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The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs. Cryo-EM structure and inhibition assays reveal a non-nucleoside inhibitor of the respiratory syncytial virus polymerase complex that acts by binding to an induced-fit pocket in the capping domain.

The Tetrahymena group I intron was one of the two first catalytic RNAs (ribozymes) to be discovered. Group I introns are sequences that can exist between exons in pre-mRNAs, that are able to self-splice and remove themselves when forming mature mRNAs in the absence of the spliceosome. The Tetrahymena cis-splicing ribozyme was engineered to accept substrate RNAs in trans. The designed trans-splicing ribozyme was termed the “spliceozyme”. The spliceozyme could in principle be used in therapeutic applications. However, there are two hurdles to overcome; the delivery of the ribozyme into cells and its efficiency once delivered. The focus of this study is to improve the efficiency of the trans-splicing ribozyme in cells by evolving the ribozyme in E. coli. In a previous study, an evolution system in cells was developed for the spliceozyme which was used to generate a clone, W11, that increased product formation. However, this evolution used high ribozyme expression levels and focused on a single splice site. To improve the efficiency and sequence generality, the spliceozyme was evolved at low expression levels at two different splice sites with different flanking sequences. The results of the spliceozyme evolution in bacterial cells showed that specific mutations are able to improve spliceozyme efficiency in bacterial cells. This suggests that further analysis, and perhaps further evolution experiments, could generate spliceozymes with improved efficiency on a broad range of substrate sequences.

Dengue virus (DENV) infection in children exhibits varied clinical presentations, wherein the role of monocytes is important in the innate immune response. In this study, laboratory-confirmed DENV pediatric patients ( n  = 120), with DENV-2 infection, were categorized into dengue fever (DF), dengue with warning signs (DWS) and severe dengue (SD) were assessed for monocyte subpopulation analysis using immunophenotyping involving CD14 and CD16 host-surface markers. Molecular docking was performed using HADDOCK 2.4 to analyze the interactions between CD14, CD16 and DENV envelope and capsid proteins. Among the cases, 84 (70%) were classified as DF and 36 (30%) as DWS & SD. Hematological and biochemical parameters indicated that thrombocytopenia and elevated hematocrit (> 40%) were significantly more common in DWS & SD, with markedly elevated liver enzymes (ALT and AST) in severe cases. Classical monocytes (CM-CD14++ CD16−) constituted 72.51% and 66.25% of the monocyte population in DF and DWS & SD cases, respectively. Intermediate monocytes (IM-CD14+ CD16+) comprised 9.89% and 30.86% in DF and DWS & SD cases, respectively. Non-classical monocytes (NCM-CD14+ CD16++) comprised 5.75% and 8.12% in DWS & SD and DF cases, respectively. In silico analysis revealed host CD16 and CD14 exhibited potential interactions with DENV capsid and envelope proteins, with binding energies − 8.9, − 10.1, − 8.6, and − 11.1 kcal/mol, respectively. IM was significantly increased in DWS & SD compared to DF ( p  < 0.05). These findings suggest that IM could act as host markers of DENV severity in children.

Xanthomonas oryzae pv. oryzae (Xoo) releases OMVs; however, the role of Xoo-OMVs in Xoo-rice interaction has not yet been studied. We investigated the early signalling events underlying Xoo-OMV perception by rice to gain insight into early plant-pathogen interactions. We observed that Xoo-OMVs are perceived by rice through a rapid and unique Ca2+ signal that is essential to evoke an immune response. By confocal imaging and proteomic analysis of the rice PM, we found that the Ca2+ signal is required for Xoo-OMV-induced nanodomain enrichment and for the aggregation of defence response-associated proteins in rice PM. The differential assembly of proteins in the PM helps the plant defend itself against Xoo. In the absence of the early Ca2+ signal, Xoo-OMVs are not perceived by the plant, resulting in a compromised immune response. Collectively, our study shows that Xoo-OMVs are recognised by rice via Ca2+ signal-induced nanodomain assembly leading to protein reorganisation in the PM to prepare the plant for an imminent pathogen invasion.

Mycobacterium tuberculosis (Mtb) lipoproteins are known to facilitate bacterial survival by manipulating the host immune responses. Here, we have characterized a novel Mtb lipoprotein LprE (LprEMtb), and demonstrated its role in mycobacterial survival. LprEMtb acts by down-regulating the expression of cathelicidin, Cyp27B1, VDR and p38-MAPK via TLR-2 signaling pathway. Deletion of lprEMtb resulted in induction of cathelicidin and decreased survival in the host. Interestingly, LprEMtb was also found to inhibit autophagy mechanism to dampen host immune response. Episomal expression of LprEMtb in non-pathogenic Mycobacterium smegmatis (Msm) increased bacillary persistence by down-regulating the expression of cathelicidin and autophagy, while deletion of LprEMtb orthologue in Msm, had no effect on cathelicidin and autophagy expression. Moreover, LprEMtb blocked phago-lysosome fusion by suppressing the expression of EEA1, Rab7 and LAMP-1 endosomal markers by down-regulating IL-12 and IL-22 cytokines. Our results indicate that LprEMtb plays an important role in mycobacterial pathogenesis in the context of innate immunity.