Human papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the human papillomavirus 16 (HPV16) life cycle. Here, we demonstrate that in HPV-negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication, and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phospho-mimic (SAMHD1 T592D) reduces E1-E2-mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells, SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1, and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes, endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here, we demonstrate that HPV16 replication converts sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) into a homologous recombination (HR) factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16-immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16-immortalized human foreskin keratinocytes (HFKs) and HPV16-positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

Ferroptosis-related lncRNA NRAV affects the prognosis of hepatocellular carcinoma via the miR-375-3P/SLC7A11 axis.

Ferroptosis has important value in cancer treatment. It is significant to explore the new ferroptosis-related lncRNAs prediction model in Hepatocellular carcinoma (HCC) and the potential molecular mechanism of ferroptosis-related lncRNAs. We constructed a prognostic multi-lncRNA signature based on ferroptosis-related differentially expressed lncRNAs in HCC. qRT-PCR was applied to determine the expression of lncRNA in HCC cells. The biological roles of NRAV in vitro and in vivo were determined by performing a series of functional experiments. Furthermore, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to confirm the interaction of NRAV with miR-375-3P. We identified 6 differently expressed lncRNAs associated with the prognosis of HCC. Kaplan-Meier analyses revealed the high-risk lncRNAs signature associated with poor prognosis of HCC. Moreover, the AUC of the lncRNAs signature showed utility in predicting HCC prognosis. Further functional experiments show that the high expression of NRAV can strengthen the viciousness of HCC. Interestingly, we found that NRAV can enhance iron export and ferroptosis resistance. Further study showed that NRAV competitively binds to miR-375-3P and attenuates the inhibitory effect of miR-375-3P on SLC7A11, affecting the prognosis of patients with HCC. In conclusion, We developed a novel ferroptosis-related lncRNAs prognostic model with important predictive value for the prognosis of HCC. NRAV is important in ferroptosis induction through the miR-375-3P/SLC7A11 axis.

Jujuboside B alleviates acetaminophen-induced hepatotoxicity in mice by regulating Nrf2-STING signaling pathway.

BACKGROUND: Jujuboside B (JuB) is the main bioactive saponin component of Chinese anti-insomnia herbal medicine Ziziphi Spinosae Semen, which has been reported to possess varied pharmacological functions. Even though it has been traditionally used to treat inflammation- and toxicity-related diseases, the effects of JuB on acetaminophen (APAP) overdose-induced hepatotoxicity have not been determined yet. METHODS: C57BL/6 J mice were pre-treated with JuB (20 or 40 mg/kg) for seven days before APAP (400 mg/kg) injection. After 24 h of APAP treatment, serum, and liver tissues were collected to evaluate the therapeutic effects. To investigate whether the Nrf2-STING signaling pathway is involved in the protective effects of JuB against APAP-induced hepatotoxicity, the mice received the DMXAA (the specific STING agonist) or ML385 (the specific Nrf2 inhibitor) during the administration of JuB, and Hematoxylin-eosin staining, Real-time PCR, immunohistochemical, and western blot were performed. RESULTS: JuB pretreatment reversed APAP-induced CYP2E1 accumulations and alleviated APAP-induced acute liver injury. Furthermore, JuB treatment significantly inhibited oxidative stress and the pro-inflammatory cytokines, as well as alleviated hepatocyte apoptosis induced by APAP. Besides, our result also demonstrated that JuB treatment upregulated the levels of total Nrf2, facilitated its nuclear translocation, upregulated the expression of HO-1 and NQO-1, and inhibited the APAP-induced STING pathway activation. Finally, we verified that the beneficial effects of JuB were weakened by DMXAA and ML385. CONCLUSION: Our study suggested that JuB could ameliorate APAP-induced hepatic damage and verified a previously unrecognized mechanism by which JuB prevented APAP-induced hepatotoxicity through adjusting the Nrf2-STING pathway.

Comprehensive analysis of candidate signatures of long non-coding RNA LINC01116 and related protein-coding genes in patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a long-term malignancy that causes high morbidities and mortalities worldwide. Notably, long non-coding RNAs (LncRNAs) have been identified as candidate targets for malignancy treatments. METHODS: LncRNA LINC01116 and its Pearson-correlated genes (PCGs) were identified and analyzed in HCC patients. The diagnostic and prognostic value of the lncRNA was evaluated using data from The Cancer Genome Atlas (TCGA). Further, we explored the target drugs of LINC01116 for clinical application. Relationships between immune infiltration and PCGs, methylation and PCGs were explored. The diagnostic potentials were then validated by Oncomine cohorts. RESULTS: LINC01116 and the PCG OLFML2B are differentially and highly expressed in tumor tissues (both P ≤ 0.050). We found that LINC01116, TMSB15A, PLAU, OLFML2B, and MRC2 have diagnostic potentials (all AUC ≥ 0.700, all P ≤ 0.050) while LINC01116 and TMSB15A have prognostic significance (both adjusted P ≤ 0.050). LINC01116 was enriched in the vascular endothelial growth factor (VEGF) receptor signaling pathway, mesenchyme morphogenesis, etc. After that, candidate target drugs with potential clinical significance were identified: Thiamine, Cromolyn, Rilmenidine, Chlorhexidine, Sulindac_sulfone, Chloropyrazine, and Meprylcaine. Analysis of immune infiltration revealed that MRC2, OLFML2B, PLAU, and TMSB15A are negatively associated with the purity but positively associated with the specific cell types (all P < 0.050). Analysis of promoter methylation demonstrated that MRC2, OLFML2B, and PLAU have differential and high methylation levels in primary tumors (all P < 0.050). Validation results of the differential expressions and diagnostic potential of OLFML2B (Oncomine) were consistent with those obtained in the TCGA cohort (P < 0.050, AUC > 0.700). CONCLUSIONS: Differentially expressed LINC01116 could be a candidate diagnostic and an independent prognostic signature in HCC. Besides, its target drugs may work for HCC therapy via the VEGF receptor signaling pathway. Differentially expressed OLFML2B could be a diagnostic signature involved in HCC via immune infiltrates.

Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor of the IRF family. IRF8 plays a key role in normal B cell differentiation, a cellular process that is intrinsically associated with Epstein-Barr virus (EBV) reactivation. However, whether IRF8 regulates EBV lytic replication remains unknown. In this study, we utilized a CRISPR/Cas9 genomic editing approach to deplete IRF8 and found that IRF8 depletion dramatically inhibits the reactivation of EBV upon lytic induction. We demonstrated that IRF8 depletion suppresses the expression of a group of genes involved in apoptosis and thus inhibits apoptosis induction upon lytic induction by B cell receptor (BCR) stimulation or chemical induction. The protein levels of caspase-1, caspase-3 and caspase-8 all dramatically decreased in IRF8-depleted cells, which led to reduced caspase activation and the stabilization of KAP1, PAX5 and DNMT3A upon BCR stimulation. Interestingly, caspase inhibition blocked the degradation of KAP1, PAX5 and DNMT3A, suppressed EBV lytic gene expression and viral DNA replication upon lytic induction, suggesting that the reduced caspase expression in IRF8-depleted cells contributes to the suppression of EBV lytic replication. We further demonstrated that IRF8 directly regulates CASP1 (caspase-1) gene expression through targeting its gene promoter and knockdown of caspase-1 abrogates EBV reactivation upon lytic induction, partially through the stabilization of KAP1. Together our study suggested that, by modulating the activation of caspases and the subsequent cleavage of KAP1 upon lytic induction, IRF8 plays a critical role in EBV lytic reactivation.

Immunoregulation with mTOR inhibitors to prevent COVID-19 severity: A novel intervention strategy beyond vaccines and specific antiviral medicines.

Coronavirus disease 2019 (COVID-19) has become a major global public health concern. The mortality rate for critically ill patients is up to 60%, and, thus, reducing the disease severity and case mortality is a top priority. Currently, cytokine storms are considered as the major cause of critical illness and death due to COVID-19. After a systematical review of the literature, we propose that cross-reactive antibodies associated with antibody-dependent enhancement (ADE) may actually be the cause of cytokine storms. It would be more difficult to develop vaccines for highly pathogenic human coronaviruses (CoVs) if ADE characteristics are taken into consideration. Therefore, it is urgent to find an effective way to prevent the occurrence of severe illness as severe acute respiratory syndrome CoV-2 specific drugs or vaccines are still in development. If the activation of memory B cells can be selectively inhibited in high-risk patients at an early stage of COVID-19 to reduce the production of cross-reactive antibodies against the virus, we speculate that ADE can be circumvented and severe symptoms can be prevented. The mammalian target of rapamycin (mTOR) inhibitors satisfy such needs and it is recommended to conduct clinical trials for mTOR inhibitors in preventing the severity of COVID-19.

Comparison on radiation effective dose and image quality of right coronary artery on prospective ECG-gated method between 320 row CT and 2nd generation (128-slice) dual source CT.

This retrospective study was to compare the image quality of right coronary artery (RCA) and effective radiation dose on prospective ECG-gated method between 320 row computed tomography (CT) and 2nd generation (128-slice) dual source CT. A total of 215 candidates underwent CT coronary angiography using prospective ECG-gated method, 120 patients enrolled in 320 row CT group, and 95 patients in dual source CT group. We divided RCA image quality scores as 1/2/3/4, which means excellent/good/adequate/not assessable and heart rates were considered, as well as the radiation dose. There is no statistically significant difference of RCA image quality of Score 1/2 between 320 row CT and 2nd generation dual source CT, but lower heart rate (<70/min) improved RCA image quality. Meanwhile, the 2nd generation dual source CT scan have significant lower radiation dose. For patients with high level heart rate variation, both prospective ECG-gated method of 320 row CT scan (Toshiba) and 2nd generation dual source CT scan (Siemens) basically provided good image quality on RCA. There is an advantage of effective radiation dose reduction in prospective ECG-gated method using the 2nd generation dual source CT scan. After the iodine contrast agent was injected into elbow vein, the threshold triggering method was used to carry out prospective gated scanning, and the acquired fault image was reconstructed by the standard post-processing software of each manufacturer. The radiation dose value is obtained through the dose report automatically generated after each scan.

Oridonin inhibits hypoxia-induced epithelial-mesenchymal transition and cell migration by the hypoxia-inducible factor-1α/matrix metallopeptidase-9 signal pathway in gallbladder cancer.

Hypoxia has crucial roles in cancer development and progression. Our previous study indicated that cell migration was increased in a hypoxic microenvironment in GBC-SD gallbladder cancer (GBC) cells. Oridonin, a bioactive diterpenoid compound that is isolated from the plant Rabdosia rubescens, has been identified as an anticancer agent in various types of cancer. However, its roles in cell proliferation, apoptosis, and migration in a hypoxic microenvironment and the associated regulatory mechanisms have not yet to be fully elucidated in GBC. The present study investigated the effect of oridonin on cell proliferation, apoptosis, the cell cycle and cell migration in GBC in vitro and in vivo. Furthermore, the role of oridonin in hypoxia-induced cell migration and its underlying mechanisms were explored in GBC. The results indicated that treatment with oridonin significantly suppressed cell proliferation and the metastatic ability of GBC-SD cells in a dose-dependent manner, increased the level of cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Further experiments demonstrated that oridonin could inhibit hypoxia-induced epithelial-mesenchymal transition and cell migration by downregulating the expression levels of hypoxia-inducible factor (HIF)-1α/matrix metallopeptidase (MMP)-9. In addition, oridonin suppressed GBC cell growth and downregulated the expression levels of HIF-1α and MMP-9 in a GBC-SD cell xenograft model. Taken together, these results suggest that oridonin possesses anticancer properties in GBC. Notably, oridonin can suppress tumor epithelial-mesenchymal transition and cell migration by targeting the HIF-1α/MMP-9 signaling pathway.

Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat.

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

Validation of the American Joint Committee on Cancer eighth edition staging system in patients undergoing hepatectomy for hepatocellular carcinoma: a US population-based study.

BACKGROUND: The American Joint Committee on Cancer (AJCC) eighth edition staging system for hepatocellular carcinoma (HCC) has incorporated several significant changes. This study aims to evaluate the newly proposed staging system and assess its strengths and weaknesses. MATERIALS AND METHODS: Using the Surveillance, Epidemiology, and End Results database, we identified patients with pathologically confirmed stage I-III HCC diagnosed between 2004 and 2014. RESULTS: After all exclusion criteria were applied, AJCC seventh and eighth edition staging was possible in 4931 patients. According to the AJCC eighth edition staging system, stages IB and II did not differ significantly in terms of overall survival (OS) and cause-specific survival (CSS) (P = 0.928 and 0.872, respectively). On the basis of the above results, we reclassified T1a, T1b, and T2 into several subgroups. According to the modified AJCC eighth edition staging system, OS and CSS among the five groups of patients differed significantly. For OS predication, the Akaike information criterion values for the AJCC seventh, eighth, and modified eighth edition staging systems were 29,288.24, 29,282.85, and 27,182.21, respectively, and the c-indices for the AJCC seventh, eighth, and modified eighth edition staging systems were 0.5716, 0.5805, and 0.6082, respectively. Regarding CSS, the Akaike information criterion values for the AJCC seventh, eighth, and modified eighth edition staging systems were 21,701.11, 21,682.12, and 20,313.26, respectively, and the c-indices for the AJCC seventh, eighth, and modified eighth edition staging systems were 0.5983, 0.6117, and 0.6436, respectively. CONCLUSIONS: This is the first large-scale validation of the AJCC eighth edition staging system for patients undergoing hepatectomy. Our study revealed that there was a lack of discrepancy in the outcomes for stage IB and II tumors for AJCC eighth edition staging system of HCC. Our modified AJCC eighth edition staging system allows for finer stratification of patients undergoing hepatectomy according to more detailed tumor size and vascular invasion classifications.

Lysosomes-targeting imaging and anticancer properties of novel bis-naphthalimide derivatives.

A series of novel N,N-bis(3-aminopropyl)methylamine bridged bis-naphthalimide derivatives NI1-NI8 containing saturated nitrogenous heterocycles were designed and synthesized, their cytotoxic activities against Hela, MCF-7, A549 and MGC-803 cells were investigated, Compounds NI1-NI4 modified with piperidine and piperazine exhibited good and selective cytotoxic activity, for instance, compounds NI1 and NI4 showed potent cytotoxic activity against Hela cells and MGC-803 cells with the IC50 values of 2.89, 060, 2.73 and 1.60 µM, respectively, better than the control drug (Amonafide). However, compounds NI5-NI8 conjugated with pyrrole derivatives showed weak cytotoxic activities against the all tested cell lines. Furthermore, their DNA binding properties, fluorescence imaging and cell cycle were investigated. Interestingly, compounds NI1 and NI4 showed fluorescence enhancement because of the strong binding with Ct-DNA, and exhibited fluorescence imaging with Hela cells on the lysosomes.

Long Noncoding RNA AFAP1-AS1 Promoted Tumor Growth and Invasion in Cholangiocarcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been shown to play important roles in a wide range of pathophysiological processes, including cancer progression. Our previous study has shown that AFAP1-AS1 was upregulated and acted as an oncogene in hepatocellular carcinoma. However, the expression and biological functions of lncRNA AFAP1-AS1 in intrahepatic cholangiocarcinoma (CCA) remains largely unknown. METHODS: The expression level of AFAP1-AS1 was measured in 56 pairs of human cholangiocarcinoma tumor tissues and corresponding adjacent normal bile duct tissues. The correlation between AFAP1-AS1 and the clinicopathological features were evaluated by chi-square test. The effects of AFAP1-AS1 on CCA cells were determined by CCK-8 assay, clone formation assay, flow cytometry and transwell assay. Finally, to determine the effect of AFAP1-AS1 on tumor growth in vivo, AFAP1-AS1 knockdowned CCLP-1 cells were subcutaneously into nude mice to evaluate tumor growth. RESULTS: In this study, we found that lncRNA AFAP1-AS1 was increased in CCA tissues and patients with high AFAP1-AS1 expression had a shorter overall survival. SiRNA-mediated AFAP1-AS1 knockdown significantly decreased cell proliferation of the CCA cells, with downregulation of C-myc and Cycling D1 in vitro. Furthermore, AFAP1-AS1 silencing inhibited cell migration partly due to decrease the expression of MMP-2 and MMP-9. In addition, CCLP-1 cells with AFAP1-AS1 knockdown were injected into nude mice to investigate the effect of AFAP1-AS1 on the tumorigenesis in vivo. CONCLUSIONS: Taken together, our findings suggested that AFAP1-AS1 might promote the CCA progression and provided a novel potential therapeutic target for CCA.

Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD002411 (http://proteomecentral.proteomexchange.org/dataset/PXD002411).

Critical role for the long non-coding RNA AFAP1-AS1 in the proliferation and metastasis of hepatocellular carcinoma.

Increasing evidence has indicated that dysregulation of long non-coding RNAs (lncRNAs) can contribute to the progression and metastasis of human cancer, including HCC. Previous studies have shown that the lncRNA AFAP1-AS1 plays a critical role in cancer. However, the roles of AFAP1-AS1 in HCC remain to be determined. In the present study, AFAP1-AS1 was found to be increased in HCC tissues, and high AFAP1-AS1 expression was associated with tumor size, TNM stage, vascular invasion, and poor prognosis. Silencing of AFAP1-AS1 significantly reduced cell proliferation, clonal growth, cell migration, and invasion and increased apoptosis in vitro. Furthermore, AFAP1-AS1 silencing markedly reduced tumor growth in a murine allograft model in vivo. The results suggested that AFAP1-AS1 is important in HCC development and serves as a therapeutic target of HCC.

Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China.

Porcine epidemic diarrhea virus (PEDV) has caused devastating impact on pig-rearing industry in China and current vaccine is not effective against the circulating PEDV variants. In the present study, the full-length genome sequence from a PEDV isolate (CH/HNQX-3/14) was determined. The complete genome sequence analysis showed that the CH/HNQX-3/14 possessed unique deletion regions in the S and ORF3 genes. It was identified as a recombinant strain using phylogenetic analysis and recombination detection program. Further analyses of the full-length sequence suggest that CH/HNQX-3/14 is a natural recombinant between the attenuated vaccine strains (CV777 and DR13) and circulating wild-type strain (CH/ZMDZY/11). The recombination occurred not only in structural protein-coding region (S1 and N genes) but also in non-structural protein-coding region (replicases 1a and ORF3 genes). These results provided new evidence that PEDV strains circulating in China underwent recombination between vaccine and field strains, suggesting that recombination contributes to the genetic diversity of PEDV. Our findings provide valuable information on PEDV evolution and underscore the need for ongoing surveillance of this economically important swine disease.

Isolation and genetic analysis of a variant porcine epidemic diarrhea virus in China.

Porcine epidemic diarrhea virus (PEDV) is having a severe effect on the pig breeding industry in central China. The mucosa and the content of the small intestine from newborn pre-weaned piglets with diarrhea were tested for the presence of PEDV by molecular and morphologic methods, and found to be positive. Negative-staining electron microscopy (EM) revealed the presence of coronavirus- like particles in the samples. The result of molecular detection by nested RT-PCR based on the amplification of the M gene was positive. Using a novel alternative method we successfully propagated the PEDV strain (CH/QX-2) in Vero cells, confirmed by ultrathin sections of the cells and Immunofluorescence assay (IFA). Phylogenetic analysis based on the partial S gene showed that the CH/QX-2 isolate was genetically closer to strains more commonly found in China, but differed genetically from two domestic strains (CH/S, 1986 and LZC, 2007), Korean strains (DR13, 2007), and the vaccine strain (CV777 vs) currently being used in China. CH/QX-2 formed a unique clade in the derived phylogenetic tree indicating that the CH/QX-2 strain currently circulating in central China is a new variant of PEDV. This study extends current knowledge on the diversity and epidemiology of PEDV.

Phosphorylation of the chromatin binding domain of KSHV LANA.

The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

Phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in central China based on the ORF3 gene and the main neutralization epitopes.

Since 2010, porcine epidemic diarrhea has re-emerged with devastating impact on the swine-raising industry in central China. To investigate the epidemic characteristics of PEDV, the complete ORF3 genes of 14 PEDV field strains from central China during 2012 to 2013 were cloned, sequenced and compared with reference strains. Phylogenetic analysis based on the complete ORF3 gene showed that the PEDVs in central China and the reference strains could be divided into three groups: G1, G2, and G3. The 14 PEDV isolates were classified as G1 and showed a close relationship to some Chinese strains isolated previously in central China and differed genetically from recent isolates from southern China, Korean strains (SM98 and DB1865, 2012), the Chinese LZC strain (2007), and the vaccine strain (CV777) being used in China. Our findings suggested that the PEDVs circulating between 2012 and 2013 in central China might have evolved from earlier strains in the local region. To determine the reason for recent vaccination failures, we also studied variations in antigenicity of field strains by analyzing the three neutralizing epitope regions in the S gene. The results showed that the neutralizing epitopes at aa 245-252 were highly conserved, but most of the amino acid changes occurred in the epitope regions aa 7-146 and 271-278. We speculate that the amino acid mutations in the neutralizing epitope regions may be associated with changes in the antigenicity of PEDV and consequently result in vaccination failure. Together, these findings may be useful for understanding the epidemiology of PEDV and may be relevant for designing of new and more efficacious vaccines.

Targeted eradication of EBV-positive cancer cells by CRISPR/dCas9-mediated EBV reactivation in combination with ganciclovir.

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment. IMPORTANCE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.

SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.

YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 small ubiquitin-like modifier (SUMO) ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 synergizes with wild-type YTHDF2, but not a SUMOylation-deficient mutant, to limit EBV lytic replication. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3, to restrict EBV replication. These results collectively uncover a unique mechanism whereby YTHDF family proteins control EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.IMPORTANCEm6A RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-EBV activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely, YTHDF1 and YTHDF3, to limit EBV replication. These findings together illuminate an important regulatory mechanism of YTHDF proteins in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.