Narciclasine inhibits vaccinia virus infection by activating the RhoA signaling pathway

Highlights

• •Significance of the study: Our findings emphasize the role of narciclasine in inhibiting vaccinia virus (VACV), probably by activating RhoA. This study lays the groundwork for developing potential drugs targeting mpox virus (MPXV).
• •Scientific question: The global spread of the MPXV poses a significant public health threat. Although tecovirimat is the primary treatment for MPXV, instances of resistance to this drug have been reported in patients. What strategies can be employed to develop new inhibitors against MPXV?
• •Evidence before this study: Previous research on narciclasine has mainly focused on its application in cancer treatment, with occasional reports on its inhibitory effects on certain viruses. Narciclasine is known to activate the RhoA signaling pathway, promoting the formation of actin stress fibers. VACV serves as the prototype for poxvirus research. Inhibition of RhoA signaling is essential for VACV morphogenesis and extracellular enveloped virus (EEV) production.
• •New findings: This study presents novel insights into narciclasine. We discovered that narciclasine inhibits VACV EEV formation and subsequent viral spread. Furthermore, our research indicates that narciclasine-induced activation of RhoA suppresses VACV replication. These results suggest that RhoA agonists could potentially inhibit VACV.

Abstract

In 2022, a sharp rise in global cases of mpox virus (MPXV) led the World Health Organization (WHO) to declare it a public health emergency of international concern. However, progress in developing drugs targeting MPXV has been slow. Here, we investigate the natural alkaloid narciclasine as a potential inhibitor of poxviruses. Our investigation demonstrates that narciclasine at 40 nmol/L (nM) to 160 nM dosages effectively blocks vaccinia virus (VACV), a representative poxvirus. Specifically, narciclasine disrupts the production of extracellular enveloped virus (EEV), which is crucial for viral spread. Narciclasine’s antiviral impact is probably attributed to its activation of the RhoA signaling pathway. This study highlights narciclasine’s potential as a promising new therapeutic candidate against poxviruses, offering prospects for its development into a potent antiviral agent that is essential for combating emerging poxvirus outbreaks.

1. Introduction

In 2022, a notable rise in global cases of mpox virus (MPXV) prompted the World Health Organization (WHO) to declare it a public health emergency of international concern [1]. In 2023, there has been a notable increase in cases across the Western Pacific region, which includes China, Japan, and the Republic of Korea [2]. This surge has sparked global concern, emphasizing the need for coordinated efforts to curb its spread and minimize its impact on global health. Tecovirimat (ST-246) is currently the primary inhibitor for mpox, targeting the poxvirus F13 protein [3]. However, recent studies have reported cases of tecovirimat resistance in mpox patients [4], [5], [6], [7]. Hence, there is an urgent need to develop new inhibitors against mpox.

Orthopoxviruses belong to a family of double-stranded deoxyribonucleic acid (DNA) viruses encompassing various species, such as variola virus, vaccinia virus (VACV), MPXV, and cowpox virus. As of 2023, the International Committee on Taxonomy of Viruses (ICTV) recognizes 22 genera and 83 species within the Poxviridae family, capable of causing a broad spectrum of diseases in humans and animals [8]. Within the Orthopoxvirus genus alone, over 12 species are identified, including highly pathogenic viruses like variola virus and MPXV. Historically, smallpox has been the deadliest infectious disease, causing an estimated 300 million deaths in the first eight decades of the 20th century alone. Despite its eradication in 1980, waning herd immunity following the cessation of smallpox vaccination and the absence of cross-protection against other Orthopoxviruses have increased susceptibility to such infections [9], [10].

Consequently, other Orthopoxviruses represent significant threats to public health. The current MPXV outbreak underscores its pandemic potential, with future outbreaks anticipated. Over time, this virus could evolve to better adapt to humans, potentially resulting in more severe consequences [11].

VACV is a laboratory-adapted Orthopoxvirus with uncertain origins. Unlike other naturally occurring Orthopoxviruses, VACV typically causes mild viral illness in humans and mice [12]. It features a large double-stranded DNA genome spanning 192 kb, encoding over 200 proteins critical for viral functions such as host cell entry, transcription, DNA and ribonucleic acid (RNA) synthesis, viral particle assembly, and evasion of host immune responses [13]. The replication cycle of VACV begins with the activation of viral genes, leading to core breakdown and DNA replication, followed by the formation of crescent-shaped membranes. VACV exists in four infectious forms: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV) [14]. IMV is a resilient and stable virion highly effective in transmitting infection between hosts. IEV is created by wrapping IMV with intracellular membranes, serving as an intermediate between IMV and CEV / EEV, facilitating efficient virus transport to the cell surface along microtubules [15]. The spread of VACV in vivo is believed to occur through EEV [14].

Narciclasine (C14H13NO7), also known as lycoricidinol, is a natural compound derived from the Narcissus species (Amaryllidaceae) (Fig. 1A) [16]. Throughout different cultures, including Chinese, North African, Central American, and Arabian, Narcissus species have been historically recognized for their potential medicinal properties in combating cancer [16]. However, narciclasine demonstrated only minimal pharmacological activity in animal tumor models. Narciclasine selectively induces apoptosis in cancer cells by activating the death receptor pathway [17]. Its mechanism of action involves specific pathways: in glioblastoma cells, narciclasine activates the small GTPase RhoA, forming actin stress fibers [18]. Meanwhile, melanoma cells bind the eukaryotic translation elongation factor eEF1A, disrupting cell division and protein synthesis [19].

Fig. 1.

Narciclasine inhibits VACV viral protein level. A) Structure of narciclasine. B) Cell viability in the presence of narciclasine. HeLa, Huh7.5.1, or BSC-1 cells were exposed to varying concentrations of narciclasine for 24 h. Cell viability was assessed using the CCK8 assay. The bar chart shows mean ± standard deviation (n = 5). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. C) – E) Narciclasine inhibits viral protein expression in HeLa, Huh7.5.1, and BSC-1 cells. HeLa (C), Huh7.5.1 (D), or BSC-1 cells were infected with A4-YFP VACV (MOI = 3) for 2 h, followed by treatment with varying concentrations of narciclasine. Western blot analysis was performed to evaluate viral protein expression, with GAPDH as a loading control. Data are presented as mean ± standard deviation from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. F) Narciclasine reduces viral protein expression in BSC-1 cells. After incubation with A4-YFP VACV for 1 h, BSC-1 cells were treated with 80 nmol/L (nM), 160 nM, or 320 nM narciclasine for 24 h and analyzed by immunofluorescence microscopy. A4-YFP VACV is shown in green. Scale bar, 275 μm. Abbreviations: VACV, vaccinia virus; BSC-1, biologics standards-cercopithecus-1; CCK8, Cell Counting Kit 8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MOI, multiplicity of infection; DMSO, dimethylsulfoxide; YFP, yellow fluorescent protein.

RhoA, a member of the small Rho-GTPase family, plays a critical role in regulating the cytoskeleton. Suppression of RhoA signaling is essential for both vaccinia morphogenesis and EEV release [20], [21], [22]. Recent studies have highlighted the potential antiviral properties of RhoA agonists. Narciclasine demonstrates potent antiviral effects on dengue virus type 4 (DENV-4) and zika virus (ZIKV) at very low concentrations [the half maximal effective concentration (EC₅₀) of 0.02 µmol/L (µM) for both viruses], with a half maximal concentration of cytotoxicity (CC₅₀) in the low micromolar range[23].

In this study, we investigate the potential of narciclasine to inhibit VACV infection in a cell culture model. Our discoveries may lead to innovative strategies for developing anti-poxvirus drugs targeting RhoA.

2. Materials and methods

2.1. Cell and virus

Biologics standards-cercopithecus-1 (BSC-1) and HeLa cells are bought from Cobioer Biosciences Co., LTD. Huh7.5.1 cell is a gift from Dr. Francis Chisari. HeLa, Huh7.5.1, and BSC-1 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM, KGL1208-500, KeyGen BioTECH, China) with high glucose, supplemented with 10% fetal bovine serum (FBS, ExCell, FSP500), 1% penicillin–streptomycin 100 × solution (HyClone, SV30010, US). All cell lines were maintained at 37 °C in a 5% CO₂ atmosphere. The Western Reserve (WR) strain of VACV and A4-yellow fluorescent protein (YFP) virus (a variant of the WR strain where the viral A4 protein is fused with YFP) were from Dr. Bernard Moss and cultured in HeLa cells using DMEM supplemented with 10% FBS.

2.2. Plasmid and chemical

The RhoA plasmid was procured from SinoBiological. Narciclasine (TargetMol, TQ0183, US) was dissolved in dimethylsulfoxide (DMSO) (Solarbio, D8371, China) to create a 10 mmol/L (mM) stock solution, which was stored at −80 °C. Before cell treatment, this stock solution was diluted further in a cell culture medium with a maximum DMSO concentration of 0.04%.

2.3. Antibodies

The antibodies utilized in this study included mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 60004–1-Ig, Wuhan) and anti-GFP (XHY038L, Beijing, China), as well as rabbit RhoA Rabbit PolyAb (Proteintech, Wuhan, China), rabbit anti-FLAG (XHY, Beijing, China), and rabbit anti-GFP (XHY, Beijing, China). Western blot detection was performed using horseradish peroxidase (HRP)-conjugated Goat anti-mouse IgG and HRP-conjugated Goat anti-rabbit IgG (Proteintech, Wuhan, China). Alexa Fluor 647-conjugated secondary antibodies are from Thermo Fisher Scientific Inc (Waltham, MA, US).

2.4. Cell viability assay

Cell viability was measured by the Cell Counting Kit 8 (CCK8) (Proteintech, PF00004) according to the manufacturer's protocol. CCK8 s enables rapid detection using the water-soluble tetrazolium salt WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)–2H-tetrazolium monosodium salt]. The principle is that dehydrogenases can reduce WST-8 in the mitochondria to generate an orange-yellow formazan dye in the presence of an electron coupling reagent. Colorimetric analysis allows for the dynamic quantification of viable cells, facilitating cell proliferation assessments or drug toxicity. To conduct the CCK8 assay, begin by inoculating each well of a 96-well plate with 100 μL of cell suspension and incubate in a humidified environment (e.g., at 37 °C with 5% CO₂). Next, add 10 μL of the CCK8 solution to each well. Allow the plate to incubate for 1 to 4 h. Finally, measure the absorbance at 450 nm using a microplate reader.

2.5. A4-YFP protein expression measurement by fluorescence microscope

BSC-1 cell cultures were grown until they reached 80% confluence, then infected with A4-YFP VACV in a medium supplemented with 2% FBS. After 2 h, the medium was replaced with one containing 10% FBS. After 12 h incubation, A4-YFP expression was visualized using an EVOS M7000 microscope (US).

2.6. GTP-RhoA activity

HeLa cells were infected with the indicated viruses and replaced with 10% medium supplemented with 40–320 nM narciclasine after 2 h and incubated for 24 h at 37 °C under 5% CO₂ before conducting the glutathione-S-transferase (GST) pulldown assay by GST-Rhotekin-RBD.

2.7. Immunofluorescence microscopy

In brief, cells were seeded on glass coverslips, washed with phosphate buffered saline (PBS), and fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. After fixation, cells were treated with a blocking solution (PBS with 10% normal goat serum) for 5 min at room temperature. Subsequently, coverslips were incubated with primary antibodies in permeabilization buffer (0.3% Triton X-100 in PBS with 10% normal goat serum) for 1 h. Following three washes with blocking solution, coverslips were incubated with Alexa Fluor 647-conjugated secondary antibodies or phalloidin-rhodamine (Beyotime, Shanghai, China) for 1 h at room temperature. After three additional washes with blocking solution, coverslips were mounted with a 4',6-diamidino-2-phenylindole‌ (DAPI)-containing mounting medium. Imaging was conducted using an Olympus-FV12-IXCOV microscope (Japan) with a 100 × oil immersion lens.

2.8. Plaque assay

BSC-1 cells were grown to 90%–100% confluence. Supernatant samples were collected and serially diluted, then applied to the cells for 2 h at 37 °C. Following inoculation, cells were washed three times with phosphate-buffered saline and overlaid with 2% agarose mixed with 2 × DMEM at a 1:1 ratio. After 72 h of incubation at 37 °C, the agarose was removed, and cells were stained with 0.1% crystal violet for 10 min. Plaques were subsequently visualized.

2.9. Statistical analysis

The statistical analyses were conducted using GraphPad Prism software version 8.0. The two-tailed Student's t-test determined significant differences between treated and control groups. All values are depicted as mean ± standard deviation. A P-value of less than 0.05 was considered statistically significant.

3. Results

3.1. Narciclasine decreased the level of VACV viral protein

Cell viability assays using the CCK8 were performed after 24 h of narciclasine treatment on HeLa or Huh7.5.1 cells to evaluate the cytotoxicity of narciclasine on cells. As shown in Fig. 1B, narciclasine (≤ 320 nM) had no significant effect on HeLa cell viability. However, it slightly reduced the viability of Huh7.5.1 cells, starting from 80 nM and higher concentrations.

To explore the potential inhibition of VACV viral protein levels by narciclasine, HeLa, Huh7.5.1, or BSC-1 cells were infected with A4-YFP VACV at a multiplicity of infection (MOI) of 3 and treated with varying concentrations of narciclasine (40, 80, 160, and 320 nM). The levels of intracellular viral proteins, assessed by A4-YFP, were quantified using Western blot analysis to evaluate narciclasine's inhibitory effects on the vaccine virus in both cell types. We demonstrated a dose-dependent decrease in VACV viral protein levels with narciclasine treatment in HeLa, Huh7.5.1, and BSC-1 cells (Fig. 1C, 1D, and 1E). Additionally, fluorescence microscopy of BSC-1 cells infected with A4-YFP VACV (MOI = 0.1) showed a reduction in fluorescence signal 24 h post-narciclasine treatment (Fig. 1F), indicating the inhibition of viral protein expression. In summary, narciclasine led to a decrease in the level of VACV viral proteins.

3.2. Narciclasine inhibits EEV production

HeLa cells were infected at high (MOI of 3) and low (MOI of 0.1) multiplicities and treated with varying concentrations of narciclasine or DMSO to assess narciclasine's antiviral activity against EEV, Viral titers in the cell culture supernatant were quantified using plaque assays in BSC-1 cells. Narciclasine demonstrated effective inhibition of VACV at high MOI (Fig. 2A), with a more pronounced inhibitory effect observed at low MOI infections, as shown in Fig. 2B. These results suggest that narciclasine may reduce EEV formation. The half maximal inhibitory concentration (IC₅₀) value for EEV produced by VACV (MOI = 3) in HeLa cells was determined to be 160.3 nM (Fig. 2A), and for MOI = 0.1, it was 81.04 nM (Fig. 2B). Actin tail formation is critical for EEV formation [15]. Next, the impact of narciclasine on actin tail formation was assessed using immunofluorescence staining with phalloidin-rhodamine. Cells infected with A4-YFP VACV and treated with narciclasine exhibited reduced actin tail length and A4-YFP signal compared to untreated cells (Fig. 2C). Taken together, these findings demonstrate that narciclasine effectively inhibits EEV formation.

Fig. 2.

Narciclasine inhibits the formation of EEV. A) – B) HeLa cells were infected with VACV at an MOI of 3 (A) or 0.1 (B), and the supernatant was collected for virus titer experiments. The IC₅₀ for inhibiting EEV was determined using Graph Prism at various concentrations. The IC₅₀ values were 160.3 nmol/L (nM) at an MOI of 3 and 81.04 nM at an MOI of 0.1, with results reported as mean ± standard deviation (n = 6). The intracellular titer of HeLa cells infected with VACV at an MOI of 3 was measured by plaque assay in BSC-1 cells. C) HeLa cells were infected with A4-YFP VACV (MOI = 1) for 1 h, followed by treatment with 40 or 80 nM narciclasine for 36 h. Immunofluorescence analysis was then performed, visualizing phalloidin-rhodamine (Red) and A4-YFP VACV (Green) fluorescence. Arrows showed actin tails. Relative actin tail length and relative fluorescence intensity of A4-YFP were calculated in at least 20 cells. Scale bar: 10 μm. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Abbreviations: EEV, extracellular enveloped virus; VACV, vaccinia virus; MOI, multiplicity of infection; IC₅₀, the half maximal inhibitory concentration; BSC-1, biologics standards-cercopithecus-1; DMSO, dimethylsulfoxide; YFP, yellow fluorescent protein; nM, nmol/L.

3.3. Narciclasine inhibits the spread of VACV

BSC-1 cells were seeded in 24-well plates and pre-incubated with narciclasine for either 24 h (Fig. 3A) or 2 h (Fig. 3B) to investigate narciclasine's effect on VACV plaque size. The cells were then infected with VACV for 1 h at 37 °C, followed by the replacement of the inoculum with a serum-free medium containing varying narciclasine concentrations. After 52 h of incubation, cells were stained with crystal violet and plaque sizes were measured using ImageJ. Statistical analysis with Graph Prism revealed the differences in plaque sizes between different narciclasine concentrations and the DMSO control (Fig. 3A and 3B). To further confirm narciclasine's inhibitory effect on VACV spread, changes in plaque size were observed microscopically. After infecting with A4-YFP VACV for 1 h, the medium was replaced with 10% FBS containing different drug concentrations and incubated for 50 h. Subsequent microscopic observations using an Invitrogen EVOS M7000 microscope showed apparent inhibition of plaque formation, with noticeable reductions in plaque size and viral protein expression correlating with increasing narciclasine concentrations (80 nM, 160 nM, 320 nM) (Fig. 3C). These findings emphasize narciclasine's inhibitory role in VACV spread.

Fig. 3.

Narciclasine reduces the spread of VACV. A) – B) Different concentrations of narciclasine were added to BSC-1 cell monolayers either 24 h (A) or 2 h (B) before VACV infection. Following infection, cells were incubated with VACV for 52 h, and the sizes of 30 plaques per group were measured and reported as mean ± standard deviation of plaque diameter. Plaque sizes were normalized quantitatively relative to the control group. C) BSC-1 cells were infected with A4-YFP VACV (MOI = 1) for 1 h, followed by incubation in a medium containing 10% FBS for 50 h. Virus foci were visualized using fluorescence microscopy, with A4-YFP VACV fluorescence depicted in green. Scale bar: 275  μm. **** indicates P < 0.0001. Abbreviations: VACV, vaccinia virus; MOI, multiplicity of infection; BSC-1, biologics standards-cercopithecus-1; FBS, fetal bovine serum; YFP, yellow fluorescent protein; DMSO, dimethylsulfoxide; nM, nmol/L.

3.4. Narciclasine reduces VACV EEV formation by activating RhoA

Previous research on glioblastoma cells has demonstrated that narciclasine significantly activates the small GTPase RhoA, leading to the formation of F-actin stress fibers in a RhoA-dependent manner [18]. The inactivation of RhoA is a crucial mechanism involved in VACV morphogenesis and the formation of VACV-induced actin tails [20], [21], [22]. To investigate whether narciclasine activates RhoA signaling in HeLa cells, we performed pulldown assays to assess RhoA activation and quantified the levels of activated RhoA (GTP-RhoA) using GST-Rhotekin-RBD [24]. Our results showed that treatment with 80 nM narciclasine indeed activated RhoA in HeLa cells (Fig. 4A). It should be noted that the basal level of GTP-RhoA in HeLa cells under DMSO conditions is already high, which is why narciclasine only resulted in a modest increase in GTP-RhoA levels.

Fig. 4.

Narciclasine exerts an antiviral effect through the activation of RhoA activity. A) HeLa cells were pre-seeded and treated with 80 nmol/L (nM) narciclasine for 6 h, with 0.04 % DMSO used as a control. Activated RhoA (GTP-RhoA) was pulled down using GST-Rhotekin-RBD and detected via RhoA antibody blotting, with GAPDH as a loading control. Data are presented as mean ± standard deviation from three independent experiments. * indicates P < 0.05. B) – C) HeLa cells transfected with HA-tagged RhoA (RhoA-HA) or mock transfection were infected with A4-YFP VACV at MOI of 0.1 (B) or 3 (C) for 24 h. Cells were fixed and stained with HA antibodies (blue), and phalloidin-rhodamine to visualize actin, with the scale bar at 10  μm. Arrows showed actin tails. Western blot analysis was performed to evaluate RhoA-HA protein expression, with GAPDH as a loading control. Relative actin tail length in Fig. 4B was quantitated and presented as mean ± standard deviation from three independent experiments. **** indicates P < 0.0001. D) HeLa cells transfected with RhoA-HA, RhoA G14V-HA, or RhoA T19N-HA were infected with VACV (MOI = 1) for 1 h, then incubated in 10% FBS medium for 50 h. Virus plaque sizes were analyzed by crystal violet staining and statistically assessed across three independent experiments. The bar chart shows mean values ± standard deviation. ns, not significant; ****, P < 0.0001. Abbreviations: VACV, vaccinia virus; MOI, multiplicity of infection; DMSO, dimethylsulphoxide; GST, glutathione-S-transferase; HA, hemagglutinin; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BSC-1, biologics standards-cercopithecus-1; YFP, yellow fluorescent protein.

To further investigate the effect of RhoA activation on VACV, HeLa cells were transfected with a plasmid expressing HA-tagged RhoA (RhoA-HA) or subjected to mock transfections for 12 h. The cells were then infected with the A4-YFP VACV WR strain at an MOI of 0.1 for 24 h. Western blot analysis confirmed the expression of RhoA-HA (Fig. 4B). Due to low transfection efficiency, the protein levels of A4-YFP remained the same between the mock and RhoA-HA transfected cells. However, when examining A4-YFP signals in individual cells expressing RhoA-HA, we found that overexpression of RhoA-HA decreased the level of A4-YFP, suggesting that narciclasine may reduce IMV formation (Fig. 4B). Additionally, actin tail length was reduced with RhoA-HA overexpression, indicating that RhoA-HA could inhibit EEV formation (Fig. 4B). Next, HeLa cells transfected with plasmids expressing RhoA-HA were infected with A4-YFP VACV at an MOI of 3. Similar to the results from the low MOI infection, overexpression of RhoA-HA also led to a decrease in both A4-YFP levels and actin tail lengths during high MOI VACV infection (Fig. 4C). These findings demonstrated that RhoA inhibited the expression of viral proteins and actin tail formation.

BSC-1 cells were seeded at 2.5 × 10⁵ cells per well in a 24-well plate one day before the experiment to assess the effect of RhoA on VACV plaque size. Following transfection with plasmids expressing RhoA-HA or mock transfections for 24 h, the cells were infected with VACV (MOI = 1) for 1 h at 37 °C. After removing the inoculum, the cells were incubated for another 50h and stained with crystal violet. Plaque sizes were measured using ImageJ, and statistical analysis performed with Graph Prism showed a decrease in plaque sizes in cells expressing RhoA-HA compared to those with mock transfection, indicating that RhoA reduces VACV plaque size (Fig. 4D).

4. Discussion

This research explored how narciclasine inhibits VACV infection and delved into its underlying mechanisms. Our findings demonstrate that narciclasine effectively suppresses VACV replication under various experimental conditions. Initially, we observed a dose-dependent decrease in VACV viral protein levels upon narciclasine treatment. Furthermore, narciclasine inhibited EEV production in HeLa cells infected with VACV, both at high (MOI = 3) and low (MOI = 0.1) multiplicities, resulting in the reductions in viral titers in the cell culture supernatant in a dose-dependent manner. The calculated IC₅₀ values confirmed narciclasine's potency against EEV formation (160.3 nM for MOI = 3; 81.04 nM for MOI = 0.1), highlighting its effectiveness even at lower infection rates. Immunofluorescence staining using phalloidin-rhodamine revealed reduced actin tail formation following narciclasine treatment, which is crucial for EEV production, suggesting that narciclasine interferes with essential cytoskeletal dynamics for efficient viral transmission.

Moreover, our study unveiled a novel mechanism by which narciclasine exerts its antiviral effects: activating RhoA. Previous studies have shown that narciclasine induces RhoA activation in glioblastoma cells, promoting the formation of F-actin stress fibers and influencing cytoskeletal regulation [18]. Expanding on these insights, we found that narciclasine-induced RhoA activation during VACV infection suppresses viral protein expression and disrupts the formation of actin tails critical for VACV EEV production, which underscores the pivotal role of RhoA signaling in modulating viral infection dynamics. Manipulating RhoA activity with narciclasine reduced EEV titer and plaque size, suggesting RhoA is a promising therapeutic target against poxvirus infections. The ability of RhoA to inhibit VACV replication suggests broader implications for this GTPase in viral pathogenesis beyond its established roles in cellular cytoskeletal dynamics [25]. Our study provides significant insights into narciclasine's antiviral properties against VACV and emphasizes the potential of targeting RhoA signaling pathways for combating poxvirus infections. Future research could delve deeper into the molecular mechanisms underlying narciclasine's activation of RhoA and its downstream effects on viral replication, potentially opening new avenues for antiviral drug development strategies.

Narciclasine's effectiveness against VACV has spurred interest in its potential against other poxviruses. Given their genetic and structural similarities [26], narciclasine's mechanisms of action may extend to related pathogens like MPXV, variola virus, and molluscum contagiosum virus. Assessing its efficacy across different poxviruses promises valuable insights into its broad-spectrum antiviral potential, informing strategies against these medically significant pathogens. Our study of narciclasine's antiviral activity against VACV underscores its unique mechanisms compared to conventional anti-poxvirus treatments. While typical therapies focus on inhibiting EEV formation, viral replication enzymes, or entry processes, narciclasine simultaneously inhibits IMV and EEV production. This dual-action approach suggests advantages in halting viral spread and reducing infection severity. Comparative studies against established antiviral therapies will be crucial to fully understand narciclasine's distinct effectiveness and potential synergies in combination treatments.

Beyond its efficacy against VACV, narciclasine's broad-spectrum antiviral activity warrants exploration against other viral pathogens. Initial findings suggest effectiveness against Japanese encephalitis, yellow fever, DENV-4, Punta Toro, and Rift Valley fever viruses, indicating potential applications across diverse viral infections [27]. Moreover, ZIKV and DENV-2 could be inhibited by narciclasine [23]. With our current study, narciclasine could inhibit both RNA and DNA viruses. Further research is needed to elucidate its performance against various virus families and uncover specific viral targets and pathways, guiding the development of narciclasine as a versatile antiviral treatment.

In conclusion, our research underscores narciclasine as a potent inhibitor of VACV infection, effectively reducing viral protein levels and EEV production in a dose-dependent manner. By activating RhoA signaling, narciclasine disrupts critical cytoskeletal dynamics essential for viral spread, highlighting its dual antiviral mechanisms. These findings advance our understanding of narciclasine's therapeutic potential against poxviruses and suggest that RhoA is a promising target for broader antiviral strategies. Further exploration into molecular interactions of narciclasine with RhoA may pave the way for innovative antiviral therapies.