Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis

Abstract

Compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV). In contrast to mammalian cells, inhibition of viral replication by A3 is absent in chicken cells, which suggests species-specific activity of A3. Correspondingly, the antiviral activity of A3 can be linked to a cellular protein, dihydroorotate dehydrogenase (DHODH), which is an enzyme in the de novo pyrimidine biosynthesis pathway. Viral replication of both RNA and DNA viruses can be restored in the presence of excess uracil, which promotes pyrimidine salvage, or excess orotic acid, which is the product of DHODH in the de novo pyrimidine biosynthesis pathway. Based on these findings, it is proposed that A3 acts by depleting pyrimidine pools, which are crucial for efficient virus replication.

Small-molecular-weight compounds with antiviral activity can act by inhibiting viral proteins or host cell proteins that are required for virus replication. Although drugs directed at viral proteins are more virus specific, they can easily lead to the selection of resistant mutants. For example, the use of adamantanes which target the M2 ion channel of influenza A viruses is now precluded due to wide-spread resistance (1, 2). By targeting host cell proteins, resistance is less likely to occur, and if that protein is necessary for replication of a variety of different viruses, broad-spectrum antiviral activity can be achieved. Such compounds are presumed to be more prone to toxicity, and it is important to identify targets that are not critical for cell growth but that are absolutely essential for the virus.

Recently, two small molecules were reported, LJ-001 (3) and dUY11 (4), that demonstrate broad antiviral activity against all enveloped viruses. These so-called rigid amphiphiles resemble phospholipids and are incorporated into viral membranes, where they modulate the membrane curvature needed for the membrane fusion event and they therefore inhibit viral entry. The virus specificity of these compounds takes advantage of structural differences in virion membranes versus cellular membranes and also the lack of repair mechanisms for viral membranes. To date, the only approved broad-spectrum antiviral that is effective against both RNA and DNA viruses is ribavirin (5), which is currently used in combination with IFN for hepatitis C therapy (6). Ribavirin is a ribosyl purine analog, the carboxamide group of which can resemble adenosine or guanosine, depending on its rotation. As a prodrug, it is sequentially phosphorylated by cellular kinases, and all its intermediates such as ribavirin mono- (RMP), di-(RDP) and triphosphate (RTP) are inhibitors of certain viral RNA-dependent RNA polymerases (7). RTP is incorporated into RNA and pairs equally well with uracil and cytosine, inducing lethal hypermutations (8), and RMP has been shown to target the cellular inosine monophosphate dehydrogenase (IMPDH) and thereby depletes intracellular pools of GTP (9, 10). This lack of GTP may explain the inhibition of DNA viruses, as well as the general cytotoxicity of ribavirin.

Favipiravir (T-705), which is currently in Phase III trials in Japan, also displays broad-spectrum antiviral activity, but only among RNA viruses. In vivo efficacy has been demonstrated for influenza viruses (A, B, and C), flaviviruses (West Nile virus, Yellow Fever virus), bunyaviruses (Punta Toro virus, Rift Valley fever), arenaviruses (Pichinde virus), and picornaviruses (foot-and-mouth disease virus) (11, 12). T-705 is converted to the ribofuranosyl triphosphate form by host cell enzymes, and it was shown that the antiviral activity of T-705 can be reversed by an excess of purines (13). Nevertheless, the mechanism of T-705 is not fully understood. It does not affect the synthesis of cellular DNA or RNA (13, 14), and may therefore target viral RNA-dependent RNA polymerases directly or may be preferentially incorporated into viral RNA, thereby causing hypermutations. Brequinar was described as a broad-spectrum antiviral agent capable of inhibiting both negative- and positive-strand RNA viruses (15), and leflunomide and a derivative, FK778, have been reported to inhibit human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) (16–19). This group of compounds all target the cellular enzyme dihydroorotate dehydrogenase (DHODH) (20–22), although leflunomide shows the weakest activity against this enzyme (23). DHODH is a key player in the pyrimidine de novo biosynthesis pathway and converts dihydroorotate (DHO) into orotate (24). Both the pyrimidine de novo biosynthesis pathway and the uracil salvage pathway channel into the production of uridine monophosphate (UMP) (25), which is the precursor for all pyrimidine nucleotides needed for RNA (UTP, CTP) and DNA (dTTP, dCTP) synthesis. Leflunomide (Arava) is a Food and Drug Administration (FDA)-approved therapy for rheumatoid arthritis, and its immunosuppressive properties are related to inhibition of T-cell proliferation, which is heavily reliant on pyrimidine pools (26). Virus replication is also dependent on large nucleotide pools, and therefore the antiviral activity of these compounds is likely due to pyrimidine depletion.

Here we report that the small-molecular-weight compound A3, which was identified in a previously reported influenza virus high-throughput screen (27), possesses broad-spectrum activity against RNA-, DNA-, and retroviruses and acts by targeting pyrimidine metabolism.

Results

Antiviral Activity of Compound A3.

Approximately 61,600 commercial, small-molecular-weight compounds were screened in duplicate in a high-throughput screen (HTS) assay described previously (27). Briefly, a compound was defined to be a strong inhibitor of influenza virus replication if the luminescence signal of an influenza virus inducible firefly luciferase reporter was decreased by at least 90% compared with that in untreated cells. Compound A3 (Fig. 1A) from the Asinex1 library displayed a very strong effect on viral replication, such that there was no luminescence detectable. The compound was further evaluated for cytotoxity, and its CC₅₀ in A549 cells was determined to be 268 μM over a 24-h incubation period (Fig. 1B). To confirm the results of the primary screen, A3 was tested at noncytotoxic concentrations in viral replication assays performed at an MOI of 0.01. A reduction in viral titers of 4 logs was detected at a concentration of 2 μM, and the IC₅₀ over a 24-h replication period was determined as 0.178 μM (Fig. 1B). This resulted in a selective index (SI = CC₅₀/ IC₅₀) of 1,505, which indicates compound A3 to be a very strong inhibitor with very little toxicity. The inhibitory effect of A3 was even more pronounced in primary human tracheal–bronchial epithelial (HTBE) cells (Fig. S1A). Viral titers were reduced by 4 logs after 24 h, and the IC₅₀ was determined to be 0.04 μM, which resulted in an SI of >2,380 (CC₅₀ >100 μM). In contrast, the same experiment performed in mouse embryo fibroblast (MEF) cells (Fig. S1B) reduced viral titers by less than 2 logs at a similar concentration of A3. The IC₅₀ was determined as 1 μM, which resulted in an SI of >100 (CC₅₀ >100 μM).

Fig. 1.

Compound A3 and its antiviral activity against influenza A/WSN/33 virus. (A) Chemical structure of compound A3 and its molecular weight (MW). (B) A549 cells were infected with influenza A/WSN/33 virus (MOI = 0.01) in the presence of increasing concentrations of compound A3. Viral titers were determined at 24 h postinfection and the IC₅₀ calculated (left-hand scale, blue curve). Mean of three replicates ± SD are shown. Cell viability (CC₅₀) was determined independently for a 24-h incubation period (right-hand scale, red curve). Mean of five replicates ± SD are shown.

A3 Inhibits Influenza Virus Polymerase Activity.

Next, we tested A3 at different time points during the viral life cycle to understand whether it acts early or late in infection. First we compared its effect on replication when added pre- or postinfection. A549 cells were infected at a high MOI with influenza A/WSN/33 virus and compound treatment started 2 h preinfection or at several time points postinfection. Inhibition was observed when A3 was present preinfection or when added up to 2 h postinfection but not later (Fig. 2A). The inhibition was further demonstrated by reduced levels of viral proteins in the presence of A3 (Fig. 2B). These data indicated that A3 was acting at early to mid stages in the viral life cycle, leading to the hypothesis that replication and transcription may be targeted.

Fig. 2.

Inhibition of influenza A/WSN/33 virus replication by A3 added at different times during the viral life cycle. (A) A549 cells were infected with influenza virus A/WSN/33 (MOI = 1). Compound A3 was present in the culture medium 2 h before infection or added to the medium at indicated time points postinfection at its CC₁₀. Viral titers were determined 24 h postinfection by plaque assay. Assay was performed in triplicate; results are presented as mean ± SD. (B) Viral protein levels (NP, M1, and M2) from infections shown in A were determined by Western blot analysis using specific antibodies.

We addressed this question by performing an influenza virus minigenome assay to determine whether influenza virus polymerase activity is affected by A3. A549 cells were transfected with expression plasmids for the influenza virus polymerase proteins (PB1, PB2, and PA), the nucleoprotein (NP), and the previously described influenza virus-specific firefly luciferase reporter (27). To normalize for transfection efficiency, a Renilla luciferase plasmid was cotransfected. Compounds were added at 4 h before transfection and were present for the duration of the assay. A3 strongly inhibits influenza virus polymerase function by 98% compared with the DMSO control, without affecting cellular gene expression as monitored by Renilla luciferase activity (Fig. 3A). Diphyllin was shown to inhibit influenza virus entry (28) and was included as a negative control, whereas ribavirin, a known polymerase inhibitor of RNA viruses, was included as a positive control. A dose–response assay indicated that influenza virus polymerase activity is inhibited to the same degree by 2 μM A3 as with 100 μM ribavirin (Fig. S2), suggesting that A3 is 50 times more potent than ribavirin. To determine whether A3 is affecting viral RNA synthesis, primer extension assays were performed on the NA segment to observe synthesis of v-, c-, and mRNA. To control for cellular replication, the levels of 5S rRNA were also monitored. A3 was shown to fully inhibit production of all three viral RNA species at a concentration of 2 μM (Fig. 3B) without decreasing levels of cellular 5S rRNA. Similar results were obtained for 100 μM ribavirin.

Fig. 3.

A3 inhibits influenza viral polymerase activity. (A) A549 cells were transfected with protein expression plasmids for influenza A/WSN/33 virus polymerase subunits PB1, PB2, PA, and nucleoprotein NP. An influenza virus-specific firefly luciferase reporter and a Renilla luciferase expression plasmid were cotransfected. Transfections were performed in the presence of DMSO or A3 at its CC₁₀. Ribavirin (replication inhibitor) is included as positive control and diphyllin (entry inhibitor) is included as negative control. Cells were harvested 24 h posttransfection, and activation of the luciferase reporter by the viral polymerase was measured. DMSO control is set to 100%. Assay was performed in triplicate; results are presented as mean ± SD. (B) A549 cells were infected with influenza A/PR/8/34 virus at an MOI of 7. Infections were performed in the presence of DMSO, ribavirin, or A3. Viral RNA was extracted 9 h postinfection and subjected to primer extension analysis to determine the levels of v-, c-, and mRNA. Host cell-derived 5S rRNA was used as an internal standard.

A3 Displays Broad-Spectrum Antiviral Activity.

A3 was shown to inhibit a number of influenza virus strains of both H1N1 and H3N2 subtypes (Fig. S3); therefore, to address the question of whether A3 displays broad-spectrum antiviral activity, we examined the replication of viruses representing several different families (Table 1). All viruses were tested in A549 cells (unless otherwise indicated) at a concentration of 10 μM A3 or less. At this concentration, A3 does not appear to be either cytostatic or cytotoxic over a 48-h period (Fig. S4). For influenza A/WSN/33 virus (representing the Orthomyxoviridae), viral titers for infections performed under multicycle replication conditions (MOI = 0.01) are decreased by 5 logs compared with a 2-log reduction for infections performed at an MOI of 1 (Fig. S5A). Sendai virus (SV52), a paramyxovirus, was inhibited by 3 logs (Fig. S5B), and an even stronger effect of A3 on viral replication was found for vesicular stomatitis virus (rhabdoviridae), the titer of which was decreased by 5 logs (Fig. S5C). The positive-sense RNA viruses, Sindbis virus (togaviridae) and hepatitis C virus (flaviviridae), were both inhibited by 2 logs in the presence of A3. Surprisingly, DNA viruses were also affected by A3, and titers of the human adenovirus 5 (hAd5) (adenoviridae) were reduced by 6 logs in the presence of A3 (Fig. S5D). It was noted that viruses that depend on DNA synthesis, such as vaccinia virus (poxviridae), adenovirus, and HIV-1 (retroviridae), were also sensitive to A3. The IC₅₀ of A3 against HIV-1 (NL4-3) is in the mid nanomolar range (205 nM, Fig. S6A), which is remarkably similar to the IC₅₀ for influenza virus (178 nM; Fig. 1). Time-of-addition experiments with A3 in conjunction with FDA-approved HIV-1 antiretroviral drugs show that A3 remains active even when added as late as 12 h after infection, whereas inhibitors of reverse transcriptase or integration lose activity when added 4–8 or 10–12 h postinfection (Fig. S6B). These findings suggest that the mechanism of action of A3 is at the step of transcription after integration but before maturation (Fig. S6B).

Table 1.

Viruses tested for their susceptibility to A3

Viruses	Group	Family	Inhibition of	Degree of inhibition
Influenza A virus (A/WSN/33)	(−) ssRNA	Orthomyxoviridae	Virus	∼5 logs (MOI = 0.01)
Influenza B virus (B/Yamagata/88)	(−) ssRNA	Orthomyxoviridae	Virus	∼1.5 logs (MOI = 1)
Newcastle disease virus (La Sota)	(−) ssRNA	Paramyxoviridae	Virus	∼2 logs (MOI = 1)
Sendai virus (SV52)	(−) ssRNA	Paramyxoviridae	Virus	∼3 logs (MOI = 1)
Vesicular stomatitis virus	(−) ssRNA	Rhabdoviridae	Virus	∼5 logs (MOI = 0.01)
Sindbis virus	(+) ssRNA	Togaviridae	Virus	∼2 logs (MOI = 1)
Hepatitis C virus*	(+) ssRNA	Flaviviridae	Virus	∼2 logs (n.a)
West Nile virus	(+) ssRNA	Flaviviridae	Replicon cell line	∼1 logs (NA)
Dengue I virus	(+) ssRNA	Flaviviridae	Replicon cell line	∼1 logs (NA)
Vaccinia virus (NYVAC)†	dsDNA	Poxviridae	Virus	∼3 logs (MOI = 1)
Adenovirus (hAd5)	dsDNA	Adenoviridae	Virus	∼6 logs (MOI = 5)
HIV-1‡,§	ssRNA RT	Retroviridae	Virus	∼2 logs (NA)

NA, not applicable.

*Tested in Huh 7.5 cells.

^†Tested at 1 μM A3

^‡Tested in TZM bl cells.

^§Tested at 2 μM A3.

Antiviral Activity of A3 Is Linked to Pyrimidine Metabolism.

Antivirals with broad-spectrum activity often affect nucleotide synthesis or incorporation. To address the question of whether A3 acts in such a way, we tested its activity in the presence of different purines and pyrimidines in plaque reduction assays. Uracil was found to be the only base that is able to reverse the inhibitory effect of A3 and to restore viral replication. Surprisingly, at these concentrations, the effect was seen only in Madin-Darby canine kidney (MDCK) cells and not in A549 cells (Fig. 4 A–C). Thus, A3 is able to inhibit viral replication efficiently in both cell lines, but its effect can be more easily compensated for by uracil in MDCK cells than in A549 cells. This result suggested a tissue or species specificity of A3, and therefore viral replication in the presence of A3 was tested in a panel of cell lines of different species. A strong inhibition of viral replication was seen among human cells (A549, 293T) and primate cells (Vero, CV1), independent of the tissue origin, and only high concentrations of uracil (100-fold) were able to partially restore viral replication (Fig. S7A). Vero cells, unlike CV1, are deficient in type I IFN, but both cell lines are derived from the same species (Cercopithecus aethiops) and tissue (kidney). This suggested that the involvement of IFN could be excluded in the antiviral actions of A3. This was further confirmed via quantitative RT-PCR, which showed no induction of IFN-β and other antiviral genes (ISG56, IRF7, RIG-I, and TNF-α) upon treatment with A3 alone or in combination with influenza virus infection in A549 cells. In nonprimate mammalian cells, complete inhibition of viral replication by A3 was seen in both MDCK and bronchial–tracheal ferret cells, but this could be easily reversed by the addition of uracil. Replication was only partially inhibited by A3 in baby hamster kidney (BHK) cells and pig kidney (PK-15) cells, and the addition of uracil had no obvious effect. In contrast, A3 had no effect on viral replication in chicken fibroblast (DF-1) cells (Fig. S7B). This further underlines the possible species specificity of A3 and suggests that A3 may target a host cell protein. Of note, attempts to generate A3-resistant influenza viruses have been unsuccessful, which is another indication that a host factor may be the target of A3.

Fig. 4.

Inhibition of influenza virus replication by A3 is reversed by excess uracil and orotate. (A) Plaque-reduction assay with influenza A/WSN/33 virus was performed in A549 (human) and MDCK (canine) cells in the presence of DMSO, 10 μM A3 alone, or A3 and indicated purine and pyrimidine bases (10-fold excess relative to A3). Percent replication was calculated and set relative to DMSO control. (B and D) A549 (human) cells and (C and E) MDCK (canine) cells were infected with influenza A/WSN/33 virus (MOI = 0.001). (B and C) Cell culture medium was supplemented with A3 (5 μM) and in addition with increasing concentrations of uracil (Ura), as indicated by fold excess relative to A3. (D and E) Cell culture medium was supplemented with A3 (10 μM) and in addition with increasing concentrations (fold excess relative to A3) of dihydroorotate (DHO) or orotate (Oro). Viral titers were determined by standard plaque assay at indicated time points.

The finding that uracil can reverse the inhibition of viral replication by A3, suggests that the pyrimidine pathway may be affected. Uracil is converted by the pyrimidine salvage pathway to uridine monophosphate (UMP), the base from which all other pyrimidines are manufactured in the cell. Besides the salvage of uracil, UMP is also generated via de novo biosynthesis. To distinguish between de novo biosynthesis and the salvage pathway, we tested all intermediates of the pyrimidine biosynthesis cycle for their ability to restore viral replication. Only orotic acid, which itself is converted in a two-step reaction by UMP synthase into UMP, was able to reverse the inhibition by A3 in both A549 and MDCK cells. Viral replication was tested in both cell lines in the presence of A3 alone or in combination with increasing concentrations of dihydroorotate (DHO) and orotate (Fig. 4 D and E). DHO is converted by DHODH into orotate, which can rescue viral replication in a dose-dependent manner in contrast to DHO. Although we cannot guarantee that DHO and orotate are equally efficient at entering cells or are equally stable, the dose–response seen with orotate provides a strong indication that DHODH activity may be targeted by A3. Leflunomide is a known weak inhibitor of DHODH and was tested in A549 and MDCK cells at a concentration of 10 μM for its effect on influenza virus replication. No inhibition was observed at this concentration, perhaps indicating that A3 is a superior inhibitor of DHODH compared with leflunomide. A sequence alignment of known DHODHs reveals ∼90% sequence identity between the human and canine DHODH proteins but only ∼73% between the human and chicken DHODH, which supports the idea of species-specific activity of A3.

Discussion

We describe the properties of a small-molecular-weight compound named A3 that inhibits a broad spectrum of viruses. At least eight different families including RNA (orthomyxoviridae, paramyxoviridae, rhabdoviridae, flaviviridae, togaviridae), DNA (poxviridae, adenoviridae), and retroviruses (retroviridae) are potently inhibited (Table 1). A time-of-addition experiment performed with influenza A virus suggested that the viral genome replication step is affected by A3. This was confirmed in minigenome assays, and the primer extension assay demonstrated that A3 treatment resulted in complete inhibition of viral RNA synthesis. Because a variety of different virus families were inhibited by A3, it appeared likely that a cellular factor that is essential for virus replication was the target. Several compounds described to have broad-spectrum antiviral activity act on cellular nucleotides, and here we demonstrate that the antiviral effect of A3 can be reversed specifically by uracil. Uracil is a key player in pyrimidine metabolism. Homeostasis of cellular UMP levels is ensured by salvage of uracil and also by the pyrimidine de novo biosynthesis pathway. It is known that different cell types rely to varying degrees on the de novo biosynthesis versus salvage pathways (29), and this could be one explanation for the observation that excess uracil can more easily restore virus replication in MDCK than in A549 cells. Alternatively, this could indicate species-specific activity of A3, particularly as the phenotype in A549 cells was also seen in other human and primate cells independent of their tissue origin. This hypothesis is supported by the complete absence of A3 antiviral activity in avian cells.

The restorative activity of uracil could indicate that A3 is affecting either the uracil salvage pathway or the pyrimidine de novo biosynthesis pathway. However, the finding that orotic acid also has the ability to reverse A3-mediated inhibition of viral replication led to the conclusion that A3 targets the pyrimidine de novo biosynthesis pathway. The de novo synthesis pathway consists of six synthetic steps; orotate is the product of the fourth step, which is catalyzed by DHODH. This strongly suggests a role for this enzyme in the inhibitory mechanism of A3 but an in vitro enzymatic assay is needed to verify this hypothesis. Interestingly, the chemical structure of A3 is distinct from those of leflunomide and brequinar, which are known inhibitors of DHODH, so perhaps the mode of DHODH inhibition may differ. DHODH has two binding sites. The substrate dihydroorotic acid (DHO) binds to the first site and is oxidized via a cosubstrate electron acceptor. After the release of orotate, ubiquinone binds to a second site, where it reoxidizes the cofactor. Leflunomide interferes with the ubiquinone binding site, and brequinar was shown to be a noncompetitive inhibitor versus DHO but competitive versus ubiquinone (20). Whether A3 interacts with the DHO or the ubiquinone binding sites or whether it competes with the cofactor binding site needs to be determined by cocrystallization. The cofactor binding site seems to be a plausible target, as different species use distinct cofactors (20). This could explain the lack of activity in avian cells and perhaps reduced activity in nonhuman/nonprimate cells, making it easier for excess uracil to reverse A3-mediated inhibition and to restore UMP levels. Our attempts to generate A3-resistant influenza viruses were most likely unsuccessful due to the fact that a lack of nucleosides is hard to overcome. Nevertheless, Qing et al. (15) recently reported a brequinar-resistant dengue virus in which a mutation in the NS5 gene (RNA-dependent RNA polymerase) conferred resistance through enhancement of viral RNA synthesis.

In summary, the broad-spectrum antiviral activity of A3 is associated with de novo pyrimidine synthesis, and this feature contrasts with the activity of ribavirin and favipiravir, both of which target the purine pathway. Further studies with A3 and its derivatives are required to investigate the in vivo efficacy of this unique structural group of antivirals.

Materials and Methods

Cell Lines, Viruses, and Plasmids.

A549, HEK 293T, TZM bl (HeLa), Vero, CV1, MDCK, BHK, PK-15, chicken fibroblast (DF1) cells, and primary human tracheal–bronchial epithelial (HTBE) cells were obtained from the American Type Culture Collection (ATCC). MEF cells were kindly provided by Benjamin tenOever (Mount Sinai School of Medicine, New York, NY). Bronchial tracheal ferret cells were kindly provided by Randy Albrecht (Mount Sinai School of Medicine, New York, NY). Human liver hepatoma (Huh 7.5) cells were kindly provided by Charles Rice (Rockefeller University, New York, NY). Vero cells expressing the replicon system of West Nile virus (WNV) and dengue I virus (DENV-I) were kindly provided by Pei Yong Shi (Novartis Institute for Tropical Diseases, Singapore). A549, HEK 293T, Huh 7.5, TZM bl, Vero, CV1, DF-1, BHK, PK-15, MEF, and ferret cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (HyClone). MDCK cells were cultured in Eagle's minimum essential medium (MEM) (Invitrogen) supplemented with 10% FBS. HTBE cells were cultured in Bronchial Epithelial Cell Growth Medium (BEGM) supplemented with the BEGM SingleQuot kit (Lonza).

Influenza viruses A/WSN/33 (H1N1) and B/Yamagata/88 were grown and titered in MDCK cells. Sindbis virus (SINV) and vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP; kindly provided by John Hiscott, McGill University, Montreal, QC, Canada) were grown and titered in Vero cells. Newcastle disease virus (NDV-LaSota) was grown in 10-d-old embryonated hens’ eggs and titered in DF1 cells. The influenza viruses A/Hong Kong/68 (H3N2), A/Victoria/3/75 (H3N2), A/Sw/Texas/98 (H1N1), A/Moscow/10/99 (H3N2), A/NY/2008 (H1N1), A/PR/8/34 (H1N1), A/Udorn/72 (H3N2), and Sendai Virus (SV52) were grown in 8-d-old embryonated hens’ eggs and titered in MDCK or Vero cells. Human adenovirus (hAd5) was grown and titered in A549 cells. Vaccinia virus (NYVAC) was grown and titered in CV1 cells. HIV-1 (NL4-3) viral stocks were produced by transfection of HEK 293T and titered as described previously (30). Hepatitis C virus (Jc1FLAG2[p7-nsGluc2a]) was kindly provided by Charles Rice (Rockefeller University, New York, NY) and grown as described previously (31).

The influenza minigenome reporter (pPolI-Luc) was previously described (27).

Compounds.

A3 was purchased from ASINEX. Diphyllin was purchased from ChemDiv. Ribavirin, leflunomide, purines (adenine, inosine, 2′-deoxyadenosine, 2′-deoxyguanosine, adenosine, guanosine, hypoxanthine), pyrimidines (thymine, cytosine, uracil), and components of the pyrimidine de novo biosynthesis (aspartic acid, carbamoyl phosphate, carbamoyl aspartate, dihydroorotic acid, orotic acid, orotidylic acid) were purchased from Sigma-Aldrich. Lamivudine, nevirapine, raltegravir, and amprenavir were obtained through the NIH AIDS Research and Reference reagent program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Cell Viability Assay.

The CellTiterGlo Cell Viability Assay (Promega) was used to detect cell viability. A549 cells were seeded into 96-well plates at 1.25 × 10³ cells per well and after 24 h incubation, the medium was replaced with 100 μL fresh DMEM containing the compounds. After a further 24-or 48-h incubation, CellTiterGlo solution was added to each well, and luminescence was measured using a Beckman Coulter DTX 880 plate reader (Beckman Coulter).

Viral Growth Assays in the Presence of Inhibitors.

Cells were seeded into six-well plates at 5 × 10⁵ cells per well or into 12-well plates at 2 × 10⁵ cells per well and incubated for 24 h at 37 °C, 5% CO₂. Four hours before infection, the cells were washed with PBS (Invitrogen), and the medium was replaced with DMEM containing the compound of interest at the indicated concentrations. Compounds were absent during the 1-h virus incubation but were present in the DMEM postinfection medium. For infections with influenza viruses, NDV-LaSota and SV52 postinfection medium also contained 1 μg/mL TPCK-treated trypsin (Sigma-Aldrich). When indicated, the medium was supplemented with uracil, dihydroorotic acid, and orotic acid. The infected cells were incubated at 37 °C with the exception of influenza B virus-infected cells, which were incubated at 33 °C. Viral titers were determined by standard plaque assay in MDCK cells. For influenza virus plaque reduction assays, the overlay medium was supplemented with the indicated purines, pyrimidines, or components of the pyrimidine de novo biosynthesis pathway. Viral replication of WNV, DENV-I, and HCV was monitored by the production of Renilla luciferase, which was measured using the Renilla Luciferase Assay Kit (Promega) according to the specifications of the manufacturer. TZM-bl reporter cells, which encode a Tat-responsive β-galactosidase indicator gene under the transcriptional control of the HIV-1 LTR, were used to assess HIV-1 infectivity in the presence of A3. β-Galactosidase activity was quantified 48 h after infection as described elsewhere (32). Infectivity values were normalized and IC₅₀ values were computed by nonlinear regressions (GraphPad PRISM). For the time-of-addition experiments, TZM-bl reporter cells were infected in duplicate with NL4-3 at an MOI of 0.1. FDA-approved HIV-1 antiretroviral drugs were added at 0, 1, 2, 4, 6, 8, 10, and 12 h postinfection at a multiple of the concentration required to inhibit 50% of the viral infectivity (IC₅₀), as follows: lamivudine (a nucleoside analog reverse transcriptase inhibitor, concentration 10 μM; 25-fold IC₅₀), nevirapine (a nonnucleoside reverse transcriptase inhibitor, concentration 1 μM; 11-fold IC₅₀); raltegravir (an integrase inhibitor, 1 μM; 75-fold IC₅₀), and amprenavir (a protease inhibitor, 1μM; 50-fold IC₅₀). A3 was used at a concentration of 1 μM (fivefold IC₅₀). β-Galactosidase activity was quantified 48 h after infection as previously described (32).

Influenza Virus Minigenome Assay.

A549 cells were seeded into 12-well plates at 2 × 10⁵ cells per well and incubated overnight at 37 °C, 5% CO₂. The cells were transfected with pCAGGS constructs for influenza A/WSN/33 virus PB1, PB2, and PA (100 ng each) and NP (200 ng), the RNA polymerase II driven Renilla luciferase reporter pRLTK (Promega) (200 ng), and the influenza virus-specific RNA polymerase I driven firefly luciferase reporter (pPolI Luc) (150 ng). Four hours before transfection, the cells were cultured in DMEM supplemented with compounds at their CC₁₀ or DMSO. The transfection was performed with Lipofectamine 2000 (Invitrogen) in OptiMEM (Invitrogen), which was also supplemented with compounds or DMSO. OptiMEM was replaced 4 h posttransfection with DMEM containing compounds or DMSO. After a 20- to 24-h incubation period, cells were harvested, and firefly luciferase and Renilla luciferase expression was determined using the Dual Luciferase Assay Kit (Promega).

Primer Extension Assay.

A549 cells were seeded into 12-well plates at 2 × 10⁵ cells per well. After incubation for 24 h at 37 °C and 5% CO₂, the cells were washed with PBS, and the medium was replaced with DMEM supplemented with DMSO, A3 (2 and 10 μM) or ribavirin (10 and 100 μM). The cells were incubated for 4 h before infection with influenza virus A/PR/8/34 (MOI = 7). RNA was extracted 9 h postinfection using the QIAamp viral RNA kit (Qiagen). Primers were synthesized for the NA segment of influenza A/PR/8/34 virus (c/m-RNA primer: 5′-tccagtatggttttgatttccg-3′ and v-RNA primer: 5′-ggactagtgggagcatcatttc-3′) and the human 5S rRNA (5′-tcccaggcggtctcccatcc-3′). For the RT reaction, they were labeled with ATP-[γ-³²P] using T4-kinase (Invitrogen) according to the specifications of the manufacturer. Viral RNA (2 μg) was reverse transcribed with the SuperScript First-Strand Synthesis System (Invitrogen) using labeled primers for v-, c/mRNA, and 5s rRNA. Samples were separated on a 6% SDS/PAGE gel that contained 5 M urea, transferred to membrane, and crosslinked. cDNA was visualized by exposure (24–72 h at −80 °C) to autoradiographic film (World Wide Medical Products [WWMP]).