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With the widespread use of plastics and nanotechnology products, nanoplastics (NPs) have become a potential threat to human health. It is of great practical significance to study and evaluate the distribution of NPs in mice as mammal models and their entry, transport, and cytotoxicity in human cell lines. In this study, we detected the tissue distribution of fluorescent polystyrene nanoplastics (PS-NPs) in mice and assessed their endocytosis, transport pathways, and cytotoxic effects in GES-1 cells. We found that PS-NPs were clearly visible in gastric, intestine, and liver tissues of mice and in GES-1 cells treated with PS-NPs. Entry of PS-NPs into GES-1 cells decreased with the inhibition of caveolae-mediated endocytosis (nystatin), clathrin-mediated endocytosis (chlorpromazine HCl), micropinocytosis (ethyl-isopropyl amiloride), RhoA (CCG-1423), and F-actin polymerization (lantrunculin A). Rac1 inhibitors (NSC 23766) had no significant effect on PS-NPs entering GES-1 cells. F-actin levels significantly decreased in CCG-1423-pretreated GES-1 cells exposed to PS-NPs. GES-1 cell ultrastructural features indicated that internalized PS-NPs can be encapsulated in vesicles, autophagosomes, lysosomes, and lysosomal residues. RhoA, F-actin, RAB7, and LAMP1 levels in PS-NPs-treated GES-1 cells were remarkably up-regulated and the Rab5 level was significantly down-regulated compared to levels in untreated cells. PS-NPs treatment decreased cell proliferation rates and increased cell apoptosis. The formation of autophagosomes and autolysosomes and levels of LC3II increased with the length of PS-NPs treatment. The results indicated that cells regulated endocytosis in response to PS-NPs through the RhoA/F-actin signaling pathway and internalized PS-NPs in the cytoplasm, autophagosomes, or lysosomes produced cytotoxicity. These results illustrate the potential threat of NPs pollution to human health.

Microplastics provide stable habitats for the colonization and survival of pathogenic microorganisms, and cooperate with microorganisms to pose a potential threat to human health. In this study, polyethylene microplastics (PE-MPs) in artificial gastric juice time-dependently decomposed and broke into small-diameter PE-MP fragments that were more stable than those in an aqueous solution. Helicobacter pylori adhered to the surfaces of the PE-MPs to form a biofilm. The gastric tissues of mice treated with PE-MPs first and mixture of PE-MPs and H. pylori were positive for H. pylori infection in the 10th and 14th weeks after treatment, whereas those infected with H. pylori first and H. pylori alone were positive only in the 14th week after treatment. PE-MPs were visible in the gastric, intestinal, and liver tissues of mice treated with PE-MPs. The average diameter of the PE-MP fragments in the liver was greater than those of fragments that entered the gastric or intestinal tissues, and the average diameter of PE-MPs in the PE-MPs only-treated mice was significantly smaller than those of PE-MPs entering the intestinal tissues of the other groups. The infiltration of inflammatory cells was most serious in the mice treated with the mixture of PE-MPs and H. pylori, or with PE-MPs first and then H. pylori. Of all the groups, the gastric organ index and MPO, IL6, and TNF-α levels were highest in the mice treated with the mixture of PE-MPs and H. pylori. These results indicate that the interaction between PE-MPs and H. pylori contributed to the rapid bacterial colonization of gastric mucosal epithelial cells, improved the efficiency of PE-MP entry into tissues, and promoted gastric injury and inflammation in mice. These findings suggest that microplastics may provide a stable habitat for H. pylori, and act synergistically with H. pylori to pose a potential threat to human health.

The ability of Helicobacter pylori to manipulate host autophagy is an important pathogenic mechanism. We found an inverse correlation between the expression of ILK and the autophagy marker protein LC3B in H. pylori-positive human samples, H. pylori-infected mice models and H. pylori-infected GES-1 cell lines. When the ILK-knockdown GES-1 cells were infected by H. pylori, CagA were significantly degraded, autophagosomes accumulation and autolysosomes formation were significantly increased, and LC3B protein levels and ratio of LC3BII to LC3BI were also remarkably upregulated. And chloroquine treatment increased LC3B levels in ILK-knockdown GES-1 cells. The expression levels of both Rac1 and RhoA were downregulated in GES-1 cells after H. pylori infection and were decreased in ILK-knockdown GES-1 cells. The mRNA and protein levels of PAK1, MLC, and LIMK were significantly decreased and cofilin mRNA and protein levels were significantly increased in GES-1 cells treated with the Rac1 inhibitor NSC 23766. The mRNA and protein levels of ROCK1, ROCK2, MLC, and LIMK1 were significantly reduced and cofilin mRNA and protein levels were significantly increased in GES-1 cells treated with the RhoA inhibitor CCG-1423. F-actin was significantly reduced in Rac1- or RhoA-inhibited GES-1 cells. F-actin depolymerization induced autophagosomes accumulation, autolysosomes formation, and the increase of LC3B levels in GES-1 cells. Therefore, these findings revealed that ILK could serve as a novel regulator to affect Rac1/PAK1 and RhoA/ROCKs signaling pathways, thereby influencing H. pylori-induced autophagy.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.

Emerging coronaviruses (CoVs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. Here, A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (TGEV) infection in cell-based assays. Moreover, A9 exhibited potent antiviral activity against the replication of various CoVs, including murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV). We further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of A9 against TGEV infection in vitro. We specifically identified p38 and JNK1, which are the downstream molecules of receptor tyrosine kinases (RTKs) required for efficient TGEV replication, as A9 targets through plaque assays, qRT-PCR and Western blotting assays. p38 and JNK1 inhibitors and RNA interference further showed that the inhibitory activity of A9 against TGEV infection was mainly mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. All these findings indicated that the RTKI A9 directly inhibits TGEV replication and that its inhibitory activity against TGEV replication mainly occurs by targeting p38, which provides vital clues to the design of novel drugs against CoVs., Highlights • We screened inhibitors against coronavirus replication using TGEV as a surrogate model through a high-throughput assay. • A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, was identified as a robust inhibitor of TGEV. • A9 also exhibited potent antiviral activity against the replication of various coronaviruses. • The inhibitory activity of A9 against TGEV replication is mainly regulated by targeting p38.