Prunella vulgaris, an herbaceous plant belonging to the Labiatae family, has long been utilized in traditional Chinese medicine for its potential pharmacological properties. This article delves into recent research revealing the antiviral potential of P. vulgaris, particularly against herpes simplex viruses (HSV), shedding light on its molecular mechanisms and promising applications.
The compounds and extracts derived from Prunella vulgaris have garnered attention due to their reported pharmacological properties, including antitumour activity, anti-inflammatory effects, antioxidative properties, and antiviral effects [1]. An earlier study had already highlighted the potential of water-extracted P. vulgaris in combatting HSV infection [2]. Furthermore, a specific extract obtained through hot water and 30% ethanol precipitation, known as PVE30, exhibited remarkable antiviral effects not only against HSV-1 and HSV-2 but also against acyclovir-resistant strains, although the details of this discovery were unpublished at the time [2].
To gain a deeper understanding of the antiviral capabilities of P. vulgaris, researchers embarked on a study aimed at unraveling the molecular mechanisms underlying the antiviral effects of PVE30 against HSV infection.
Herpes simplex viruses, specifically HSV-1 and HSV-2, are notorious human pathogens characterized by linear double-stranded DNA genomes. HSV-1 typically triggers oropharyngeal infections, while HSV-2 is known for inducing genital infections. The initial step of HSV entry into host cells hinges on the interaction between the virus’s envelope glycoproteins and cell surface heparan sulfate (HS) receptors.
Disrupting this binding interaction presents a promising avenue for developing new antiviral drugs. Past research had revealed that the removal of HSV envelope glycoproteins B and gC had a severe impact on virus attachment and infectivity [3, 4], while cells deficient in HS demonstrated significantly reduced susceptibility to infection [5, 6].
Apart from thwarting viral entry, another strategy for developing anti-HSV drugs involves regulating virus replication. Toll-like receptors (TLRs) play a pivotal role in controlling HSV infections by recognizing viral proteins and nucleic acids. HSV glycoprotein B, as a pathogen-associated molecular pattern (PAMP), is recognized by the TLR2 receptor, setting off a cascade of events that activate the NF-κB pathway [7].
NF-κB, a transcriptional regulator, plays a crucial role in modulating host gene expression, influencing cell survival, differentiation, inflammation, and antiviral responses. HSV infections are associated with persistent NF-κB activation, which enhances virus replication. Notably, inhibiting virus entry or employing mutated forms of viral ICP4 or ICP27 prevented NF-κB translocation [8].
Furthermore, TLR3 recognizes the double-stranded RNA intermediates generated during HSV-1 replication, leading to the activation of receptor-interacting kinase-3 (RIPK3)-dependent necroptosis, which curbs viral propagation and contributes to the host’s immune defense against infection [9, 10].
The recent study has brought to light the potent antiviral properties of PVE30, isolated from Prunella vulgaris. PVE30 effectively impedes HSV attachment to host cells by interacting with HSV surface glycoproteins B and gC. This interaction results in a diminished production of progeny viruses. Additionally, PVE30 suppresses HSV infection by inhibiting TLR-mediated NF-κB activation and blocking HSV-1-induced necroptosis. This multifaceted approach showcases PVE30 as a promising antiviral agent capable of effectively thwarting HSV infection [11].
A schematic of the mechanism of PVE30 in inhibiting HSV
Discussion
Naturally occurring sulfated polysaccharides have exhibited a broad-spectrum antiviral effect, targeting various stages of the virus life cycle, including adsorption, invasion, uncoating, transcription, replication, assembly, and release [23]. In a previous study, it was suggested that the Prunella vulgaris polysaccharide extracted by hot water could inhibit HSV through multiple mechanisms, including competition for cell receptors and other as-yet-unknown mechanisms that operate after the virus has penetrated the host cells [2]. Building on this foundation, the current investigation focused on elucidating the molecular mechanisms through which PVE30, a specific extract from Prunella vulgaris, exerts its antiviral effects against HSV-1 and HSV-2.
PVE30 displayed notable antiviral activity against both HSV-1 and HSV-2, with EC50 values of 33.36 ± 0.77 μg/mL and 26.61 ± 0.86 μg/mL, respectively, during the post-infection stage. Particularly, PVE30 exhibited remarkable efficacy during the attachment stage, inhibiting virus attachment to host cells with EC50 values of 4.53 ± 0.21 μg/mL and 4.61 ± 0.40 μg/mL for HSV-1 and HSV-2, respectively (Table 3). This discussion section aims to delve into the mechanisms by which PVE30 impedes viral attachment and replication.
Heparan sulfate (HS), a cellular glycosaminoglycan (GAG) chain of heparan sulfate proteoglycans (HSPGs), plays a pivotal role in the attachment and entry of various viruses, including HSV [15], HIV-1 [26], and SARS-CoV-2 [27]. In the case of HSV, HS serves as an initial attachment receptor, binding to virion surface glycoprotein C (gC) and glycoprotein B (gB), thereby facilitating HSV invasion [15].
To investigate whether PVE30’s inhibitory effect on viral attachment was related to interfering with the interaction between HS receptors and gB or gC, a heparin bead pull-down assay was conducted. The results indicated that the gB and gC proteins of the virus could bind to heparin-coupled magnetic beads, and treatment with PVE30 disrupted this interaction (Fig. 2A and B). Furthermore, PVE30-treated HSV particles displayed significantly reduced infectivity (Fig. 2D), suggesting that PVE30 directly deactivates virions by interacting with HSV surface glycoproteins gB and gC.
The mechanism underlying PVE30’s inhibition of viral replication was further explored. PVE30 was found to downregulate the expression of the immediate-early (IE) gene UL54 and its product, ICP27 protein (Fig. 3A–C). ICP27 plays a crucial role in lytic infection, primarily inhibiting precursor mRNA splicing and promoting the nuclear export of viral transcripts post-transcriptionally [18, 28]. Consequently, the observed attenuation of early (E) and late (L) viral gene product expression following PVE30 treatment was attributed to the downregulation of IE genes (Fig. 3B and C). PVE30 affects the entire virus replication cycle, resulting in a reduction in the production of viral progeny (Fig. 3D and E).
The activation of NF-κB has been shown to prolong host cell survival, facilitating virus replication and increasing viral progeny production [19, 29]. NF-κB activation occurs in two waves during HSV infection. The initial wave is triggered by the binding of gD to herpesvirus entry mediator A (HveA), while the second wave, occurring 3-4 hours post-infection, is induced by the synthesis of IE viral proteins and is substantial and persistent [19].
In this context, the role of NF-κB signaling in the presence of PVE30 during HSV infection was investigated. PVE30 alleviated HSV-1-triggered NF-κB activation by inhibiting IKKβ phosphorylation, upregulating IκBα protein expression, and downregulating p65 protein expression and nuclear translocation (Fig. 4A and C). Furthermore, PVE30 reduced the secretion of inflammatory cytokines IL-6 and TNF-α from host cells upon HSV-1 infection (Fig. 5A).
HSV infection is known to activate NF-κB signaling through Toll-like receptors (TLRs), leading to cytokine production [9]. TLR2 recognizes glycoprotein B and gH/gL to initiate NF-κB activation and proinflammatory cytokine induction, while TLR3 activates NF-κB to upregulate IL-6 and TNF-α in HSV-1-infected cells [7, 30]. The study suggests that PVE30 may inhibit TLR2 and TLR3 signaling by suppressing HSV-1-induced TRAF6-mediated NF-κB activation, thus interfering with viral replication (Fig. 5B and C).
Balancing cell death, proliferation, and differentiation is critical during microbe infection-associated death [22]. HSV-1 replication can trigger necroptosis through the sensing of viral dsRNA intermediates by TLR3, leading to necroptosis by forming the RIPK3/TRIF complex [10, 32]. The study clarified that PVE30 decreased the percentage of HSV-1-induced necroptosis and reduced the protein expression levels of p-MLKL (Fig. 6A and C), indicating that PVE30 also inhibits HSV-induced necroptosis. Since the protein level of MyD88 remained unaltered following HSV-1 infection, PVE30’s effect on viral infection-related elevation in mRNA levels of TLR2 and TLR3 was noted (Fig. 5B and C). It is plausible that PVE30 inhibits HSV-induced necroptosis through the TLR3 signaling pathway, recognized as a MyD88-independent TLR signaling pathway.
Conclusion
In summary, this study has demonstrated that PVE30, derived from Prunella vulgaris polysaccharide, acts as a potent antiviral agent against HSV infection. PVE30 inhibits viral attachment by competing with heparan sulfate (HS) and directly inactivates virions. Furthermore, PVE30 effectively restricts viral replication by targeting NF-κB and TLR signaling pathways and prevents HSV-1-induced necroptosis (Fig. 7).
In conclusion, the findings of this study underscore the multifaceted mechanisms of action that make PVE30 a promising antiviral agent for combatting HSV infections. Further research and clinical studies are warranted to fully harness the therapeutic potential of PVE30 against HSV and potentially other viral diseases.
reference link : https://cmjournal.biomedcentral.com/articles/10.1186/s13020-023-00865-y#Sec22
