Interactions between proteins and small molecule metabolites play vital roles in regulating protein functions and controlling various cellular processes. The activities of metabolic enzymes, transcription factors, transporters and membrane receptors can all be mediated through protein–metabolite interactions (PMIs). Compared with the rich knowledge of protein–protein interactions, little is known about PMIs. To the best of our knowledge, no existing database has been developed for collecting PMIs. The recent rapid development of large-scale mass spectrometry analysis of biomolecules has led to the discovery of large amounts of PMIs. Therefore, we developed the PMI-DB to provide a comprehensive and accurate resource of PMIs. A total of 49 785 entries were manually collected in the PMI-DB, corresponding to 23 small molecule metabolites, 9631 proteins and 4 species. Unlike other databases that only provide positive samples, the PMI-DB provides non-interaction between proteins and metabolites, which not only reduces the experimental cost for biological experimenters but also facilitates the construction of more accurate algorithms for researchers using machine learning. To show the convenience of the PMI-DB, we developed a deep learning-based method to predict PMIs in the PMI-DB and compared it with several methods. The experimental results show that the area under the curve and area under the precision-recall curve of our method are 0.88 and 0.95, respectively. Overall, the PMI-DB provides a user-friendly interface for browsing the biological functions of metabolites/proteins of interest, and experimental techniques for identifying PMIs in different species, which provides important support for furthering the understanding of cellular processes. The PMI-DB is freely accessible at http://easybioai.com/PMIDB.

Background: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, leading to an acute encephalitis and damage to the central nervous system (CNS). The mechanism of JEV pathogenesis is still unclear. DNA microarray analyses have been recently employed to detect changes in host gene expression, which is helpful to reveal molecular pathways that govern viral pathogenesis. In order to globally identify candidate host genes associated with JEV pathogenesis, a systematic mRNA profiling was performed in spleens and brains of JEV-infected mice. Results: The results of microarray analysis showed that 437 genes in spleen and 1119 genes in brain were differentially expressed in response to JEV infection, with obviously upregulated genes like pro-inflammatory chemokines and cytokines, apoptosis-related proteases and IFN inducible transcription factors. And the significant pathways of differentially expressed genes are involved in cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity, antigen processing and presentation, MAPK signaling, and toll-like receptor signaling, etc. The differential expression of these genes suggests a strong antiviral response of host but may also contribute to the pathogenesis of JEV resulting in encephalitis. Quantitative RT-PCR (RT-qPCR) assay of some selected genes further confirmed the results of microarray assay. Conclusions: Data obtained from mRNA microarray suggests that JEV infection causes significant changes of mRNA expression profiles in mouse spleen and brain. Most of differentially expression genes are associated with antiviral response of host, which may provide important information for investigation of JEV pathogenesis and therapeutic method.

Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge.In vitrostudies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number ofde novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of thede novo-synthesized leader RNA.IMPORTANCERabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level ofde novo-synthesized leader RNA. These findings may have important implications in the development of efficient rabies vaccines.

Rabies virus (RABV) induces encephalomyelitis in humans and animals. One of the major problems with rabies is that the infected individuals most often do not develop virus neutralizing antibodies (VNA). In this study we have investigated the host immune response to RABV infection in dogs, using a live-attenuated (TriGAS) or a wild-type (wt) (DRV-NG11) RABV isolated from a rabid dog. The experimental infection of dogs with TriGAS induced high levels of VNA in the serum, whereas wt RABV infection did not. Dogs infected with TriGAS developed antibodies against the virus including its glycoprotein, whereas dogs infected with DRV-NG11 only developed rabies antibodies that are presumably specific for the nucleoprotein, (N) and not the glycoprotein (G). We show that infection with TriGAS induces early activation of B cells in the draining lymph nodes and persistent activation of DCs and B cells in the blood. On the other hand, infection with DRV-NG11 fails to induce the activation of DCs and B cells and further reduces CD4 T cell production. Further, we show that intrathecal (IT) immunization of TriGAS not only induced high levels of VNA in the serum but also in the CSF while intramuscular (IM) immunization of TriGAS induced VNA only in the serum. In addition, high levels of total protein and WBC were detected in the CSF of IT immunized dogs, indicating the transient enhancement of blood-brain barrier (BBB) permeability, which is relevant to the passage of immune effectors from periphery into the CNS. IM infection of dogs with TriGAS induced the production of serum VNA whereas, IT immunization of TriGAS in dogs induces high levels of VNA in the periphery as well as in the CSF and transiently enhances BBB permeability. In contrast, infection with wt DRV-NG11 resulted in the production of RABV-reactive antibodies but VNA and antibodies specific for G were absent. As a consequence, all of the dogs infected with wt DRV-NG11 succumbed to rabies. Thus the failure to activate protective immunity is one of the important features of RABV pathogenesis in dogs. Remarkable advances have been made in elucidating the pathogenesis of rabies. One of the hallmarks of rabies infection is that human patients do not develop VNA. In this present study, immune responses were investigated in dogs after infection with a highly attenuated RABV (TriGAS) or a highly pathogenic, truly wt RABV (DRV-NG11). DRV-NG11 was isolated from the brain of a rabid dog and has not been passaged in laboratory animals or in cell culture. IM infection of dogs with TriGAS induced early activation of DCs and B cells in the blood, and production of VNA in the periphery, whereas, IT infection with TriGAS induced a high level of VNA not only in the serum but also in the CSF. On the contrary, infection of dogs with DRV-NG11 failed to elicit VNA. As a result, none of the dogs infected with wt DRV-NG11 survived. The ability of DRV-NG11 to replicate and spread without triggering an immune response to glycoprotein is evidently an important facet of its pathogenicity.

Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma cells (NA) after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly down-regulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin (IL)-1β expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins, and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotypes DENV-1 and DENV-2 also decreased miR-466d-3p expression. Results from helicase blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of Flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation. IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA binding sites because we demonstrated that miRNA degradation was abolished if arginine at R226 and R202 was mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino-acid sites that could be mutated for a JEV vaccine.

Lab-attenuated rabies virus (RABV) is a highly cellular adaptation and less pathogenic than wild-type RABV. However, the molecular mechanisms that regulate the cellular adaptation and pathogenicity remain obscure. In this work, we isolated a wild-type RABV (CNIM1701) from a rabid bovine in northern China. The original CNIM1701 was lethal in adult mice and restricted replication in cell cultures. After 20 serial passages in the brains of suckling mice, the virus was renamed CNIM1701-P20, which was safe in adult mice and replicated well in cell cultures. In addition, sequence comparison analysis of the original CNIM1701 and CNIM1701-P20 identified 2 amino acid substitutions on G protein (Lys83 → Arg83 and Pro367 → Ser 367) related to pathogenesis and cellular adaptation. Using site-directed mutagenesis to exchange Lys83 with Arg83 and Pro367 with Ser 367 in the G protein of the RABV SAD strain, the pathogenicity of rSAD-K83R was significantly decreased. Our data indicate that the decreased pathogenicity of rSAD-K83R is due to increasing the expression of RABV-G, which also induced a higher level of apoptosis in infected cells. Furthermore, the K83 mutation induced high expression of MMP-2 and MMP-9 on DCs and promoted blood–brain barrier (BBB) permeability. These results demonstrate that the pathogenesis of RABV is partially dependent on G expression and BBB permeability, which may help in the design and development of highly safe, live-RABV vaccines.