Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots.

The barley stripe mosaic virus (BSMV) triple gene block 1 (TGB1) protein is required for virus cell-to-cell movement. However, little information is available about how these activities are regulated by post-translational modifications. In this study, we showed that the BSMV Xinjiang strain TGB1 (XJTGB1) is phosphorylated in vivo and in vitro by protein kinase CK2 from barley and Nicotiana benthamiana. Liquid chromatography tandem mass spectrometry analysis and in vitro phosphorylation assays demonstrated that Thr-401 is the major phosphorylation site of the XJTGB1 protein, and suggested that a Thr-395 kinase docking site supports Thr-401 phosphorylation. Substitution of Thr-395 with alanine (T395A) only moderately impaired virus cell-to-cell movement and systemic infection. In contrast, the Thr-401 alanine (T401A) virus mutant was unable to systemically infect N. benthamiana but had only minor effects in monocot hosts. Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections. However, virus derivatives with single glutamic acid substitutions at Thr-395 and Thr-401 developed nearly normal systemic infections in the monocot hosts but were unable to infect N. benthamiana systemically, and none of the double mutants was able to infect dicot and monocot hosts. The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability. Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions

Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1

<p>Abstract</p> <p>Background</p> <p><it>Rice black-streaked dwarf virus </it>(RBSDV), a member of the genus <it>Fijivirus </it>within the family <it>Reoviridae</it>, can infect several graminaceous plant species including rice, maize and wheat, and is transmitted by planthoppers. Although several RBSDV proteins have been studied in detail, functions of the nonstructural protein P6 are still largely unknown.</p> <p>Results</p> <p>In the current study, we employed yeast two-hybrid assays, bimolecular fluorescence complementation and subcellular localization experiments to show that P6 can self-interact to form punctate, cytoplasmic viroplasm-like structures (VLS) when expressed alone in plant cells. The region from residues 395 to 659 is necessary for P6 self-interaction, whereas two polypeptides (residues 580-620 and 615-655) are involved in the subcellular localization of P6. Furthermore, P6 strongly interacts with the viroplasm-associated protein P9-1 and recruits P9-1 to localize in VLS. The P6 395-659 region is also important for the P6-P9-1 interaction, and deleting any region of P9-1 abolishes this heterologous interaction.</p> <p>Conclusions</p> <p>RBSDV P6 protein has an intrinsic ability to self-interact and forms VLS without other RBSDV proteins or RNAs. P6 recruits P9-1 to VLS by direct protein-protein interaction. This is the first report on the functionality of RBSDV P6 protein. P6 may be involved in the process of viroplasm nucleation and virus morphogenesis.</p

Detection and characterization of spontaneous internal deletion mutants of Beet Necrotic yellow vein virus RNA3 from systemic host Nicotiana benthamiana

Abstract Background Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far. Results Systemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom. Conclusion Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana

Injectable kartogenin and apocynin loaded micelle enhances the alleviation of intervertebral disc degeneration by adipose-derived stem cell.

Cell transplantation has been proved the promising therapeutic effects on intervertebral disc degeneration (IVDD). However, the increased levels of reactive oxygen species (ROS) in the degenerated region will impede the efficiency of human adipose-derived stem cells (human ADSCs) transplantation therapy. It inhibits human ADSCs proliferation, and increases human ADSCs apoptosis. Herein, we firstly devised a novel amphiphilic copolymer PEG-PAPO, which could self-assemble into a nanosized micelle and load lipophilic kartogenin (KGN), as a single complex (PAKM). It was an injectable esterase-responsive micelle, and showed controlled release ability of KGN and apocynin (APO). Oxidative stimulation promoted the esterase activity in human ADSCs, which accelerate degradation of esterase-responsive micelle. Compared its monomer, the PAKM micelle possessed better bioactivities, which were attributed to their synergistic effect. It enhanced the viability, autophagic activation (P62, LC3 II), ECM-related transcription factor (SOX9), and ECM (Collagen II, Aggrecan) maintenance in human ADSCs. Furthermore, it is demonstrated that the injection of PAKM with human ADSCs yielded higher disc height and water content in rats. Therefore, PAKM micelles perform promoting cell survival and differentiation effects, and may be a potential therapeutic agent for IVDD

ALG3 as a prognostic biomarker and mediator of PD-1 blockade resistance in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer-related death globally, characterized by high heterogeneity and drug resistance, which significantly impacts clinical outcomes. The tumor microenvironment (TME) plays a critical role in HCC initiation and progression, with immune cell infiltration and immune checkpoint expression closely linked to tumor prognosis. N-glycosylation of proteins modulates immune responses within the TME. ALG3, a key N-glycosylation enzyme, is involved in protein glycosylation. Although ALG3 expression has been studied in various tumors, its role in regulating the immune microenvironment and its prognostic significance in HCC remain unclear.MethodsThis study comprehensively evaluates ALG3 expression in HCC and its relationship with the immune microenvironment using various techniques. First, bioinformatics analysis of HCC-related data from the TCGA database was performed to investigate ALG3 expression patterns in tumor tissues and its correlation with clinical features. Multiplex immunohistochemistry (mIHC) was then used to validate ALG3 expression in HCC tissue samples and examine its relationship with immune cell infiltration. Additionally, cell experiments and 3D human organoid-based culture models were employed to further assess the role of ALG3 in the HCC immune microenvironment.ResultsThe results showed significant overexpression of ALG3 in HCC tissues, with high expression correlating significantly with poor tumor prognosis. Further analysis revealed that high ALG3 expression was associated with reduced infiltration of CD8+ T cells and CD68+ macrophages in both tumor and stromal areas, while positively correlating with increased infiltration of FOXP3+ regulatory T cells (Tregs). Notably, ALG3 expression levels were also positively correlated with PD-L1 expression in HCC tissues.ConclusionsALG3 may serve as a potential prognostic biomarker and an immunotherapy target in HCC

One-Step Multiplex RT-PCR Method for Detection of Melon Viruses

This study presents a one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) method for the simultaneous detection of multiple viruses affecting melon crops. Viruses such as Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), Tobacco mosaic virus (TMV), Papaya ring spot virus (PRSV), and Melon yellow spot virus (MYSV) pose a great threat to melons. The mixed infection of these viruses is the most common observation in the melon-growing fields. In this study, we surveyed northern Xingjiang (Altay, Changji, Wujiaqu, Urumqi, Turpan, and Hami) and southern Xingjiang (Aksu, Bayingolin, Kashgar, and Hotan) locations in Xinjiang province and developed a one-step multiplex RT-PCR to detect these melon viruses. The detection limits of this multiplex PCR were 103 copies/&mu;L for ZYMV and MYSV and 102 copies/&mu;L for WMV, SqMV, PRSV, CMV, and TMV. The detection results in the field showed 242 samples were infected by one or more viruses. The multiplex RT-PCR protocol demonstrated rapid, simultaneous, and relatively effective detection of viruses such as WMV, CMV, ZYMV, SqMV, TMV, PRSV, and MYSV. The technique is designed to identify these melon viruses in a single reaction, enhancing diagnostic efficiency and reducing costs, thus serving as a reference for muskmelon anti-virus breeding in Xinjiang