Scientific Reports
· ISSN 2045-2322
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