Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

Bacillus lacisalsi sp. nov., a moderately haloalkaliphilic bacterium isolated from a saline-alkaline lake.

An alkaliphilic and moderately halophilic strain, designated YSP-3T, characterised by optimal growth at pH 9.0 and at 8.0% (w/v) NaCl, was isolated from Yangshapao Lake, Jilin Province, China. Cells of this strain is Gram-positive, straight rods and form a central or sub-terminal ellipsoidal endospore. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it was grouped in the genus Bacillus with Bacillus aurantiacus K1-5T and Bacillus populi FJAT-45347T as the close relative (97.5 and 97.2% 16S rRNA gene sequence similarity, respectively). Genomic relatedness between strain YSP-3T and its close relative was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity with the values of 70.3-85.1%, 19.7-20.1% and 71.5-71.6%, respectively. Comparative genomics analysis showed that strain YSP-3T has distinct amino acid bias and significantly differences from foreign invasion events during evolution relative to the reference strains. Cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant polar lipids are phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol. The predominant quinone is menaquinone-7. The major fatty acids of strain YSP-3T are anteiso-C15:0, iso-C15:0, iso-C16:0, anteiso-C17:0 and Iso-C14:0. DNA G + C content of strain YSP-3T is 48.3 mol%. Based on genomics analysis, physiological, biochemical and chemotaxonomic data, strain YSP-3T represent a novel species, for which the name Bacillus lacisalsi sp. nov. is proposed. The type strain is YSP-3T (  = ACCC 60365T = KCTC 33934T).

Draft Genome Sequence of Salipaludibacillus keqinensis ACCC 60430T, an Aerobic Halophilic Bacterium Isolated from a Salt Lake.

The haloalkaliphilic bacterium Salipaludibacillus keqinensis ACCC 60430T, which grows optimally at 8.0% (wt/vol) Na+ and pH 9.0, was isolated from Keqin Lake in Qiqihaer, China. The draft genome includes 4,006 predicted genes and 3,784 coding sequences (CDSs). Genomic analysis showed that various genes may explain the mechanism of salt and alkali resistance.

Salipaludibacillus keqinensis sp. nov., a moderately halophilic bacterium isolated from a saline-alkaline lake.

A novel Gram-stain positive, short rod, forming sub-terminal endospores of ellipsoidal shape, halophilic, alkaliphilic and aerobic bacterium, designated strain KQ-12T, was isolated from a saline-alkaline lake in China, and characterised by a polyphasic taxonomic approach. The isolate grew at 4-40 °C (optimum, 25 °C), at pH 8.0-10.0 (pH 9.0) and in the presence of 0-16% (w/v) NaCl (8%). 16S rRNA gene sequence similarity of KQ-12T to species in the genera Salipaludibacillus ranged from 96.6 to 98.1%. Phylogenetic trees indicated that the strain should be assigned to the genus Salipaludibacillus. The polar lipids of KQ-12T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid and its major cellular fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C15:0, and C16:0. The isoprenoid quinone was MK-7. These key chemotaxonomic properties also confirmed the affiliation of the strain to the genus Salipaludibacillus. However, some physiological, biochemical properties, low average nucleotide identity and low digital DNA-DNA hybridization relatedness values enabled the strain to be differentiated from closely related species of the genus Salipaludibacillus. Thus, KQ-12T can be classified as a novel species in the genus Salipaludibacillus, for which the name Salipaludibacillus keqinensis sp. nov. is proposed. The type strain is KQ-12T ( =  ACCC 60430T   =  KCTC 33935T).

Draft Genome Sequence of Bacillus sp. Strain YSP-3, a Halophilic, Alkaliphilic Bacterium Isolated from a Salt Lake.

The halophilic, alkaliphilic bacterium Bacillus sp. strain YSP-3 was isolated from a salt lake. It grows optimally at 8% (wt/vol) NaCl (pH 9.0). The draft genome is composed of 4,006 predicted genes. Genomic analysis showed that various genes are potentially involved in the adaptation mechanisms for osmotic stress and pH homeostasis.

Chicken RNA-binding protein T-cell internal antigen-1 contributes to stress granule formation in chicken cells and tissues.

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.

Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA-1 and TIA-1-related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA-1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV-induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA-1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA.-Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein.

Newcastle disease virus (NDV) encodes a highly phosphorylated P protein; however, the phosphorylation sites have not been identified, and the relationship between phosphorylation and protein function is still unclear. In this study, we bioinformatically predicted 26 amino acid residues in the P protein as potential phosphorylation sites. Furthermore, we treated infected cells with kinase inhibitors to investigate NDV propagation and found that protein kinase C (PKC) is involved in the NDV life cycle and that PKC-activated phosphorylation functions in NDV replication. Using an NDV minigenome assay, we found that expression of a reporter protein decreased when the minigenome system contained P mutants lacking T44, S48, T271, S373 and especially T111. The phosphorylation status of S48, T111, S125 and T271 was determined by Phos-tag SDS-PAGE analysis. Coimmunoprecipitation assays showed that the binding activity of NP and the P-T111A mutant was stronger than that of NP and the wild-type P, suggesting that P-T111 is involved in NP-P interaction. This study sheds light on the mechanism by which P protein phosphorylation affects NDV replication and transcription.

Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling.

Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN.