Newcastle disease virus forms inclusion bodies with features of liquid-liquid phase separation.

Formation of inclusion bodies (IBs) is a hallmark of infections with negative-strand RNA viruses. Although the Newcastle disease virus (NDV) IBs had been observed in the 1950s, the characteristics of NDV IBs remained largely unknown. Here, we show that NDV infection triggers the formation of IBs that contain newly synthesized viral RNA. The structures of NDV IBs, observed by electron microscopy, were not membrane-bound. Fluorescence recovery after photobleaching a region of NDV IBs occurred rapidly, and IBs were dissolved by 1,6-hexanediol treatment, demonstrating they exhibited properties consistent with liquid-liquid phase separation (LLPS). We find the nucleoprotein (NP) and phosphoprotein (P) are sufficient to generate IB-like puncta, with the N arm domain and N core region of NP and the C terminus of P playing important roles in this process. In summary, our findings suggest that NDV forms IBs containing viral RNA, and provide insights into the formation of NDV IBs.

9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3ß and HSP90ß.

The paramyxoviruses represent a large family of human and animal pathogens that cause significant health and economic burdens worldwide. However, there are no available drugs against the virus. ß-carboline alkaloids are a family of naturally occurring and synthetic products with outstanding antiviral activities. Here, we examined the antiviral effect of a series of ß-carboline derivatives against several paramyxoviruses, including Newcastle disease virus (NDV), peste des petits ruminants virus (PPRV), and canine distemper virus (CDV). Among these derivatives, 9-butyl-harmol was identified as an effective antiviral agent against these paramyxoviruses. Further, a genome-wide transcriptome analysis in combination with target validation strategies reveals a unique antiviral mechanism of 9-butyl-harmol through the targeting of GSK-3ß and HSP90ß. On one hand, NDV infection blocks the Wnt/ß-catenin pathway to suppress the host immune response. 9-butyl-harmol targeting GSK-3ß dramatically activates the Wnt/ß-catenin pathway, which results in the boosting of a robust immune response. On the other hand, NDV proliferation depends on the activity of HSP90. The L protein, but not the NP protein or the P protein, is proven to be a client protein of HSP90ß, rather than HSP90α. 9-butyl-harmol targeting HSP90ß decreases the stability of the NDV L protein. Our findings identify 9-butyl-harmol as a potential antiviral agent, provide mechanistic insights into the antiviral mechanism of 9-butyl-harmol, and illustrate the role of ß-catenin and HSP90 during NDV infection. IMPORTANCE Paramyxoviruses cause devastating impacts on health and the economy worldwide. However, there are no suitable drugs with which to counteract the viruses. We determined that 9-butyl-harmol could serve as a potential antiviral agent against paramyxoviruses. Until now, the antiviral mechanism of ß-carboline derivatives against RNA viruses has rarely been studied. Here, we found that 9-butyl-harmol exerts dual mechanisms of antiviral action, with its antiviral activities being mediated by two targets: GSK-3ß and HSP90ß. Correspondingly, the interaction between NDV infection and the Wnt/ß-catenin pathway or HSP90 is demonstrated in this study. Taken together, our findings shed light on the development of antiviral agents against paramyxoviruses, based on the ß-carboline scaffold. These results present mechanistic insights into the polypharmacology of 9-butyl-harmol. Understanding this mechanism also deepens the host-virus interaction and reveals new drug targets for anti-paramyxoviruses.

A bivalent ß-carboline derivative inhibits macropinocytosis-dependent entry of pseudorabies virus by targeting the kinase DYRK1A.

Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV agents, we screened a total of 107 ß-carboline derivatives and found 20 compounds displaying antiviral activity against PRV. Among them, 14 compounds showed better antiviral activity than acyclovir. We found that compound 45 exhibited the strongest anti-PRV activity with an IC50 value of less than 40 nM. Our in vivo studies showed that treatment with 45 significantly reduced the viral loads and protected mice challenged with PRV. To clarify the mode of action of 45, we conducted a time of addition assay, an adsorption assay, and an entry assay. Our results indicated that 45 neither had a virucidal effect nor affected viral adsorption while significantly inhibiting PRV entry. Using the FITC-dextran uptake assay, we determined that 45 inhibits macropinocytosis. The actin-dependent plasma membrane protrusion, which is important for macropinocytosis, was also suppressed by 45. Furthermore, the kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) was predicted to be a potential target for 45. The binding of 45 to DYRK1A was confirmed by drug affinity responsive target stability and cellular thermal shift assay. Further analysis revealed that knockdown of DYRK1A by siRNA suppressed PRV macropinocytosis and the tumor necrosis factor alpha-TNF-induced formation of protrusions. These results suggested that 45 could restrain PRV macropinocytosis by targeting DYRK1A. Together, these findings reveal a unique mechanism through which ß-carboline derivatives restrain PRV infection, pointing to their potential value in the development of anti-PRV agents.

The canonical Wnt/ß-catenin signaling pathway facilitates pseudorabies virus proliferation and enhances virus-induced autophagy.

Pseudorabies virus (PRV) is a swine herpesvirus with a broad host range that causes significant economic losses worldwide. The Wnt/ß-catenin signaling pathway is reportedly involved in multiple viruses' proliferation. In this study, we demonstrated that PRV infection significantly activated the Wnt/ß-catenin signaling and promoted the nuclear translocation of ß-catenin. Applying specific chemical inhibitors (FH535 and iCRT14) caused a remarkable decrease in PRV titers in various cell lines. Knockdown of ß-catenin by siRNA also reduced the proliferation of PRV. On the contrary, treatment with lithium chloride (LiCl), an inhibitor of GSK3ß, stimulated the Wnt/ß-catenin signaling pathway and enhanced the PRV proliferation. Similarly, overexpression of ß-catenin promoted PRV proliferation and reversed the antiviral effect of FH535. Moreover, LiCl promoted PRV-induced autophagy, whereas FH535 and iCRT14 showed converse effects. These findings suggest that PRV infection stimulates the canonical Wnt/ß-catenin signaling pathway, facilitating PRV proliferation and regulating virus-induced autophagy. These data also provide potential targets for developing antiviral agents against PRV.

1-Formyl-ß-carboline Derivatives Block Newcastle Disease Virus Proliferation through Suppressing Viral Adsorption and Entry Processes.

Newcastle disease virus (NDV) is one of the highly contagious pathogens causing devastating economic effects on the global poultry industry. In the present study, three 1-formyl-ß-carboline derivatives (compounds 6, 7, and 9) were found to be potent inhibitors of different genotypes of NDV with IC50 values within 10 µM, which are similar to ribavirin. The virus titers were decreased by the presence of 1-formyl-ß-carboline derivatives in a dose-dependent manner, and the inhibition rate was found to exceed 90% at the concentration of 20 µM. These compounds mainly suppressed the adsorption and entry processes of NDV lifecycle. Through DARTS, CETSA, and RBC binding assay, these compounds were identified as novel HN inhibitors, which could directly interact with the NDV HN protein to affect the adsorption of NDV. Furthermore, they could inhibit the entry of NDV through suppressing the PI3K/Akt pathway rather than the ERK pathway. The PI3K/Akt pathway was proved to be involved in NDV entry. Our findings reveal a unique mechanism through which 1-formyl-ß-carboline derivatives restrain NDV infection. Moreover, these compounds represent suitable scaffolds for designing novel HN inhibitors.

Evasion of Host Antiviral Innate Immunity by Paramyxovirus Accessory Proteins.

For efficient replication, viruses have developed multiple strategies to evade host antiviral innate immunity. Paramyxoviruses are a large family of enveloped RNA viruses that comprises diverse human and animal pathogens which jeopardize global public health and the economy. The accessory proteins expressed from the P gene by RNA editing or overlapping open reading frames (ORFs) are major viral immune evasion factors antagonizing type I interferon (IFN-I) production and other antiviral innate immune responses. However, the antagonistic mechanisms against antiviral innate immunity by accessory proteins differ among viruses. Here, we summarize the current understandings of immune evasion mechanisms by paramyxovirus accessory proteins, specifically how accessory proteins directly or indirectly target the adaptors in the antiviral innate immune signaling pathway to facilitate virus replication. Additionally, some cellular responses, which are also involved in viral replication, will be briefly summarized.