Vero cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FND500, ExCell Bio, Shanghai, China) in a humidified 37°C 5% CO₂ incubator. HSV-1 HF and HSV-1 GFP, initially obtained from Professor Erguang Li of Nanjing University, were propagated in Vero cells. HSV-1 McKrae and HSV-1 F were kindly provided by Dr. Kai Hu, Department of Ophthalmology, Nanjing Drum Tower Hospital. HSV-1 blue, a thymidine kinase (TK) mutant derived from HSV-1 KOS, was a gift from Dr. Tao Peng of State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, and an ACV-resistant clinical HSV-1 strain HSV-1 153 was a kind gift from Prof. Yifei Wang, Institute of Biomedicine, College of Life Science and Technology, Jinan University.

Harringtonine was purchased from MCE (Cat#: HY-N0862, NJ, United States) and dissolved in Dimethyl Sulphoxide at a concentration of 10mM. ACV was purchased from Selleck (Cat#: S1807, Houston, TX, United States). Antibodies against gD (Cat#: sc-69802), ICP4 (Cat#: sc-69809), and HVEM (Cat#: sc-365971) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States), and anti-GAPDH antibody was purchased from Cell Signaling Technology (CST, MA, United States).

The anti-HSV-1 activity of HT or ACV was determined by In-cell Western assay. Vero cells grown in 96-well plates were treated with indicated concentrations of the test compounds for 30min at 37°C. The cells were then infected with HSV-1 multiplicity of infection (MOI=1) with the presence of a test compound. After further incubation for 24h, the supernatant was removed, and the cells were measured by In-cell Western assay of HSV-1 gD protein expression. The 50% inhibitory concentration (IC₅₀) for antiviral activity was calculated as the concentration of antiviral compounds that reduce the virus-induced infection by 50% comparing to the virus infection without compound treatment.

According to the manufacturer’s instructions, the In-cell Western assay was carried out using the Odyssey Imaging System (Li-COR Biosciences, NE, United States). Vero cells cultured in a 96-well plate were either mock-infected or infected with HSV-1 at a given MOI when they reached 80% cell density. After treatment, they were fixed with 4% paraformaldehyde for at least 30min. Then, the cells were permeabilized with 0.5% Triton X-100 for 15min and blocked with blocking buffer (4% non-fat dry milk) for 90min and incubated with gD antibody diluted in blocking buffer (1:500) at 4°C overnight. After being washed three times with PBST, the cells were stained with IRDye IgG (1:2,000) for 1h following 1h DRAQ5 staining and scanned with an Odyssey Infrared Imager. The relative amount of gD protein expression was obtained by normalizing to endogenous DRAQ5 in all experiments. Viral inhibition (%) was calculated as follows: [1−fluorescence_gD/fluorescence_control] * 100%, where “fluorescence_gD” indicates gD protein expression of the infected cells with the compound treatment and “fluorescence_control” indicates gD protein expression of the infected cells without the compound treatment.

Vero cells grown in the 96-well plate were inoculated in triplicate with HSV-1 HF (MOI=1). HT at 0.5μM was treated at −2 (2h before viral inoculation), 0 (instantaneous viral inoculation), 2, 4, 6, 8, 12, and 24h followed by infection with HSV-1. Viral protein level was determined in 96-well plate by In-cell Western assay at the indicated time points.

Herpes simplex virus type 1 HF (1*10⁷ PFU) was diluted in 1ml DMEM at 37°C with or without HT at the indicated concentrations for 2h. After the treatment, the mixture was diluted to 1,000-fold to obtain an equivalent of 10⁴ PFU in 1ml DMEM. HT also was diluted to a non-inhibitory concentration against HSV-1 at this step. The diluted mixture was added to confluent Vero cell monolayers in a 96-well plate and incubated at 37°C for 24h. At the same time, a group of HT at the indicated concentrations was incubated at 37°C for 2h, which was then directly added to the confluent Vero cell monolayer without dilution. After incubation for 24h, the supernatant was removed, and the infected cells were measured by In-cell Western assay.

Vero cells were infected with HSV-1 (MOI=1) with or without HT. About 2h post-infection (hpi), the supernatant was removed, and the new DMEM containing 2% FBS was added, and the culture was maintained for 24h. Then, 10-fold serially dilutions of the virus suspension were used to infect monolayer Vero cells in a 96-well plate for 72h. Each dilution was tested with eight replicates in each experiment, and the wells were observed and scored for the presence or absence of cytopathic effect (CPE). The 50% tissue culture infectious dose (TCID₅₀) was calculated based on the Reed–Muench method (Capelli et al., 2020).

The cells were lysed by adding RIPA lysis buffer supplemented with protease inhibitors in an ice bath for 10min. The lysates were centrifuged at 12,000×g for 10min at 4°C, and the supernatants containing total proteins were prepared. Total protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL, United States). The proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. Proteins were detected by the respective primary antibodies overnight at 4°C and then IRDye IgG (1:10,000 dilution) for 1h. Bands were visualized under Li-COR Odyssey Infrared Imager (Li-COR).

Total RNA was extracted using TRIzol reagent (Life Technologies), and equivalent amounts of RNA (1μg) from each sample were subjected to reverse-transcription using a PrimeScript®RT reagent kit (Takara, Japan) according to the manufacturer’s protocol. Quantitative real time-PCR (qPCR) was performed using the ABI SYBR Green Master Mix (Life Technologies), followed by detecting an ABI Prism 7500 Sequence Detection System. GAPDH was used for the normalization of mRNA, and analysis was carried out using the 2^−ΔΔCt method. The sequences of primers used in this study are listed in Table 1.

Vero cells were seeded in 96-well plates and incubated at 37°C, 5% CO₂ overnight. The supernatant was then removed, and a new medium with increasing doses of HT or ACV was added. After treatment for 48h, the medium was replaced with 100μl of fresh medium containing 10μl of Cell Counting Kit 8 (CCK-8) reagent (Dojindo Laboratories, Kumamoto, Japan) for 2h. OD values at 450nm were measured by a TECAN Infinite M200 microplate reader (Männedorf, Switzerland). The cell viability values for treated cells were normalized with those of untreated cells. The 50% cytotoxic concentration (CC₅₀) was calculated as the concentration of compounds that reduce 50% cell viability comparing to the cells without compound treatment.

Vero cells were seeded on coverslips in 12-well plates. Cells were uninfected or infected with HSV-1 GFP (MOI=1) in the presence of HT. After 24h, cells were fixed with 4% paraformaldehyde for 30min at RT, permeabilized with 0.1% Triton X-100 for 15min, and blocked with 2% BSA for 1h. Cellular proteins were immunolabeled with anti-HVEM antibody overnight at 4°C and then Alexa Fluor 594 (red)-labeled secondary antibody (Life Technologies) for 1h. Nuclei were stained with DAPI (blue). Finally, images were obtained using an Olympus FluoView FV10i confocal microscope (Tokyo, Japan).

Statistical analysis was performed with one-way ANOVA or Student’s t-test as appropriate, using GraphPad Prism 5 (Version 5.01, GraphPad Software). The level of significance was set at ^*p<0.05, ^***p<0.01, or ^***p<0.001.

Vero cells were treated with increasing concentrations of HT and then infected with five different HSV-1 strains, including two ACV-resistant isolates, to determine the effect of HT on HSV-1 infection. ACV was chosen as a positive control in the antiviral experiment. After 24h, the antiviral effects of HT and ACV were determined by In-cell Western assay, and the IC₅₀ values were calculated. As shown in Table 2, HT IC₅₀ values range from 0.1081 to 0.1584μM. HT showed higher antiviral activity for all five HSV-1 strains at the same concentration of ACV and HT than ACV. These results suggested that HT could efficiently inhibit HSV-1 infection. The cytotoxicity of HT and ACV in Vero cells was detected by CCK8 assay (Figure 1B), and the CC₅₀ value of HT in Vero cells was 239.6μM, as shown in Table 2. The selective index (SI) of HT against HSV-1 HF, HSV-1 F, HSV-1 Mckrae, HSV-1 blue, and HSV-1 153 was 2216.5, 1871.9, 1973.6, 1815.2, and 1512.6 in Vero cells, respectively.

Next, we further evaluated the effect of HT on the HSV-1 HF strain. Vero cells were infected with HSV-1 at 1 MOI in the presence of increasing concentrations of HT for 24h. Then, gD protein was measured by In-cell Western assay. As shown in Figure 2A, HT significantly inhibited HSV-1 infection at 0.25–2.5μM concentrations. A progeny virus yield assay was performed to evaluate the effect of HT on HSV-1 virus proliferation. Vero cells were infected with HSV-1 at 1 MOI in the presence of 0, 0.1, and 0.5μM HT for 2h, and the inoculum was replaced with a medium containing HT at the corresponding concentrations for another 24h. The supernatant was collected and added to Vero cell monolayers at 10-fold serially dilutions. Then, we determined the virus titers by a TCID₅₀ assay. As shown in Figure 2B, in untreated-HSV-1-infected cells, the virus titers of HSV-1 were 5.08±0.23 log₁₀ PFU/ml, while in the presence of HT at 0.1 and 0.5μM, the virus titers of HSV-1 were 4.21±0.42 log₁₀ PFU/ml and 1.63±0.29 log₁₀ PFU/ml, respectively. HT at 0.1μM resulted in 0.87 log reduction of HSV-1 titers, and HT at 0.5μM resulted in 3.45 log reduction of HSV-1 titers. HT significantly reduced HSV-1 titers in a dose-dependent manner.

Viral gene and protein expression were also determined to examine the inhibitory activity of HT. Vero cells were infected with HSV-1 HF in the presence of HT at increasing concentrations. After 24h, cells were collected, and cellular proteins were extracted for Western Blot and RNA for qPCR. HSV-1 immediate-early protein ICP4 (infected cell protein 4) and HSV-1 late protein gD (glycoprotein D) were detected in the HSV-1-infected-Vero cells. The results showed that HT reduced the protein levels of viral ICP4 and gD in a dose-dependent manner (Figure 2C). Similar results of ICP4 mRNA and gD mRNA were also obtained in Figure 2D, correlated with Figure 2C.

The most potent antiviral drug currently used to treat HSV-1 is ACV, a nucleoside analog (De Clercq and Field, 2006). Firstly, to compare the antiviral effects of HT with ACV, Vero cells were treated with the same concentrations of HT and ACV, and then they were infected with HSV-1 HF (MOI=1) for 24h. Western Blot assay revealed that viral proteins ICP4 and gD were decreased by more than 50 and 80% in HT-treated-HSV-1-infected cells, respectively. However, the proteins of ICP4 and gD were reduced by only 20% in ACV-treated-HSV-1-infected cells (Figure 3A). Vero cells were also infected with HSV-1 GFP at 1 MOI in the presence of HT and ACV, respectively. After 24h, HT-treated cells and ACV-treated cells were imaged by a fluorescence microscope. We found that the fluorescence intensity of HT-treated cells was markedly weaker than that of ACV at all corresponding concentrations, which indicated that HT inhibited HSV-1 replication more potently than ACV (Figure 3B).

We further evaluated the antiviral effect of HT against ACV-resistant strains, including HSV-1 blue and HSV-1 153 (Wang et al., 2011). In the presence of HT or ACV, Vero cells were infected with HSV-1 blue or HSV-1 153 for 24h, and the viral protein gD was analyzed by In-cell Western assay, and the bands’ density was scanned by a densitometer. As shown in Figure 3C, the IC₅₀ and IC₉₀ values of HT inhibition of HSV-1 blue infection were 0.1320 and 0.7832μM, respectively, and the IC₅₀ and IC₉₀ values for HSV-1 153 were 0.1584 and 1.0134μM, respectively. As expected, both viruses resisted ACV inhibition at a concentration up to 5μM, consistent with previous reports (Li et al., 2019). These results demonstrated that HT not only more potently inhibited HSV-1 replication but also suppressed ACV-resistant strains HSV-1 blue and HSV-1 153.

To investigate whether HT directly inactivates HSV-1 virions, we performed a virucidal assay. In group a, HSV-1 was co-incubated with HT at inhibitory concentrations of 1.0, 2.5, and 5.0μM, respectively, at 37°C for 2h, and the mixtures were diluted to non-inhibitory concentrations of 0.001, 0.0025, and 0.005μM of HT and were added to Vero cells. In group b, HSV-1 was incubated alone at 37°C for 2h before being diluted to the same viral inoculum as group a, and then the virions were added to Vero cells with HT at 0.001, 0.0025, 0.005μM. In group c, HSV-1 infection was performed as group b, but the concentration of HT was adjusted to viral inhibitory concentrations of 1, 2.5, and 5μM, respectively. In-cell Western assay was performed to detect the viral gD protein expression after 24h. There was no difference between groups a and b, which showed that the infectivity of HT-virus co-incubation was similar to that of HT and virus without co-incubation (Figure 4A). This result suggested that the pre-treatment of the virus with HT had no virucidal effect on HSV-1 virions. Next, to explore the potential mechanism of HT antiviral activity, we conducted the time-of-addition assay. HT at 0.5μM was added to Vero cells at various time points of −2, 0, 2, 4, 6, 8, 12, and 24hpi. After treatment for another 2h, viral protein gD was detected by In-cell Western assay. As shown in Figure 4B, HSV-1 replication was almost completely inhibited when HT was added at −2, 0, and 2hpi. However, with the time lag of the drug, the antiviral effect gradually decreased. This result suggested that HT may act at an early stage of HSV-1 replication.

The early steps of HSV-1 infection include adsorption, entry, and intracellular transport. Membrane fusion starting from gD binding to the specific cellular receptors is a critical step of HSV-1 entry into host cells (Nicola et al., 2005). We investigated the effect of HT on four major gD receptors, including HVEM, nectin-1, nectin-2, and 3-O-S HS. 3-OST-3 is the particular enzyme that generates the modified form of 3-OS HS (Shukla et al., 1999). Vero cells were treated with HT at the indicated concentrations for 4h, and the mRNA levels of HVEM, nectin-1, nectin-2, and 3-OST-3 were measured by qPCR analysis. Results showed that HVEM mRNA levels were significantly reduced after treatment with different concentrations of HT. However, the mRNA levels of nectin-1 and nectin-2 were not affected significantly in the presence of HT. At the highest concentration of 0.5μM but not at the lower concentrations of 0.1 and 0.2μM, the mRNA level of 3-OST-3 was reduced by the treatment of HT. Only HVEM was downregulated in HT-treated cells in a dose-dependent manner, and it showed a decrease of HVEM about 40% at 0.1μM, 70% at 0.2μM, and 85% at 0.5μM compared to untreated cells (Figure 5A). After HSV-1 infection, the mRNA level of HVEM was upregulated, as shown in Figure 5B. While in the presence of HT, HVEM was decreased in HSV-1-infected-cells, and HT also showed a dose-dependent suppression of HVEM (Figure 5B). We further examined the protein levels of HVEM after HT treatment by Western Blot analysis. HT at 0.2μM reduced 20% of HVEM protein, and HT at 0.5μM reduced 40% of HVEM compared to the untreated cells. Similar results of WB showed that HVEM was upregulated with the infection of HSV-1. After treatment with HT, HVEM was suppressed in HSV-1-infected-cells compared to untreated cells (Figure 5C).

Moreover, HT-induced downregulation of HVEM protein was further confirmed by immunofluorescence assay. As shown in Figure 5D, the fluorescence intensity of HVEM was reduced in either HSV-1-infected or uninfected cells after HT treatment. These results together suggested that inhibition of HVEM by HT may interrupt viral membrane fusion and virus entry. Taken together, HT blocked the entry receptor HVEM of HSV-1, thereby affecting the viral replication.