Structural biology of innate immunity.

Innate immune responses depend on timely recognition of pathogenic or danger signals by multiple cell surface or cytoplasmic receptors and transmission of signals for proper counteractions through adaptor and effector molecules. At the forefront of innate immunity are four major signaling pathways, including those elicited by Toll-like receptors, RIG-I-like receptors, inflammasomes, or cGAS, each with its own cellular localization, ligand specificity, and signal relay mechanism. They collectively engage a number of overlapping signaling outcomes, such as NF-κB activation, interferon response, cytokine maturation, and cell death. Several proteins often assemble into a supramolecular complex to enable signal transduction and amplification. In this article, we review the recent progress in mechanistic delineation of proteins in these pathways, their structural features, modes of ligand recognition, conformational changes, and homo- and hetero-oligomeric interactions within the supramolecular complexes. Regardless of seemingly distinct interactions and mechanisms, the recurring themes appear to consist of autoinhibited resting-state receptors, ligand-induced conformational changes, and higher-order assemblies of activated receptors, adaptors, and signaling enzymes through conserved protein-protein interactions.

Widespread and Functional RNA Circularization in Localized Prostate Cancer.

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.

The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex.

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of ß-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

Distinct Effects of SARS-CoV-2 Protein Segments on Structural Stability, Amyloidogenic Potential, and α-Synuclein Aggregation.

Amyloidosis is characterized by the abnormal accumulation of misfolded proteins, called amyloid fibrils, leading to diverse clinical manifestations. Recent studies on the amyloidogenesis of SARS-CoV­2 protein segments have raised concerns on their potential link to post-infection neurodegeneration, however, the mechanisms remain unclear. Herein, we investigated the structure, stability, and amyloidogenic propensity of a nine-residue segment (SK9) of the SARS-CoV-2 envelope protein and their impact on neuronal protein α-synuclein (αSyn) aggregation. Specifically, the amino acid sequence of the SK9 wildtype has been modified from a basic and positively charged peptide (SFYVYSRVK), to a nearly neutral and more hydrophobic peptide (SAAVASAVK, labelled as SK9 var1), and to an acidic and positively charged peptide (SFYVYSRVK, labelled as SK9 var2). Our findings reveal that the SK9 wildtype exhibited a pronounced amyloidogenic propensity due to its disordered and unstable nature, while the SK9 variants possessed more ordered and stable structures preventing the amyloid formation. Significantly, the SK9 wildtype demonstrated distinct effect on αSyn aggregation kinetics and aggregate morphology to facilitate the formation of αSyn aggregates with enhanced resistance against enzymatic degradation. This study highlights the potential of modifying short peptide sequences to fine-tune their properties, providing insights into understanding and regulating viral-induced amyloid aggregations.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity.

The compact CRISPR/Cas9 system, which can be delivered with their gRNA and a full-length promoter for expression by a single adeno-associated virus (AAV), is a promising platform for therapeutic applications. We previously identified a compact SauriCas9 that displays high activity and requires a simple NNGG PAM, but the specificity is moderate. Here, we identified three compact Cas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Of these three Cas9 orthologs, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. Importantly, we generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We finally engineered SlugCas9 with mutations to generate a high-fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

Structural basis for guide RNA trimming by RNase D ribonuclease in Trypanosoma brucei.

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.

Efficacy and safety of recombinant human endostatin during peri-radiotherapy period in advanced non-small-cell lung cancer.

Background: This study aimed to retrospectively investigate the efficacy and safety of recombinant human endostatin (Rh-endostatin) combined with radiotherapy in advanced non-small-cell lung cancer (NSCLC). Methods: Patients with unresectable stage III and IV NSCLC who treated with radiotherapy were enrolled. Patients who received Rh-endostatin infusion throughout the whole peri-radiotherapy period formed the Endostar group, and those who received no Rh-endostatin infusion were the control group. Results: The median progression-free survival was 8.0 and 4.4 months (hazard ratio: 0.53; 95% CI: 0.32-0.90; p = 0.019) and median overall survival was 40.0 and 13.1 months (hazard ratio: 0.53; 95% CI: 0.28-0.98; p = 0.045) for the Endostar and control groups, respectively. The Endostar group exhibited a numerically lower rate of radiation pneumonitis relapse, radiation pneumonitis death and pulmonary fibrosis. Conclusion: Rh-endostatin infusion throughout the peri-radiotherapy period enhanced radiosensitivity and showed better survival outcomes and a tendency toward fewer radiation-related pulmonary events in patients with NSCLC.

Recombinant human endostatin (Rh-endostatin/Endostar) combined with chemotherapy has been approved as first-line standard treatment in patients with advanced non-small-cell lung cancer (NSCLC) in China. This study aimed to retrospectively investigate the efficacy and safety of Rh-endostatin combined with radiotherapy in advanced NSCLC. Patients with unresectable stage III and IV NSCLC who treated with radiotherapy were enrolled. Patients who received Rh-endostatin infusion throughout the whole peri-radiotherapy period were the Endostar group, and those receiving no Rh-endostatin infusion were the control group. Results showed that the median progression-free survival was 8.0 and 4.4 months, and median overall survival was 40.0 and 13.1 months, for the Endostar and control groups, respectively. The Endostar group had a lower rate of radiation pneumonitis relapse, radiation pneumonitis death and pulmonary fibrosis. In conclusion, Rh-endostatin infusion throughout the peri-radiotherapy period enhanced radiosensitivity and showed better survival outcomes and a tendency toward fewer radiation-related pulmonary events in patients with NSCLC.

Structural studies reveal a ring-shaped architecture of deep-sea vent phage NrS-1 polymerase.

NrS-1 is the first known phage that can infect Epsilonproteobacteria, one of the predominant primary producers in the deep-sea hydrothermal vent ecosystems. NrS-1 polymerase is a multidomain enzyme and is one key component of the phage replisome. The N-terminal Prim/Pol and HBD domains are responsible for DNA polymerization and de novo primer synthesis activities of NrS-1 polymerase. However, the structure and function of the C-terminus (CTR) of NrS-1 polymerase are poorly understood. Here, we report two crystal structures, showing that NrS-1 CTR adopts one unique hexameric ring-shaped conformation. Although the central helicase domain of NrS-1 CTR shares structural similarity with the superfamily III helicases, the folds of the Head and Tail domains are completely novel. Via mutagenesis and in vitro biochemical analysis, we identified many residues important for the helicase and polymerization activities of NrS-1 polymerase. In addition to NrS-1 polymerase, our study may also help us identify and understand the functions of multidomain polymerases expressed by many NrS-1 related phages.

Structural analysis and functional study of phosphofructokinase B (PfkB) from Mycobacterium marinum.

Phosphofructokinase B (PfkB) belongs to the ribokinase family, which uses the phosphorylated sugar as substrate, and catalyzes fructose-6-phosphate into fructose-1,6-diphosphate. However, the structural basis of Mycobacterium marinum PfkB is not clear. Here, we found that the PfkB protein was monomeric in solution, which was different from most enzymes in this family. The crystal structure of PfkB protein from M. marinum was solved at a resolution of 2.21 Å. The PfkB structure consists of two domains, a major three-layered α/ß/α sandwich-like domain characteristic of the ribokinase-like superfamily, and a second domain composed of four-stranded ß sheets. Structural comparison analysis suggested that residues G236, A237, G238, and D239 could be critical for ATP catalysis and substrate binding of PfkB. Our current work provides new insights into understanding the mechanism of the glycolysis in M. marinum.

Bioinformatics analysis of the expression and role of microRNA-221-3p in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, associated with a high rate of morbidity and mortality. However, the target genes of miR-221-3p and the underlying mechanism involved in HNSCC are still not clear. Therefore, in the current study, we studied the role of miR-221-3p in the HNSCC. METHODS: Tissues collected from 48 control and 21 HNSCC patients were processed to check the differential expression of miR-221-3p by RT-qPCR. Overexpression of microRNA-221-3p (miR-221-3p) is significantly correlated to the onset and progression of HNSCC. We also conducted the meta-analysis of the cancer literature from the cancer genome atlas (TCGA) and the Gene Expression Omnibus (GEO) database to estimate the expression of miR-221-3p in HNSCC. The miR-221-3p target genes in the HNSCC were predicted with the miRWalk and TCGA databases, and functionally annotated via the Gene Ontology. Finally, Spearman's analysis was used to determine the role of the related target genes in important pathways involved in the development of HNSCC. RESULTS: We observed a significantly higher expression of miR-221-3p in HNSCC compared to the normal with a summary receiver operating characteristic (sROC) of 0.86(95% Cl: 0.83,0.89). The KEGG and GO comprehensive analysis predicted that miR-221-3p might be involved in the development of HNSCC through the following metabolic pathways, viz. Drug metabolism - cytochrome P450 UGT1A7 and MAOB may be important genes for the role of miR-221-3p. CONCLUSION: Based on bioinformatics analysis, our results indicate that miR-221-3p may be used as a non-invasive and hypersensitive biomarker in the diagnosis. Thus, it can be concluded that miR-221-3p may be an extremely important gene locus involved in the process of the deterioration and eventual tumorigenesis of HNSCC. Hopefully, additional work will validate its usefulness as a target for future clinical research.

Clinical features and death risk factors in COVID-19 patients with cancer: a retrospective study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has spread around the world. This retrospective study aims to analyze the clinical features of COVID-19 patients with cancer and identify death outcome related risk factors. METHODS: From February 10th to April 15th, 2020, 103 COVID-19 patients with cancer were enrolled. Difference analyses were performed between severe and non-severe patients. A propensity score matching (PSM) analysis was performed, including 103 COVID-19 patients with cancer and 206 matched non-cancer COVID-19 patients. Next, we identified death related risk factors and developed a nomogram for predicting the probability. RESULTS: In 103 COVID-19 patients with cancer, the main cancer categories were breast cancer, lung cancer and bladder cancer. Compared to non-severe patients, severe patients had a higher median age, and a higher proportion of smokers, diabetes, heart disease and dyspnea. In addition, most of the laboratory results between two groups were significantly different. PSM analysis found that the proportion of dyspnea was much higher in COVID-19 patients with cancer. The severity incidence in two groups were similar, while a much higher mortality was found in COVID-19 patients with cancer compared to that in COVID-19 patients without cancer (11.7% vs. 4.4%, P = 0.028). Furthermore, we found that neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) were related to death outcome. And a nomogram based on the factors was developed. CONCLUSION: In COVID-19 patients with cancer, the clinical features and laboratory results between severe group and non-severe group were significantly different. NLR and CRP were the risk factors that could predict death outcome.

Clinical characteristics and a decision tree model to predict death outcome in severe COVID-19 patients.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) spreads rapidly among people and causes a pandemic. It is of great clinical significance to identify COVID-19 patients with high risk of death. METHODS: A total of 2169 adult COVID-19 patients were enrolled from Wuhan, China, from February 10th to April 15th, 2020. Difference analyses of medical records were performed between severe and non-severe groups, as well as between survivors and non-survivors. In addition, we developed a decision tree model to predict death outcome in severe patients. RESULTS: Of the 2169 COVID-19 patients, the median age was 61 years and male patients accounted for 48%. A total of 646 patients were diagnosed as severe illness, and 75 patients died. An older median age and a higher proportion of male patients were found in severe group or non-survivors compared to their counterparts. Significant differences in clinical characteristics and laboratory examinations were found between severe and non-severe groups, as well as between survivors and non-survivors. A decision tree, including three biomarkers, neutrophil-to-lymphocyte ratio, C-reactive protein and lactic dehydrogenase, was developed to predict death outcome in severe patients. This model performed well both in training and test datasets. The accuracy of this model were 0.98 in both datasets. CONCLUSION: We performed a comprehensive analysis of COVID-19 patients from the outbreak in Wuhan, China, and proposed a simple and clinically operable decision tree to help clinicians rapidly identify COVID-19 patients at high risk of death, to whom priority treatment and intensive care should be given.

Indications for and contraindications of immune checkpoint inhibitors in cancer patients with COVID-19 vaccination.

The COVID-19 pandemic has lasted over 1 year and will not disappear in a short time. There is no specific remedy against the virus as yet. Vaccination is thus far one of the most important strategies for preventing COVID-19. Cancer patients with COVID-19 have a higher mortality because of immunosuppression. Immune checkpoint inhibitors (ICIs) are a novel anticancer strategy for blocking inhibitory pathways, which are related to the immune response. There is a question regarding whether COVID-19 vaccination and ICI treatment impact each other in cancer patients. This review explores both sides of the relationship between ICI treatment and COVID-19 vaccination and suggests good efficacy and safety of ICI treatment after COVID-19 vaccination as well as little impact on the virus protection and toxicity associated with COVID-19 vaccination during ICI treatment.

Lay abstract The COVID-19 pandemic has lasted over 1 year. Vaccination is a promising strategy for preventing COVID-19. Cancer patients are prone to infection with COVID-19, and these patients have high mortality. Immune checkpoint inhibitors (ICIs) are a novel anticancer strategy. Whether COVID-19 vaccination and ICI treatment impact each other in cancer patients remains unknown. This review explores both sides of the relationship between ICI treatment and COVID-19 vaccination and suggests good efficacy and safety of ICI treatment after COVID-19 vaccination as well as little impact on the virus protection and toxicity associated with COVID-19 vaccination during ICI treatment.

Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA.

BACKGROUND: Very few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133). METHODS: We overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation. RESULTS: miPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass. CONCLUSION: miPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

The Trp183 is essential in lactonohydrolase ZHD detoxifying zearalenone and zearalenols.

Lactonohydrolase ZHD can detoxify oestrogenic mycotoxin zearalenone and zearalenols through hydrolysis and decarboxylation. The detail mechanism, especially the role of Trp183, which interacts with substrate through p-π interaction and one hydrogen bond, is still unknown. The Trp183 mutants abolished activity to ZEN, α-ZOL and ß-ZOL, except that W183F mutant retained about 40% activity against α-ZOL. In two W183F-reactant complex structures the reactants still bind at the active position and it suggested that this p-π interaction takes responsible for the reactants recognization and allocation. Further, the ZHD-productant complex structures showed that the resorcinol ring of hydrolysed α-ZOL and hydrolysed ß-ZOL move a distance of one ring as compare to the resorcinol ring of reactant α-ZOL and ß-ZOL. The same movement also found in comparison of hydrolysed ZEN and ZEN. In the structure of W183F complex with hydrolysed α-ZOL the resorcinol ring of hydrolysed α-ZOL doesn't move as compare to the resorcinol ring of reactant α-ZOL. It suggested the Trp183 coordinated hydrogen bond takes responsible for the movement of the hydrolysed product. These functional and structural results suggested that Trp183 is essential for ZHD detoxifying zearalenone and zearalenols.

Unique 5'-P recognition and basis for dG:dGTP misincorporation of ASFV DNA polymerase X.

African swine fever virus (ASFV) can cause highly lethal disease in pigs and is becoming a global threat. ASFV DNA Polymerase X (AsfvPolX) is the most distinctive DNA polymerase identified to date; it lacks two DNA-binding domains (the thumb domain and 8-KD domain) conserved in the homologous proteins. AsfvPolX catalyzes the gap-filling reaction during the DNA repair process of the ASFV virus genome; it is highly error prone and plays an important role during the strategic mutagenesis of the viral genome. The structural basis underlying the natural substrate binding and the most frequent dG:dGTP misincorporation of AsfvPolX remain poorly understood. Here, we report eight AsfvPolX complex structures; our structures demonstrate that AsfvPolX has one unique 5'-phosphate (5'-P) binding pocket, which can favor the productive catalytic complex assembly and enhance the dGTP misincorporation efficiency. In combination with mutagenesis and in vitro catalytic assays, our study also reveals the functional roles of the platform His115-Arg127 and the hydrophobic residues Val120 and Leu123 in dG:dGTP misincorporation and can provide information for rational drug design to help combat ASFV in the future.