GS-441524 inhibits African swine fever virus infection in vitro

African swine fever virus (ASFV) is a highly infectious and lethal swine pathogen that causes serious socio- economic consequences in endemic countries for which no safe and effective vaccine is currently available. GS-441524, a 1-cyano-substituted adenine C-nucleoside ribose analogue, inhibits viral RNA transcription by competing with natural nucleosides (ATP, TTP, CTP, and GTP) and effectively inhibits viral RNA-dependent RNA polymerase activity. However, whether GS-441524 can inhibit the replication of DNA viruses is unknown. In this study, we confirmed that GS-441524 inhibits ASFV infection in porcine alveolar macrophages (PAMs) in a dose- dependent manner; GS-441524 significantly inhibited ASFV replication at different time points after ASFV infection, particularly at the early stages of viral replication. Notably, GS-441524 did not increase the levels of antiviral cytokines or ATP in PAMs. However, an increase in the concentration of natural ATP in PAMs promoted the replication of ASFV and attenuated the inhibitory effect of GS-441524 in a dose-dependent manner. Our results suggest that GS-441524 is an effective antiviral against ASFV.

African swine fever virus (ASFV) is a single-molecule linear double- stranded DNA virus belonging to the order of double-stranded DNA vi- ruses (Gaudreault et al., 2020) and the African classical swine fever virus family and genus (Malogolovkin and Kolbasov, 2019). It is the only DNA arbovirus identified to date (Chapman et al., 2011). Its genome contains 151–167 open reading frames (Galindo and Alonso, 2017), and viral replication occurs in the cytoplasm of host cells. The diameter of the virus is 175–215 nm (Dixon et al., 2013; Ya´n˜ez et al., 1995). Notably,mature virus particles (outside cells) consist of a membrane covering the nucleocapsid and have a hexagonal appearance. They contain more than 160 virus-encoded proteins (L K Dixon et al., 2019), including structural proteins and enzymes required for gene transcription and RNA pro- cessing that are essential for re-infection (Rodríguez and Salas, 2013). Antiviral agents against ASFV are limited; therefore, new agents must be developed.In recent years, the antiviral activity of natural small-molecule drugs has been increasingly investigated. GS-441524, a 1-cyano-substituted adenine C-nucleoside ribose analogue (Amirian and Levy, 2020), is phosphorylated into nucleoside monophosphate by cell kinases and then transformed into an active triphosphate metabolite (NTP structural analogue). In the context of viral RNA synthesis, active NTP analogs compete with natural nucleosides (ATP, TTP, CTP, and GTP) (Yan and Muller, 2020). Different RNA polymerases have different selectivities for GS-441524, and viral RdRp cannot distinguish NA-TPs from endogenous NTPs. Thus, when active GS-441524 metabolites are inserted into a viral RNA strand, the viral RNA transcription process is halted (Agostini et al., 2018; Brown et al., 2019; Lo et al., 2017).

The RNA polymerases of mitochondria in eukaryotic cells have very strong selectivity; however, viral RNA polymerase cannot distinguish GS-441524 from natural nucleotides. Therefore, GS-441524 does not affect cell transcription but inhibits viral transcription (Bimonte et al., 2020; Salvi and Patankar, 2020). This drug exhibits strong antiviral activity against various viruses including severe acute respiratory syn- drome caused by coronavirus (Cho et al., 2020), Ebola virus, and feline coronavirus (causative agent of feline infectious peritonitis). For instance, in an in vitro model, GS-441524 inhibited the replication of type II feline infectious peritonitis (fipv-wsu-79-1146) in CrFK cells; the EC50 was low (0.78 μM), and no cytotoxicity was observed at the highest tested concentration (100 μM) (Murphy et al., 2018; Pedersen et al., 2019; Tchesnokov et al., 2019). Although GS-441524 has been reported to be active against various RNA viruses, no studies have evaluated whether GS-441524 affects DNA viruses. Here, we tested the antiviral potential of GS-441524 against ASFV in vitro.

2.Material and methods
2.1.Cells and viruses
ASFV genotype II can replicate in porcine alveolar macrophages (PAMs), peripheral blood mononuclear cells, and bone marrow cells (Carrillo et al., 1994). We used PAMs in the present study, which were obtained from 4-week-old specific pathogen-free pigs (Borca et al., 2019). The ASFV strain used was GZ201801 (GenBank accession num- ber MT496893.1), with virus titers calculated using the Reed Muench method. All operations involving ASFV in the present study were carried out in a biosafety level-3 laboratory at South China Agricultural Uni- versity (Guangzhou, China).

2.2.Antibodies, chemicals, and reagents
The mouse monoclonal antibody P30 was deposited in the Depart- ment of Infectious Diseases, School of Veterinary Medicine, South China Agricultural University. Fluorescein isothiocyanate-goat anti-mouse IgG (A0568), horseradish peroxidase-labeled goat anti-mouse IgG (A0216), an anti-β-actin mouse monoclonal antibody (AF003), a 3,3N-diamino- benzidine tertrahydrochloride (DAB) Horseradish Peroxidase Color Development Kit (P0203), CCK8 cytotoxicity kit (C0037), and bicin- chonininc acid protein concentration determination kit (P0012s) were purchased from Beyotime Biotechnology (Shanghai, China). GS-441524 was provided by Gilead Sciences (Foster City, CA, USA). GS-441524 was diluted to 15 mg/mL in 5% ethanol, 30% propylene glycol, 45% PEG
400, and 20% water (pH 1.5).

2.3.Cell cytotoxicity assay
The CCK8 assay was used to determine the cytotoxicity of GS- 441524. Briefly, PAMs were distributed into 96-well plates, and GS- 441524 was added [in RPMI1640 medium with 10% fetal bovine serum (FBS)] to final concentrations of 800, 400, 200, 100, 50, and 20 μM. The experiments were included with three replicates and a blank control was included. Cells were incubated for 72 h, and then CCK8 was added and incubated for 1 h; the absorbance was measured at 450. Cytotoxicity was calculated as per the following formula: 1- (OD drug/ OD blank). The drug concentration leading to 50% cytopathy (CC50) was calculated using GraphPad Prism 8 software (GraphPad, Inc., La Jolla, CA, USA).

2.4.Anti-entry assays
To evaluate virus attachment, PAMs in a 12-well cell culture plate (2× 105 cells/well) were incubated with ASFV [multiplicity of infection (MOI) = 0.1] and different concentrations of GS-441524 at 4 ◦C for 1 h to allow virus binding but prevent virus internalization. The virus solution was then discarded, and the cells were washed three times with pre-cooled phosphate-buffered saline (PBS). Virus infection was then detected by real-time PCR.To evaluate virus internalization, the cells were plated and incubated with ASFV as described in the previous paragraph. Next, the cells were washed three times with cold PBS. Different concentrations of GS-441524 were added to each sample. The cell plates were incubated at 37 ◦C for 2 h. The drugs were then discarded, and the cells were washed three times with PBS, after which RPMI1640 medium containing 10% FBS was added to the cells. The cell plates were then incubated at 37 ◦C for 72 h. Finally, virus infection was detected by real-time PCR.

2.5.Viricidal assay
To evaluate viral replication, the PAMs were spread into 6-well plates (2 × 106 cells/well) and 12-well plates (2 × 105 cells/well). After the cells had completely adhered to the plate wall, ASFV solution (MOI = 0.1) was added and incubated in a 37 ◦C incubator for 2 h to ensure that the virus was completely adsorbed and penetrated. The virus solution was discarded, and different concentrations of GS-441524 were added to each sample. A negative control without the drug was included, and the virus was placed with the PAMs. The cells were incubated at 37 ◦C for 48 h. Virus infection was detected by indirect immunofluo- rescence, real-time PCR, western blotting, and virus titer assay.

2.6.Time-of-addition assay
ASFV solution (MOI = 0.1) was added to adherent PAMs and placed in a 37 ◦C incubator for 2 h to ensure complete viral penetration. The virus solution was then discarded, and 10% FBS culture medium was added (0 h). As the replication cycle of ASFV is 18 h, GS-441524 (100 μM) was added to different wells at different time points (0, 3, 6, 9, 12 or 18 h). Negative control cells were not treated with the. The cells were cultured at 37 ◦C for 48 h, and then frozen and thawed at —20 ◦C three times for real-time PCR analysis.

2.7.Time-of-infection assay
ASFV solution (MOI = 1) was added to adherent PAMs and incubated at 4 ◦C for 1 h to ensure the virus was completely adsorbed by the cells.
After discarding the virus and washing plates with PBS three times, 10% FBS in medium or 100 μM GS-441524 (0 h) was added. The cells were then cultured at 37 ◦C and collected at 0, 1, 3, 6, 9, 12, and 16 h after infection for real-time PCR analysis of CP204L and B646L.

2.8.Quantification of antiviral cytokines and ATP
Four different groups were used: an ASFV infection group (MOI = 0.1), ASFV infection (MOI = 0.1) and GS-441524 (100 μM) treatment group, GS-441524 (100 μM) treatment group, and negative control group. After 48 h of incubation, the RNA was extracted from each group of PAMs, and the levels of IFN-α, IFN-β, GAPDH, and IL-6 were deter- mined by RT-PCR. The ATP content in each group was determined using an ATP assay kit (Beyotime Biotechnology Shanghai, China) according to the manufacturer’s instructions.

2.9.Effect of ATP on GS-441524-induced inhibition of ASFV
Ten different groups were used: ASFV, GS-441524 (100 μM) and ATP groups (1–50 μM). Briefly, ASFV-infected (MOI = 0.1) cells were treated with GS-441524 and different concentrations of ATP for 48 h. Next, DNA and RNA were extracted from the cells in each group and analyzed by reverse-transcription real-time PCR.

The cells were then fixed with 4% paraformaldehyde at 20 ◦C and permeabilized with 1% (v/v) TritonX-100/PBS for 30 min. The cells
were then blocked with 2% bovine serum albumin for 1 h. An anti-P30 (1:200) monoclonal antibody was used as the primary antibody, and fluorescein isothiocyanate goat anti-mouse IgG was used as the sec- ondary antibody; the nucleus were counter-stained with 2-(4-amino- phenyl)-6-indolecarbamidine (DAPI). Immunofluorescence was detected using a Nikon DMI 4000b fluorescence microscope (Tokyo, Japan).

2.11.Quantitative real-time PCR (qPCR)
Total RNA was extracted from the cells using a total RNA rapid extraction kit (Fastagen, China, Shanghai) according to the manufac- turer’s instructions; RNA in each sample was reverse-transcribed into cDNA using a reverse transcription kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. The obtained cDNAs were used as templates for qPCR on a CFX96 real-time polymerase chain reaction system (Bio-Rad, Hercules, CA, USA) using TB green premix ex Taq II (TLI RNaseH plus) or premix ex-Taq (probe Q-PCR) (Takara). mRNA expression was assessed in each sample in triplicate, and GAPDH was used as an endogenous negative control. The expression of each target gene was calculated using the 2—ΔΔCT method. The sequences of the primers and probes used in this study are listed in Table 1.

2.12.Western blotting
The cells were washed twice with cold PBS and then lysed with RIPA lysis buffer (Beyotime) containing 1% protein inhibitor (Apexbio, Houston, TX, USA). A bicinchoninic acid kit was used to determine the total protein concentration of each sample to ensure consistency in protein loading. The protein samples were separated by 12% alkyl sul- fate 10% polyacrylamide gel electrophoresis and transferred onto pol- yvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% bovine serum albumin and incubated with an anti-P30 (1:1000) monoclonal antibody. Horseradish peroxidase-labeled goat anti-mouse IgG (1:1000) was used as the sec- ondary antibody. A DAB Horseradish Peroxidase Color Development Kit was used to visualize proteins on PVDF membranes incubate with sec- ondary antibodies. A Tanon-5200 multi infrared imaging system (Shanghai Tianneng Technology Co., Ltd.) was used to analyze the protein staining on PVDF membranes.

2.13.Statistical analysis
All data are representative of at least two independent experiments,in which measurements were performed in triplicate; data are shown as the mean ± standard deviation. All target genes were compared with the reference gene GADPH, and relative expression was calculated by the 2-△△Ct method. The results were analyzed using GraphPad Prism 8.0.1 software. Statistical relevance was assessed by one-way analysis of variance, followed by Tukey’s post-hoc analysis: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 refer to the different levels of statistical significance. 3.Results 3.1.Cytotoxicity of GS-441524 towards PAMs The cytotoxicity of GS-441524 toward PAMs was investigated in a CCK8 assay. High concentrations of GS-441524 showed an obvious cytotoxic effect towards PAMs (Fig. 1A); for example, at 400 μM, the cell survival rate was only 30%. However, when the concentration of GS- 441524 was less than 200 μM, the cell survival rate was more than 85%, with no obvious difference in cell morphology compared to in the untreated group. Notably, the CC50 of GS-441524 was calculated as 287.51 μM (Fig. 1B). Importantly, to reduce cytotoxic effects towards PAMs, 200 μM was selected as the maximum concentration of GS- 441524. The molecular formula of GS-441524 is shown in Fig. 1C. 3.2.Effect of GS-441524 on viral attachment and internalization The relative expression levels of B646L and GAPDH in the group treated with GS-441524 were measured and compared with those in the control group (The concentration of GS-441524 was 0). The results showed that ASFV B646L transcription was not inhibited by GS-441524. This suggests that the drug did not affect virus attachment or its inter- nalization into PAMs (Fig. 2A, and B, respectively); regardless of the concentration of GS-441524, the expression of ASFV B646L did not significantly differ from that in the untreated group. 3.3.Effect of GS-441524 on viral replication Next, the effect of GS-441524 on viral replication was investigated; PAMs were infected with ASFV at 37 ◦C for 2 h and treated with different concentrations of GS-441524. This incubation time was used to ensure complete viral adsorption and entry, which enabled analysis of only the effect on viral replication. Remarkably, the virus titers decreased significantly with increasing drug concentrations, with 0.4, 1.1, 2.3, and 3.3 log 10 reductions in the presence of 20, 50, 100, and 200 μM ASFV, respectively (Fig. 3A). In line with these results, ASFV B646L mRNA levels also decreased significantly with increasing drug concentrations; in fact, 100 μM GS-441524 downregulated B646L mRNA levels by more than 50% compared to in the untreated group (Fig. 3B). The same results were observed when we evaluated the expression of ASFV P30 protein by indirect immunofluorescence (Fig. 3C). A clear fluorescence signal was observed, and the fluorescence density was very high in the un- treated group. In contrast, the fluorescence intensity was significantly decreased after drug treatment and was no longer detected at a drug concentration of 200 μM (Fig. 3C). Notably, the inhibition rate of viral replication was dose-dependent, with an EC50 of 73.2 μM, as calculated using the ImageJ software (NIH, Bethesda, MD, USA) (Fig. 3D). Importantly, the same results were observed by western blot analysis; the expression of the viral P30 protein expression was dose-dependently. Fig. 1. Cytotoxicity and CC50 of GS-441524 on PAMs. (A) Cytotoxicity of GS-441524 toward PAMs was analyzed by CCK8 assay. The relative viability of PAMs cultured without GS-441524 was set to 100%. Each value represents the average of three independent experiments. (B) CC50 of GS-441524 in PAMs was calculated.(C) Chemical structure of ginsenoside GS-441524. Statistical significance is denoted by ****P < 0.0001. Fig. 2. Effects of GS-441524 on viral attachment (A) and internalization (B). ASFV B646L mRNA levels were analyzed by RT qPCR. downregulated, with extremely low levels at a dose of 200 μM (Fig. 3E). These results indicate that GS-441524 inhibits the synthesis of ASFV mRNA and proteins during replication. 3.4.Effect of GS-441524 on the ASFV replication cycle The effects of GS-441524 on the replication of ASFV were further analyzed. We added 100 μM GS-441524 to the culture medium at 0, 1, 3, 6, 9, 12, and 18 h after viral entry into the PAMs. As expected, addition of GS-441524 significantly inhibited the replication of ASFV and viral replication was significantly downregulated, particularly at early time points. Results in PAMs incubated with the virus for 2 h at 37 ◦C were recorded as 0 h results. Addition of GS-441524 at 0 and 1 h showed the most obvious inhibitory effect on viral replication, indicating that GS- 441524 is more effective at early stages of replication (Fig. 4). These results indicate that GS-441524 can block the early, middle, and late stages of ASFV infection. 3.5.GS-441524 inhibits the transcription of early and late ASFV genes GS-441524 significantly inhibited the early transcription of CP204L and B646L in ASFV. GS-441524 obviously inhibited the transcription of the early gene CP204L at 1 h post-infection (hpi), showing more sig- nificant effects over time. GS-441524 did not inhibit the transcription of B646L until 6 hpi (Fig. 5). This may be because the transcription of B646L did not start in the early and late stages of ASFV replication. These results indicate that GS-441524 significantly inhibited the tran- scription of ASFV early and late genes. 3.6.Effect of GS-441524 on antiviral cytokine and ATP levels IFN-α, IFN-β, TNF-α, and IL-6 are important anti-ASFV cytokines (Takamatsu et al., 2013). Additionally, ATP is expected to thought with GS-441524 during viral transcription Therefore, to further explore the anti-ASFV mechanism of GS-441524, the effects of GS-441524 on the levels of important antiviral cytokines and ATP were analyzed. Unex- pectedly, there was no difference in the levels of the four antiviral cy- tokines evaluated (IFN-α, IFN-β, TNF-α, and IL-6) between cells treated with GS-441524 and control (non-treated) cells; this was also observed in the absence or presence of ASFV (Fig. 6A–D). Therefore, although we observed a significant increase in the expression of antiviral cytokines in the presence of ASFV, this was virus-specific rather than drug-specific (Fig. 6D); in other words, the mechanism of action of GS-441524 is not related to the levels of IFN-α, IFN-β, TNF-α, and IL-6.In contrast, there was a significant difference in ATP concentration in infected cells not treated with versus treated with GS-441524 (Fig. 6E). These results suggest that the levels of ATP vary in response to both viral infection and GS-441524 treatment, as the treatment alone condition also suggests (Fig. 6E). Fig. 3. Effect of GS-441524 on ASFV replication. ASFV-infected PAMs (MOI = 0.1) were cultured at 37 ◦C for 2 h, and then treated with different con- centrations of GS-441524. (A) Titers of ASFV decreased significantly in a dose-dependent manner after GS-441524 treatment. (B) ASFV B646L mRNA levels were analyzed by RT qPCR. (C) Antiviral ac- tivity of GS-441524 against ASFV strains was also determined in PAMs by immunofluorescence assay. Briefly, PAMs were seeded into 12-well plates, and then incubated with RPMI1640 supplemented with the indicated concentrations of GS-441524. ASFV was detected by immunofluorescence using a mouse anti-P30 antibody at 48 h post-infection; the nuclei were counter-stained with DAPI (blue). (D) EC50 of GS-441524 in ASFV-infected PAMs. (E) Expression of P30 in the presence of different concentrations of GS-441524 was also evaluated by western blotting.Statistical significance is denoted by * P < 0.05, ***P < 0.001, and ****P < 0.0001. Fig. 4. GS-441524 acts at early stages post-infection to decrease ASFV RNA levels. PAMs were infected with ASFV (MOI = 0.1) and treated with 100 μM GS- 5734 at the indicated times post-infection. ASFV B646L mRNA levels were analyzed by RT qPCR. Statistical significance is denoted by ***P < 0.001 and ****P < 0.0001. 3.7.Low-dose ATP promotes replication of ASFV and attenuates the inhibitory effect of GS-441524 on ASFV Interestingly, we found that 1, 5, 10, and 20 μM ATP promoted the replication of ASFV and weakened the inhibition of ASFV. In fact, when the concentration of ATP was 5, 10, and 20 μM, inhibition of ASFV was significantly reduced in a dose-dependent manner. Different results were observed when the concentration of ATP reached 50 μM; however, inhibition of ASFV was still significantly reduced. These results suggest that ATP promotes the synthesis of ASFV mRNA (Fig. 7). Importantly, this is supported by the increase in viral levels in untreated cells in the presence of ATP (Fig. 7). As a natural nucleoside, ATP participates in the transcription of viral replication (Fig. 7). 4.Discussion ASFV was first reported in Kenya in 1921. At the end of 2018, ASF emerged for the first time in Shenyang, China (L. K. L. K. Dixon et al., 2019; Tao et al., 2020), causing considerable losses to China’s pig in- dustry. Currently, there are no safe and effective vaccines against ASFV available on the market. Therefore, it is very important to identify new antiviral drugs for controlling ASFV. Remdesivir, a broad-spectrum antiviral drug, has shown extensive inhibitory potential against various RNA viruses by inhibiting viral replication (Gordon et al., 2020). However, no studies have reported the effect of Remdesivir on DNA Fig. 5. Effect of GS-441524 on ASFV replication at different time points. PAMs were infected with ASFV (MOI = 1) for 1 h at 4 ◦C and then cultured in fresh medium supplemented with 100 μM GS-441524. The expression levels of ASFV CP204L and B646L in PAMs were detected by RT-qPCR analysis at the indicated time points. Statistical significance is denoted by * P < 0.05, **P < 0.01, ***P< 0.001, and ****P < 0.0001 viruses. Here, we demonstrated that GS-441524 inhibited a nucleocy- toplasmic large DNA virus, ASFV, in vitro.In this study, PAMs were used as host cells. Our results showed that the CC50 of GS-441524 on PAMs was 287.5 μM; therefore, we used doses of ≤200 μM. ASFV infects cells through receptor-mediated endocytosis; therefore, infection depends on three main processes: adsorption, internalization, and replication (Sa´nchez et al., 2017). Interestingly, we observed that GS-441524 did not impact the adsorption and internali- zation of ASFV but inhibited its replication. At a concentration of 20 μM,GS-441524 reduced the infection ability of ASFV, as detected at both the transcription and protein levels. Additionally, with increasing drug concentrations, the replication and infection ability of ASFV became increasingly weaker; the EC50 of GS-441524 was 73.2 μM. Earlier addition of the drug had a more obvious inhibitory effect on ASFV, which is consistent with the mechanism of GS-5734 (Agostini et al., 2018), indicating that the drug acted on the whole transcription process of ASFV. The early transcription gene CP204L of ASFV was inhibited at 1 hpi, whereas the late expression gene B646L was inhibited at 6 hpi. This indicates that once a viral gene begins to be transcribed in large quantities, it is inhibited by GS-441524. Studies have shown that IFN-α, IFN-β, TNF-α, and IL-6 are important anti-ASFV cytokines. We found that GS-441524 did not increase the levels of the abovementioned antiviral cytokines in PAMs; in contrast, ASFV significantly increased the levels of these four cytokines. This is consistent with the results of previous studies (Go´mez del, Razzuoli, Zakaryan et al. (Go´mez del Moral et al., 1999; Razzuoli et al., 2020; Zakaryan et al., 2015). These findings indicate that the GS-441524-mediated inhibition of ASFV replication is not dependent on an immunomodulatory effect. Many studies have focused on GS-441524, but none have reported that GS-441524 can improve the level of antiviral cytokines (Jung et al., 2020; Li et al., 2021). Studies have shown that viral stomatitis virus infection can increase the level of ATP in cells (Zhang et al., 2017). Our results showed that ASFV infection significantly increased the level of ATP in cells compared to that in the mock group. ATP is necessary for viral transcription. Up-regulation of ATP production caused by ASFV infection may be the environmental condition required by ASFV to create a more suitable environment for its own proliferation. This is consistent with the promotion of ASFV repli- cation by low ATP concentrations. How GS-441524 increased the level of ATP remains unclear, but 1.1 μM ATP could not inhibit ASFV. The nucleoside analogue GS-441524 can bind to viral RNA by competing with the natural nucleoside ATP, resulting in the termination of viral transcription. Delayed chain termination was shown to be a plausible mechanism of action of remdesivir against Ebola virus (Tchesnokov et al., 2019). The triphosphate form of GS-441524 (RDV-TP) competes with ATP, the natural substrate of RNA polymer- ase. The selectivity value of RDV-TP obtained here through a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. GS-441524 can effectively inhibit Middle East respiratory syndrome coronavirus in this manner (Gordon et al., 2020). It is thought that increasing the concentration of the natural nucleoside ATP weakens the antiviral effect of GS-441524. Therefore, we further explored the levels of ATP in and effect of ATP supplementation on ASFV-infected GS-441524-treated cells. We found that GS-441524 inhibited ASFV transcription at all time points; thus, we kept the time of ATP addition the same as that of GS-441524 addition. Interestingly, the results showed that supplementation with low con- centrations of ATP promoted ASFV replication and reduced the inhibi- tory effects of GS-441524 on ASFV replication in a dose-dependent manner. This was particularly true at 1–20 μM GS-441524 and less so at 50 μM GS-441524. These results indicate that GS-441524 inhibits ASFV replication by competing with ATP for transcription.Some studies have shown that small-molecule drug nucleoside ana- logs are potential antiviral drugs against ASFV. Iododeoxyuridine, two types of raifs (auy11 and cm1uy11), and other nucleoside analogs showed obvious inhibitory effects against ASFV (Arabyan et al., 2019; Hakobyan et al., 2018). This study confirmed that nucleoside analogs can be used as targets of anti ASFV drugs and provided theoretical support for the development of nucleoside analogs as anti-ASFV drugs. We also found that GS-441524 may inhibit nuclear cytotoxic large DNA viruses. 5.Conclusions Here, we show that GS-441524 significantly inhibits the replication of the DNA virus ASFV. As GS-441524 did not impact the expression of antiviral cytokines, the inhibitory effect of GS-441524 on ASFV in PAMs likely depends on competition with natural nucleosides and consequent termination of ASFV transcription. Importantly, our data support that nucleoside analogs can be used as anti-ASFV drugs and potentially other DNA viruses.