T-705 (favipiravir) inhibition of arenavirus replication in cell culture.
ABSTRACT A number of New World arenaviruses (Junín [JUNV], Machupo [MACV], and Guanarito [GTOV] viruses) can cause human disease ranging from mild febrile illness to a severe and often fatal hemorrhagic fever syndrome. These highly pathogenic viruses and the Old World Lassa fever virus pose a significant threat to public health and national security. The only licensed antiviral agent with activity against these viruses, ribavirin, has had mixed success in treating severe arenaviral disease and is associated with significant toxicities. A novel pyrazine derivative currently in clinical trials for the treatment of influenza virus infections, T-705 (favipiravir), has demonstrated broad-spectrum activity against a number of RNA viruses, including arenaviruses. T-705 has also been shown to be effective against Pichinde arenavirus infection in a hamster model. Here, we demonstrate the robust antiviral activity of T-705 against authentic highly pathogenic arenaviruses in cell culture. We show that T-705 disrupts an early or intermediate stage in viral replication, distinct from absorption or release, and that its antiviral activity in cell culture is reversed by the addition of purine bases and nucleosides, but not with pyrimidines. Specific inhibition of viral replication/transcription by T-705 was demonstrated using a lymphocytic choriomeningitis arenavirus replicon system. Our findings indicate that T-705 acts to inhibit arenavirus replication/transcription and may directly target the viral RNA-dependent RNA polymerase.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: The His274-->Tyr274 (H274Y) mutation confers oseltamivir resistance on N1 influenza neuraminidase but had long been thought to compromise viral fitness. However, beginning in 2007-2008, viruses containing H274Y rapidly became predominant among human seasonal H1N1 isolates. We show that H274Y decreases the amount of neuraminidase that reaches the cell surface and that this defect can be counteracted by secondary mutations that also restore viral fitness. Two such mutations occurred in seasonal H1N1 shortly before the widespread appearance of H274Y. The evolution of oseltamivir resistance was therefore enabled by "permissive" mutations that allowed the virus to tolerate subsequent occurrences of H274Y. An understanding of this process may provide a basis for predicting the evolution of oseltamivir resistance in other influenza strains.Science 06/2010; 328(5983):1272-5. · 31.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Argentine hemorrhagic fever is a systemic viral disease caused by Junin virus, with a mortality of 15-30% in untreated individuals. Current specific therapy is highly effective in reducing mortality, and consists of the early administration of immune plasma in defined doses of specific neutralizing antibodies per kg of body weight. However, several reasons suggest the need to investigate alternative therapies. Ribavirin, a broad spectrum antiviral agent, is effective in the treatment of other viral hemorrhagic fevers, and the studies done with Junin virus infections to date indicate that this drug may also have a beneficial effect in Argentine hemorrhagic fever.Antiviral Research 02/1994; 23(1):23-31. · 3.93 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.Proceedings of the National Academy of Sciences 04/2006; 103(12):4663-8. · 9.74 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2011, p. 782–787
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 2
T-705 (Favipiravir) Inhibition of Arenavirus Replication in Cell Culture?†
Michelle Mendenhall,1Andrew Russell,1Terry Juelich,2Emily L. Messina,3,4Donald F. Smee,1
Alexander N. Freiberg,2Michael R. Holbrook,2‡ Yousuke Furuta,5Juan-Carlos de la Torre,6
Jack H. Nunberg,3and Brian B. Gowen1*
Institute for Antiviral Research and Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah1;
Department of Pathology, University of Texas Medical Branch, Galveston, Texas2; Montana Biotechnology Center3and Division of
Biological Sciences,4The University of Montana, Missoula, Montana; Research Laboratories, Toyama Chemical Company,
Ltd., Toyama, Japan5; and Viral Immunobiology Laboratory, Department of Immunology and
Microbial Science, The Scripps Research Institute, La Jolla, California6
Received 3 September 2010/Returned for modification 5 November 2010/Accepted 16 November 2010
A number of New World arenaviruses (Junín [JUNV], Machupo [MACV], and Guanarito [GTOV] viruses)
can cause human disease ranging from mild febrile illness to a severe and often fatal hemorrhagic fever
syndrome. These highly pathogenic viruses and the Old World Lassa fever virus pose a significant threat to
public health and national security. The only licensed antiviral agent with activity against these viruses,
ribavirin, has had mixed success in treating severe arenaviral disease and is associated with significant
toxicities. A novel pyrazine derivative currently in clinical trials for the treatment of influenza virus infections,
T-705 (favipiravir), has demonstrated broad-spectrum activity against a number of RNA viruses, including
arenaviruses. T-705 has also been shown to be effective against Pichinde arenavirus infection in a hamster
model. Here, we demonstrate the robust antiviral activity of T-705 against authentic highly pathogenic
arenaviruses in cell culture. We show that T-705 disrupts an early or intermediate stage in viral replication,
distinct from absorption or release, and that its antiviral activity in cell culture is reversed by the addition of
purine bases and nucleosides, but not with pyrimidines. Specific inhibition of viral replication/transcription by
T-705 was demonstrated using a lymphocytic choriomeningitis arenavirus replicon system. Our findings
indicate that T-705 acts to inhibit arenavirus replication/transcription and may directly target the viral
RNA-dependent RNA polymerase.
Several New World arenaviruses, including Junín (JUNV),
Machupo (MACV), and Guanarito (GTOV) viruses, as well as
the related Old World Lassa virus, are among a phylogeneti-
cally diverse group of negative-sense RNA viruses that cause
severe viral hemorrhagic fevers (VHFs) in regions of the world
where they are endemic (9). The National Institutes of Health
has classified these viruses as category A agents because of the
threat they pose to the U.S. population (20). Despite the bio-
defense and public health risks associated with these highly
pathogenic viruses, there are no FDA-licensed arenavirus vac-
cines and current antiarenaviral therapy is limited to an off-
label use of ribavirin (1-?-D-ribofuranosyl-1,2,4-triazole-3-car-
boxamide), which has had only mixed success in the treatment of
severe infections and is associated with significant toxicity in hu-
mans (4, 15, 27). Therefore, it is important to develop novel and
effective antiviral drugs to combat arenaviral hemorrhagic fevers.
T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide)
is a pyrazine derivative with broad antiviral activity against
RNA viruses, including influenza viruses (6, 16, 24, 25), flavi-
viruses (13, 19), bunyaviruses, and several nonpathogenic
arenaviruses (10–12). Moreover, studies employing the ham-
ster Pichinde virus (PICV) infection model of acute arenaviral
disease have demonstrated that T-705 can be used effectively
to treat advanced infections in animals (10). However, T-705
has not yet been tested against highly pathogenic human
Evidence indicates that T-705 is ribosylated and phosphory-
lated to the active T-705-4-ribofuranosyl-5?-triphosphate form
(T-705RTP) that inhibits influenza virus infection by interfer-
ing with viral RNA replication and transcription through inhi-
bition of the virus RNA-dependent RNA polymerase (RdRp)
(7). The broad activity of T-705 against a number of RNA
viruses suggests that this inhibitor may target a conserved func-
tional element in the viral polymerase. The ability of T-705 to
specifically target the viral replication machinery may minimize
the possibility of in vivo toxicity. In contrast, ribavirin also
inhibits cellular IMP dehydrogenase (IMPDH), a key enzyme
in guanosine biosynthesis, and thereby perturbs cellular nucle-
otide pools. In the present study, we explored the mechanism
of action of T-705 in cell culture and assessed the in vitro
activity of T-705 against three highly pathogenic arenaviruses.
MATERIALS AND METHODS
Viruses. JUNV, Candid 1 strain (JUNV-C), and GTOV, strain S-26764, were
provided by Robert Tesh at the World Reference Center for Emerging Viruses
and Arboviruses (WRCEVA; University of Texas Medical Branch [UTMB],
Galveston, TX). JUNV, Romero strain (JUNV-R), and MACV, strain Carvallo,
were kindly provided by Tom Ksiazek (Special Pathogens Branch, Centers for
Disease Control and Prevention, Atlanta, GA). Virus stocks of JUNV-R,
MACV, and GTOV were grown in Vero (African green monkey kidney) cells.
* Corresponding author. Mailing address: Institute for Antiviral Re-
search and Department of Animal, Dairy, and Veterinary Sciences,
Utah State University, Logan, UT 84322-5600. Phone: (435) 797-3112.
Fax: (435) 797-3959. E-mail: email@example.com.
‡ Present address: NIH Integrated Research Facility, Division of
Clinical Medicine, Fort Detrick, Frederick, MD.
† Supplemental material for this article may be found at http://aac
?Published ahead of print on 29 November 2010.
All work with JUNV-R, MACV, and GTOV was performed under biosafety
level 4 (BSL4) containment at the Robert E. Shope Laboratory at UTMB.
Tacaribe virus (TCRV), strain TRVL 11573 (ATCC, Manassas, VA), was
passaged once in baby hamster kidney (BHK) cells and three times in Vero cells.
The attenuated JUNV-C was passaged once in BSC-1 cells and once in Vero
cells. Purified stocks were prepared for both TCRV and JUNV-C by sucrose
cushion ultracentrifugation. Infected Vero cells culture lysates were clarified by
low-speed centrifugation (4,500 ? g), and the supernatants were overlaid onto a
20% (wt/vol) sucrose solution (TN buffer; 0.05 M Tris-HCl, pH 7.4, and 0.1 M
NaCl) and centrifuged at 100,000 ? g for 1 h in an SW28 rotor (Beckman
Coulter, Brea, CA). The virus pellets were resuspended in phosphate-buffered
saline (PBS), aliquoted, and stored at ?80°C until use.
Antiviral compounds, nucleotides, and nucleosides. T-705 was provided by the
Toyama Chemical Company, Ltd. (Toyama, Japan). Ribavirin was from MP
Biomedical (Santa Ana, CA). Adenine, adenosine, guanine, guanosine, 2-deoxy-
guanosine, inosine, hypoxanthine, xanthine, cytosine, cytidine, thymine, thymi-
dine, uracil, uridine, and uric acid were from Sigma (St. Louis, MO), and
2-deoxyadenosine, 2-deoxycytidine, and xanthosine were from ICN Nutritional
Biochemicals (Cleveland, OH).
Virus yield reduction assays. For experiments evaluating drug inhibition of
JUNV-R, MACV, or GTOV replication, Vero E6 (African green monkey kid-
ney) cell cultures were infected with a multiplicity of infection (MOI) of 0.1 in
duplicates in the presence of serially 2-fold diluted (1,000 to 4 uM) T-705 or
ribavirin. Supernatants from infected cells were harvested at 4 days postinfection
(d p.i.) for MACV, 6 d p.i. for JUNV-R, or 10 d p.i. for GTOV.
Viral titers for drug-treated JUNV-R infections were determined by plaque
assay. Vero E6 cells were infected with serial 10-fold dilutions of virus for 1 h at
37°C. Cell monolayers were then overlaid with 0.5% SeaKem ME agarose (Cam-
brex, East Rutherford, NJ) in minimal essential medium (MEM) supplemented
with 2% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S).
Infected cells were cultured for 6 days, at which time a second overlay containing
1% neutral red was added. PFU were counted 18 to 24 h after addition of the
second overlay, and the 90% and 50% effective concentrations (EC90and EC50,
respectively) were calculated by regression analysis.
GTOV titers were also measured by plaque assay. Vero cell monolayers were
infected with serial 10-fold dilutions of GTOV for 1 h at 37°C. After infection,
cells were overlaid with 0.5% methyl cellulose in MEM supplemented with 2%
FBS and 1% P/S. After a 10-day culture period, the overlay was removed, and
cells were fixed with 10% buffered formalin for 20 min and stained with 1%
crystal violet (Sigma). PFU were counted, and the EC90and EC50were calcu-
lated by regression analysis.
MACV titers were measured by a focus-forming unit (FFU) assay. Vero E6
monolayers were infected with serial 10-fold dilutions of virus for 1 h at 37°C.
Following infection, cells were overlaid with 0.8% tragacanth (Sigma) in MEM
supplemented with 2% FBS and 1% P/S. After infected cells were cultured for
4 days, the overlay was removed, and cells were fixed with 10% buffered formalin
for 30 min and then refrigerated overnight. Fixed cells were permeabilized in
70% ethanol for 20 min and washed with PBS. Primary antibodies were diluted
in PBS with 5% milk and 1% Tween 20. MACV-infected cells were incubated
with primary antibody, JUNV-C antisera (kindly provided by R. Tesh,
WRCEVA, UTMB), and incubated overnight at room temperature. The primary
antibody was removed, and the plates were washed once with PBS. The second-
ary antibody, goat anti-mouse IgG labeled with horseradish peroxidase (HRP;
Southern Biotech, Birmingham, AL), was diluted in PBS with 1% bovine growth
serum and added to plates for 1 to 5 h at room temperature, and then the plates
were washed with PBS. AEC substrate chromogen (DakoCytomation, Carpin-
teria, CA) was added for 15 min at room temperature. The reaction was stopped
with distilled water, and fluid was removed from the wells. FFU were counted,
and the EC90and EC50were calculated as described above.
Time-of-addition and reversal of antiviral activity assays. In time-of-addition
and reversal of antiviral activity assays, Vero monolayers (70% confluent) were
first inoculated with TCRV or JUNV-C. Cells and virus were incubated at 37°C
for 1 h to allow virus adsorption. The inoculum was removed, monolayers were
washed twice, and test medium (MEM containing 2% FBS and 50 ?g/ml gen-
tamicin) was added to the wells.
Two time-of-addition methods were employed. In method 1, monolayers were
infected with TCRV or JUNV-C at an MOI of 0.2 (time zero), and T-705 was
added at 1, 2, 4, 6, 8, 10, 12, or 15 h p.i. to give a final concentration of 200 ?M.
Cells were incubated at 37°C, and culture supernatants were collected at 24 h p.i.
for virus yield determination by cell culture infectious dose assay (11). Briefly,
each sample was serially diluted in 10-fold increments and plated on Vero cells
in 96-well microplates. Plates were incubated for 7 days, and viral cytopathic
effect (CPE) was determined for calculation of 50% endpoints (50% cell culture
infectious dose [CCID50]) as previously described (21).
In the second method, cell monolayers in triplicate wells were infected with an
MOI of 0.05, and cells were treated by adding T-705 to a final concentration of
400 ?M for the indicated periods (?2 to 0, 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to
15, and 15 to 18 h p.i.). Test medium was replaced, and incubation was contin-
ued. Cells were incubated at 37°C, supernatants were collected 24 h p.i., and virus
yields were determined.
Reversal of antiarenaviral T-705 activity by the addition of a molar excess of
purine and pyrimidine bases and nucleosides was investigated with Vero cells
infected with an MOI of 0.2 of TCRV or JUNV-C. T-705 was added to a final
concentration of 200 ?M; each competitive agent was added to triplicate wells to
a final concentration of 400 ?M. Cells were incubated at 37°C until 48 h p.i., at
which time supernatants were collected and virus yields determined.
LCMV MG rescue assay. The lymphocytic choriomeningitis arenavirus
(LCMV) minigenome (MG) rescue assay was used as previously described (17,
18). Briefly, BHK-21 cells were transfected with one plasmid that directs syn-
thesis of an LCMV MG RNA expressing the firefly luciferase (fLuc) reporter
gene in an antisense orientation together with two polymerase II expression
plasmids encoding the L polymerase (pC-L) and nucleoprotein (pC-NP), re-
quired for MG replication and expression. The plasmid mixture was transfected
at a 1:2:1 ratio of MG-fLuc–pC-L–pC-NP using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA). To assess potential cytotoxic effects of T-705 and ribavirin, the
cells were also cotransfected with the pRL-CMV plasmid (Promega, Madison,
WI) expressing the Renilla luciferase (RLuc) reporter gene under the control of
cellular, rather than viral, transcription machinery. Four hours later, cells were
reseeded into 96-well microculture dishes and incubated for 44 h with replicate
serial dilutions of T-705 or ribavirin. Cells were then lysed, and fLuc and RLuc
activities were detected using a dual reporter assay kit (Promega) and Spectra-
Max L luminometer (Molecular Devices, Sunnyvale, CA).
Reversal of T-705 and ribavirin activity in the LCMV replicon system by the
addition of purine or pyrimidine bases and nucleosides was also investigated with
BHK cells using the LCMV replicon system. T-705 or ribavirin was added to cells
at a final concentration of 200 or 100 ?M, respectively, and each base/nucleoside
was added to a final concentration of 400 ?M. Cells were lysed 48 h posttrans-
fection and assayed for bioluminescence.
T-705 activity against hemorrhagic fever-causing arenavi-
ruses. T-705 has been shown to inhibit the replication of
several nonpathogenic arenaviruses but has not to date been
tested for activity against the highly pathogenic viruses
known to cause VHFs. Therefore, we evaluated the inhibi-
tory activity of T-705 in JUNV-R, MACV, and GTOV in-
fection. As shown in Table 1, T-705 was effective against
GTOV, JUNV-R, and MACV at inhibitory concentrations
similar to those reported for JUNV-C and other nonpatho-
genic arenaviruses (11). Ribavirin was also effective against
the three viruses, but to a lesser degree, as reflected by
higher inhibitory concentrations (Table 1) and right-shifted
dose-response curves (Fig. 1) relative to T-705. Evidence of
cytotoxicity by either compound was not observed at the
TABLE 1. In vitro inhibitory effects of T-705 and ribavirin against
EC90? SD EC50? SD EC90? SD EC50? SD
Plaque 43 ? 20
21 ? 19
53 ? 11
15 ? 12
12 ? 11
14 ? 5
303 ? 228
71 ? 81
122 ? 13
239 ? 213
49 ? 71
68 ? 21Focus-forming
aData are the means and standard deviations (SD) from 3 or 4 separate
experiments using Vero E6 cells. Values for 90 and 50 percent effective concen-
trations (EC90and EC50, respectively) are expressed in ?M.
VOL. 55, 2011 T-705 INHIBITION OF ARENAVIRUS REPLICATION IN VITRO 783
T-705 time-of-addition effect on arenavirus multiplication in
cultured cells. Time-of-addition studies were conducted to
assess the stage of arenaviral replication at which T-705
imparts its antiviral activity. Inhibitor was added at various
times p.i., and the reduction in virus production relative to
the untreated culture was assessed at 24 h p.i. In untreated
cultures, infectious TCRV and JUNV-C particles could be
detected in the supernatant by 14 h (not shown), suggesting
an eclipse period of approximately 14 h. TCRV replication
was inhibited when drug was added up to 6 h p.i. and left on
throughout the 24-h incubation period (Fig. 2A, left). With
JUNV-C, inhibition was seen when T-705 was withheld until
as late as 8 h p.i. (Fig. 2A, right). Robust inhibition was
observed generally in cultures treated within 6 to 8 h of
infection. As T-705 is likely metabolized by the cell to form
T-705RTP (26), these times represent minimal estimates for
T-705 sensitivity. Nonetheless, the data suggest that T-705
acts at early or middle stages of the virus life cycle.
To investigate the timing of inhibition by T-705 in more
detail, we conducted experiments wherein cells were ex-
posed to the drug for short periods within the 24-h time
frame of the experiment. The most robust inhibition of
TCRV and JUNV-C replication was observed upon T-705
treatment during postinfection periods of 3 to 6 h, 6 to 9 h,
9 to 12 h, and 12 to 15 h (Fig. 2B). Little or no inhibition was
seen when T-705 was added from ?2 to 0 h, 0 to 3 h, or 15
to 18 h p.i. Taken together, these studies suggest a window
for T-705 inhibition within the early and intermediate stages
of virus replication, following virus entry and prior to virus
assembly and budding.
FIG. 1. T-705 inhibition of highly pathogenic arenaviruses in Vero E6 cells. The dose-response curves shown reflect the effects of T-705 (solid
line) and ribavirin (hashed line) on GTOV (A), JUNV-R (B), and MACV (C) replication. The mean EC90s and EC50s are presented in Table 1,
and the error bars represent standard deviations.
FIG. 2. Time of addition of T-705 to arenavirus-infected Vero cells. (A) For method 1, Vero cells were infected with TCRV or JUNV-C and
at indicated times (x axis) T-705 was added to infected cells at a final concentration of 200 ?M. Infected cells exposed to MEM lacking T-705 (virus
control [VC]) were included for comparison. Supernatants were collected 24 h p.i., and virus yields were determined. (B) For method 2, Vero cells
were infected with TCRV or JUNV-C, and at indicated time intervals (x axis) T-705 or MEM vehicle was added to infected cells at a final
concentration of 400 ?M. Supernatants were collected and virus yields determined as described above. Data shown are representative of three or
four independent experiments per method for each virus, and error bars represent standard deviations for triplicate samples.
784MENDENHALL ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Effects of purines at molar excess concentration on T-705-
mediated anti-TCRV and -JUNV-C activity. Based on a previ-
ous study demonstrating that the antiviral action of T-705 in
influenza virus-infected cells could be reversed by the addition
of purines or purine nucleosides, but not by pyrimidines (7), we
investigated the requirements for the reversal of T-705 activity
in arenavirus infection. As seen in Fig. 3, TCRV and JUNV-C
production could be rescued from T-705 action by the addition
of a molar excess of purines, including adenine, adenosine,
2-deoxyadenosine, guanine, guanosine, 2-deoxyguanosine, in-
osine, and hypoxanthine. In contrast, compounds generated in
purine catabolism (xanthine and uric acid) and xanthosine did
not reverse the action of T-705. Likewise, the pyrimidine
nucleobases (cytosine, thymine, and uracil) and nucleosides
(cytidine, 2-deoxycytidine, thymidine, and uridine) had little or
no impact on T-705 antiarenavirus activity.
Effect of T-705 on the activity of an LCMV MG. Previous
studies have shown that arenavirus replication can be modeled
using a recombinant plasmid replicon system comprising the
viral RdRp (L), the nucleoprotein (N), and an RNA MG (5,
17, 18, 23). To specifically investigate the effects of T-705 on
viral replication and transcription, we made use of the LCMV
replicon system. In this assay, RdRp-dependent replication of
the antigenomic viral RNA is evidenced by expression of a
firefly luciferase (fLuc) reporter gene in the MG RNA. Inhi-
bition of fLuc expression in cells transfected with the three-
plasmid replicon would be consistent with a disruption of
RdRp function. As shown in Fig. 4A, transcription from the
LCMV replicon system was inhibited by T-705 (EC50of 29
?M). Cell-driven expression of a cotransfected Renilla lucifer-
ase (RLuc) plasmid, which provides a measure of the effects on
cellular transcription, was minimally affected at the highest
concentrations of T-705 tested (Fig. 4B). This result demon-
strates the specificity and apparent absence of general cytotox-
icity by T-705. Ribavirin was also shown to inhibit fLuc expres-
FIG. 3. Reversal of T-705 antiarenavirus activity in Vero cells infected with TCRV (left) or JUNV-C (right). Vero cells were infected with
TCRV or JUNV-C, and medium containing 200 ?M T-705 and 400 ?M of the indicated compound (x axis) was added to infected cells.
Supernatants were collected 48 h p.i., and virus yields determined. Patterned gray bars indicate purines, and unpatterned gray bars indicate
pyrimidines or their respective nucleosides or derivatives. Black bars indicate virus controls. Results shown are representative of two independent
experiments for each virus, and error bars represent standard deviations for triplicate samples.
FIG. 4. T-705 inhibition of LCMV replicon system. BHK cells
transfected with the LCMV replicon system (fLuc) and control
(RLuc) plasmids were exposed to serial dilutions of T-705 or riba-
virin. After 44 h in culture, the cells were lysed, and fLuc and RLuc
luminescence was detected by dual luciferase reporter assay. fLuc
expression reflects L- and N-dependent replication and transcrip-
tion from the LCMV MG (A), and RLuc reporter activity is rep-
resentative of cellular transcription and is a measure of cell viability
(B). Results are compiled from three independent experiments and
are presented as percentages of untreated controls. Error bars
represent standard deviations. RLU, relative light units.
VOL. 55, 2011 T-705 INHIBITION OF ARENAVIRUS REPLICATION IN VITRO 785
sion by the LCMV replicon (EC50of 13 ?M), but considerable
cytotoxicity (50% cytotoxic concentration [CC50] of ?100 ?M)
was also observed (Fig. 4B). This cytotoxic effect likely con-
tributes to the unusually steep dose-response curve observed
for ribavirin (Fig. 4A) and artifactually reduces its EC50.
Consistent with our antiviral studies, the inhibitory action of
T-705 was also reversed by purines and purine nucleosides
when assessed using the LCMV replicon system. Significant
rescue from inhibition was provided by adenine, adenosine,
2-deoxyadenosine, 2-deoxyguanosine, inosine, and hypoxan-
thine (Fig. 5). Guanine and guanosine also reversed the effect
of T-705, albeit to a lesser extent. The pyrimidines, as well as
xanthine, xanthosine, and uric acid, were again inactive in this
assay. Inhibition by ribavirin was also partly reversed by gua-
nine and guanosine. In contrast to T-705, however, the addi-
tion of adenine or adenosine did not prevent inhibition by
ribavirin. This is consistent with the known inhibitory effect of
ribavirin on the cellular IMPDH, which is not involved in
adenosine biosynthesis. This observation suggests that the tar-
get for inhibition by T-705 is distinct from that of ribavirin,
which appears to act predominantly to inhibit IMPDH and the
synthesis of GMP.
T-705 has demonstrated remarkably broad in vitro activity
against a range of RNA viruses (see Table S1 in the supple-
mental material). For many of these viruses, treatment options
are severely limited, and in the case of influenza virus, oselta-
mivir resistance remains a concern (1). In particular, therapeu-
tic options for treating severe arenaviral hemorrhagic fever
cases are restricted to the use of ribavirin (2) or, in the case of
Argentine hemorrhagic fever, to transfusion of immune
plasma. Safer and more effective countermeasures are clearly
needed (4, 15). T-705 is currently being evaluated in clinical
trials in Japan and the United States for use in the treatment
of influenza virus infections (8). FDA approval for the safe use
of T-705 for influenza virus infection would facilitate its devel-
opment for other RNA virus treatment indications. Here, we
have demonstrated for the first time that T-705 is active against
the highly pathogenic human arenaviruses JUNV-R, MACV,
and GTOV and provided evidence that suggests that T-705
may act as a purine nucleoside analog specifically targeting
A recent study exploring the mechanism of action of T-705
against influenza virus infection suggests that the viral poly-
merase is the principal target of the active T-705 metabolite
T-705RTP (7). We hypothesize that T-705 is also able to in-
hibit arenavirus multiplication by targeting the virus polymer-
ase complex. It has been shown that influenza virus replication
is inhibited by T-705 at an early or middle stage of infection
and that purines but not pyrimidines are able to competitively
reverse anti-influenza virus activity (7). In the present study, we
observed analogous results in arenavirus infection.
In our studies of the reversal of T-705 inhibition, nearly all
purine-based compounds showed a significant effect on T-705
activity. The notable exceptions were uric acid, xanthine, and
xanthosine. Uric acid is the end product of purine degradation
and would thus not be expected to affect inhibition by T-705.
The biological consequences of xanthine and xanthosine me-
tabolism are poorly defined. Indeed, all biosynthetic and cat-
abolic purine pathways in the cell are highly interconnected
and tightly regulated, making it difficult to ascribe a specific
mechanism for the reversal of T-705 inhibition. However, in in
vitro assays of influenza virus RdRp activity, GTP has been
shown to be competitive with T-705RTP (7). Further biochem-
ical studies are needed to test the leading hypothesis that
T-705 acts as a nucleoside analog to inhibit the arenaviral
RdRp. Additional information from the analysis of T-705 re-
sistance will also be helpful in identifying the precise viral
Inhibition of the LCMV MG system indicates that T-705
interferes with virus transcription and/or replication. The mo-
lecular mechanism for inhibition, however, is not known and
may include effects on L, NP, or MG. Cellular transcription, as
measured by the RLuc reporter, was unaffected. In contrast,
FIG. 5. Reversal of T-705 LCMV replicon inhibition. Following transfection as described in the legend to Fig. 4, 400 ?M of the indicated
compounds (x axis) was added to the cells with 200 ?M T-705 (A) or 100 ?M ribavirin (B). Cells were lysed after a 44-h culture period. Patterned
gray bars indicate purines and their respective nucleosides or derivatives, and unpatterned gray bars indicate pyrimidines. Data are presented as
percentages of untreated replicon controls (black bars). Results shown are representative of two independent experiments, and error bars represent
standard deviations calculated from a minimum of 4 replicates. RLU, relative light units.
786MENDENHALL ET AL.ANTIMICROB. AGENTS CHEMOTHER.
ribavirin demonstrated significant inhibition of cellular pro-
cesses at concentrations only slightly greater than those that
inhibit the LCMV replicon. This is consistent with its known
inhibition of IMPDH (7, 28) and its recognized in vivo toxicity
(3, 22). The ability of hypoxanthine to reverse inhibition by
T-705, but not by ribavirin, provides additional evidence that
T-705 does not inhibit cellular IMPDH (29). The specific in-
hibitory activity of T-705 against South American VHF viruses
and its apparent lack of cellular toxicity bode well for further
development of T-705 in the treatment of severe arenaviral
J.H.N. is grateful to Ana Sanchez (The Scripps Research Institute)
and Sudhakar Agnihothram (The University of Montana) for facilitat-
ing implementation of the minigenome rescue assay.
This work was funded by subawards to B.B.G. and J.H.N. as part of
National Institutes of Health (NIH) grant U54 AI-065357 (Rocky
Mountain RCE; J. Belisle, principal investigator [PI]) and supported in
part by NIH contract N01 AI-30048 (D.F.S., PI). J.-C.D.L.T. was
supported by NIH grant R01 AI-075298.
Y.F. is an employee of the Toyama Chemical Co., Ltd., the manu-
facturer of T-705. All other authors declare no conflict of interest.
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