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.

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.

Significant correlations between heavy metals and prokaryotes in the Okinawa Trough hydrothermal sediments.

Prokaryotes play crucial roles in hydrothermal vent ecosystems, yet their interactions with heavy metals are not well understood. This study explored the diversity of prokaryotic communities and their correlations with heavy metals and nutrient elements in hydrothermal sediments from Okinawa Trough. A total of 117 bacterial genera in 26 bacterial phyla and 10 archaeal classes in 3 archaeal phyla were identified, including dominant prokaryotic phyla Planctomycetes, Acidobacteria, Verrucomicrobia, and Euryarchaeota. Furthermore, Fe (39.61 mg/g), Mn (2.84 mg/g) and Ba (0.36 mg/g) were found to be the most abundant heavy metals in the Okinawa hydrothermal sediments. Notably, the concentrations of Zn, Ba, Mn, total organic carbon, and total nitrogen significantly increased, whereas the total sulfur concentration distinctively decreased at sampling sites farther from hydrothermal vents. These changes corresponded with reductions in prokaryotic abundance and diversity. Most heavy metals, including Mn, Fe, Co, Cu and As, presented significant positive correlations with a number of prokaryotic genera in the nearby sediment samples. In contrast, both positive and negative correlations with prokaryotes were observed in remote sediment. The keystone taxa include Magnetospirillum, GOUTA19, Lysobacter, Kaistobacter, Treponema, and Clostridium were detected through prokaryote interspecies interactions. The functional predictions revealed significant genes involved in carbon fixation, nitrogen/sulfur cycling, heat shock protein, and metal resistance pathways. Structural equation modeling confirmed that metal and nutrient elements directly influence the composition of prokaryotic communities, which in turn affects the relative abundance of functional genes.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

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.

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.

Development of Coriolis mass flowmeter with digital drive and signal processing technology.

Coriolis mass flowmeter (CMF) often suffers from two-phase flowrate which may cause flowtube stalling. To solve this problem, a digital drive method and a digital signal processing method of CMF is studied and implemented in this paper. A positive-negative step signal is used to initiate the flowtube oscillation without knowing the natural frequency of the flowtube. A digital zero-crossing detection method based on Lagrange interpolation is adopted to calculate the frequency and phase difference of the sensor output signals in order to synthesize the digital drive signal. The digital drive approach is implemented by a multiplying digital to analog converter (MDAC) and a direct digital synthesizer (DDS). A digital Coriolis mass flow transmitter is developed with a digital signal processor (DSP) to control the digital drive, and realize the signal processing. Water flow calibrations and gas-liquid two-phase flowrate experiments are conducted to examine the performance of the transmitter. The experimental results show that the transmitter shortens the start-up time and can maintain the oscillation of flowtube in two-phase flowrate condition.