The infection of HSV-1 can not only induce skin herpes, but also lead to fatal diseases such as gingival stomatitis and encephalitis in severe cases [87–89]. Viral replication cycle is a complex process, including entry, uncoating, genomic amplification and expression, assembly and release, etc.. Inhibition of any of these steps can effectively interfere with viral infection. AgNPs have a broad spectrum of antiviral effects against a variety of viruses. However, the specific inhibitory mechanism of AgNPs against each type of viruses has not been fully clarified [91, 92]. In this study, we confirmed that AgNPs could directly or indirectly inhibit HSV-1 through a variety of ways, and the combined management of AgNPs and ACV ( a clinical anti-herpes drug) could enhance the lethality of HSV-1, indicating that AgNPs might be a potential anti-herpes material.
It has been found that AgNPs could reduce plaque formation caused by monkeypox virus, and inhibit the production of progeny viruses of Tacaribe virus . In this study, we proved that AgNPs could restrain the plaque formation of HSV-1 and production of the progeny viruses, exerting good inhibitory effect on the cytopathic changes caused by HSV-1. Taken together, these findings suggest that the inhibitory effect of AgNPs against different viruses might be universal in vitro. The inhibition of AgNPs on plaque formation and virus development might be attributed to the capacity that it could directly affect the viral invasion by reducing the production of progeny viruses in different ways, or indirectly interfere with the viral damage to cells by oppressing the activity of progeny viruses.
When HSV-1 infects host cells, the genome will undergo biological processes such as replication, transcription and translation. AgNPs could enter cells through endocytosis , and the DNA of the cells would lose the ability to replicate after receiving Ag+ treatment [96, 97]. Our results verified that AgNPs could downregulate the viral genomic load, indicating that AgNPs might directly interact with the viral DNA to interfere with the replication process, or disturbing the enzymes or proteins associated with viral DNA replication to inhibit genomic load. Furthermore, we found that AgNPs had the ability to bind directly to viral DNA. It has been reported that AgNPs could also interact with the double-stranded DNA of hepatitis B virus , which is consistent with the results of this study. We speculate that AgNPs are prone to adsorb negatively charged nucleic acids due to the large amount of positive charges on their surface. In addition, structures like large or small furrows in DNA molecules might also be potential binding sites for AgNPs.
The genes of HSV-1 are mainly composed of three main groups: IE, E, and L, which are sequentially controlled and expressed in a cascading manner . The expression of IE genes plays an important role in initiating the subsequent transcription of HSV-1. Regulated by protiens of IE genes, E genes begin to be successively expressed, whose products are involved in the replication of HSV-1 genome. The L genes are expressed in the late stage of viral infection, whose products are mainly structural proteins of HSV-1. We discovered that AgNPs could restrain the genetic transcription of these stages, including IE gene (ICP4, ICP22, ICP27, ICP47), E gene (ICP8, UL12, UL30, UL42), and L gene (gB, gD, gH, VP16). It has been revealed that AgNPs could also reduce viral replication by interfering with RNA polymerase dependent on Tacaribe virus and prevent viral unencapsulation in the endosome . Combined with the findings of this study, it is reasonable to believe that AgNPs might interfere with the process of gene expression of different viruses in host cells. We assume that AgNPs could down-regulate the mRNA level of the corresponding genes of HSV-1, due to the abilities that AgNPs could disturb the DNA synthesis of the virus, thus indirectly affecting the transcription level of the related genes, and that AgNPs could hinder the action of the enzymes of viral genome transcription, thus affecting the mRNA production. In addition, AgNPs might also directly interact with the RNA formed by transcription or interfere with the environment of viral genome transcription, ultimately reducing the expression of viral mRNA.
ICP4, a transcriptional agonist, is an important regulatory protein expressed by IE gene, which is necessary to activate the expression of HSV-1’s E gene and L gene . ICP8 is a single-stranded DNA binding protein expressed by the E gene (UL29), involved in regulating the replication of HSV-1’s genome and the transcription of L gene . gB is an enveloped glycoprotein expressed by HSV-1 L gene (UL27), which can recognize receptors on host cells and enable HSV-1 to adsorb to membranes of the host cells. gB is also important to mediate the penetration and the diffusion between cells . In the present experiments t investigating the expression levels of these three proteins after AgNPs treatment, all the levelsl were found to be descended. We believe that AgNPs could be highly likely to indirectly affect the expression level of downstream proteins by down-regulating the genomic load of HSV-1 and the transcription level of the corresponding genes. Meanwhile, the presence of AgNPs might also interfere with the enzymes and regulatory factors required for HSV-1 related protein synthesis, thus directly impacting the formation of viral proteins. In the study of AgNPs against foodborne viruses, it has been found that AgNPs at 10 nm could reduce viral titers in a dose-dependent manner and significantly inhibit the expression of viral capsid proteins detected by Western bloting . Therefore, the effect of AgNPs against the synthesis of viral protein might be a key step for its broad-spectrum inhibition of viral replication.
In the study of curcumin-modified AgNPs (cAgNPs) against respiratory syncytial virus (RSV), it was found that cAgNPs could directly interact with the virions to prevent RSV from infecting host cells . Likewise, in the research of naked AgNPs against influenza virus, AgNPs could destroy influenza virus envelope glycoprotein, thereby preventing viral infection . Electron microscopic observation has revealed that AgNPs could absorb to the surface of the virion of Peste des petits ruminants virus and interact with the virus core . Under the observation of TEM, AgNPs could also destroy the structure of adenosine virus type 3 in vitro . We observed the direct interaction between AgNPs and HSV-1, and found that after their interaction, the virus infectivity and morphology were significantly changed. Tanken together, these results indicat that AgNPs can directly bind to different types of virions to inhibit the infectivity of the viruses, which is probably an important reason for the broad-spectrum antiviral properties of AgNPs. The explaination to AgNPs’ interacting with different viruses may be that AgNPs can nonspecifically adsorb to the cell surface, and cause oxidative damage on the capsids and envelope proteins, therefore, destroying the normal form of the viruses and suppressing the viral activity.
Previous studies have shown that AgNPs with a diameter of 1-10 nm could interact with HIV gp120 to inhibit viral adsorption to the host cells . Moreover, AgNPs with a diameter of about 10 nm also could effectively inhibit the binding and penetration of monkeypox virus . The results of this study also confirmed that AgNPs could inhibit the absorption and penetration of HSV-1. The infection of virus depends on the appropriate combination between the viruses and the host cells, and is closely related to the integrity of the viral particles . Based on our results, we speculate that the effect of AgNPs on the adsorption and penetration of HSV-1 could be attributed to the destruction of the envelope glycoprotein by AgNPs, thereby breaching the stable binding of HSV-1 to the membrane of host cells, and expediting competition between AgNPs and viruses for receptors on cell surface, resulting in reduction of the entry of HSV-1 into the cells.
By exploring the influence of AgNPs on viral release and cell-to-cell spread in the late stage of HSV-1 infection, we found that the release ratio and plaque size of HSV-1 were significantly reduced after the treatment of AgNPs, indicating that AgNPs could restrict HSV-1 transmission. The products of L genes, such as gB, gD, gH and VP16 ect., not only construct the structural proteins of HSV-1, but also participate in the assembly and the cell-to-cell transmission of progeny viruses . As observed in this study, reduced expression of gB, gD, gH and VP16 might contribute to AgNPs’ indirect inhibition of the release and cell-to-cell spread of HSV-1. In addition, AgNPs could directly interact with the HSV-1 virions in vitro, causing less infectivity of the virus; AgNPs can also bind to the mature virions that have been assembled in the host cells to prevent the spread and transmission of the virus.
It has been shown that MAPK signaling pathway is involved in regulating viral replication and cytokine production , and the activation of NF-κB and MAPK signaling pathways in host cells plays a key role in HSV replication [110, 111]. After infecting host cells, HSV can activate the expression of NF-κB, a key transcription factor in the inflammatory pathway, and induce the production of downstream pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α [81, 82], which can not only promote the replication of HSV itself, but also cause excessive inflammatory response and further damage to host cells. Previous Studies have reported that AgNPs could inhibit the production of pro-inflammatory cytokines [112–115]. In particular, curcumin-coated AgNPs could significantly reduce HIV replication by inhibiting the activity of NFκB-p50 and the downstream expression of IL-1β, TNF-α and IL-6 . Our results also revealed that AgNPs could reduce the expression levels of TNF-α, IL-1β, IL-6 and IL-8 induced by HSV-1. Since TNF-α, IL-1β, IL-6 and IL-8 are the hallmarks of cell chemotaxis and pro-inflammatory response, it is suggested that AgNPs could not only alleviate the host cell damage caused by the inflammatory response by inhibiting the production of pro-inflammatory cytokines, but also indirectly hinder viral replication by blocking HSV-1-activated cytokines. It has been disclosed that AgNPs could impose broad-spectrum and highly effective killing effect on common pathogenic bacteria and a variety of fungi, such as Pseudomonas aeruginosa and Staphylococcus aureus . Moreover, AgNPs could exert anti-inflammatory activity by controlling the production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in vivo and in vitro [114, 118]. Therefore, compared with the traditional nucleoside antiviral drugs, AgNPs might not only have the effect of anti-HSV-1, but also play roles in sterilization, antipruritis and anti-inflammatory effects in the lesions, which suggested that development of AgNPs as antiviral agents have higher clinical value.
Acyclovir is the first clinically effective drug discovered and used worldwide for the treatment of HSV and VZV infections , which requires activation of viral thymidine kinases and cellular kinases, and ultimately inhibits viral DNA synthesis . However, the therapeutic resistance of some HSV and VZV strains to ACV became an urgent problem for the clinic [120, 121]. Although the specific mechanism of the inhibition remains to be further studied, our experiments give evidences that AgNPs can inhibit HSV-1 transcription and DNA synthesis and the effect of AgNPs do not depend on the activity of viral kinase, which is different from action of ACV. AgNPs may escape the resistance of HSV-1 and hopefully become a stable anti-herpes agent. In this study, we also found that AgNPs treatment coupled with ACV were more effective than either treatment alone againt HSV-1 infection. Although the underlying mechanism requires further investigation, combination therapy might lessen the clinical dose of ACV to HSV-1, thereby reducing the adverse effects of ACV.
In conclusion, this study suggests that AgNPs could directly or indirectly restrict the generation, formation and development of viral plaque in vitro, thus showing effective anti-HSV-1 infection activity, which might be attributed to the mechanism that AgNPs could inhibit genetic replication and expression of HSV-1, block viral entry and transmission between cells, and directly interact with the virus particles to disrupt the normal structure of the viruses. We also found that AgNPs could inhibit the inflammatory response activated by HSV-1 infection and enhance the anti-HSV-1 activity of ACV. These results not only confirmed that AgNPs were effective and broad-spectrum antiviral agents, but also provided experimental and theoretical basis for further study of AgNPs as antiviral agents. Further studies should focus on the in-depth and specific molecular mechanism of AgNPs against HSV-1 and the development of safer and more efficient AgNPs products. In addition, the pharmacodynamics and toxicology in animal models also deserve further exploration to determine the safety parameters of AgNPs as well as to investigate how AgNPs effectively inhibit HSV-1 infection in vivo.