NSUN2 mediates distinct pathways to regulate enterovirus 71 replication.

Increasing evidences suggest that the methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on viral RNAs, which are essential for the replication of various viruses. Despite the function of m5C deposition is well characterized, other potential roles of NSUN2 in regulating viral replication remain largely unknown. In this study, the m5C modified residues catalyzed by NSUN2 on enterovirus 71 (EV71) RNAs were mapped. NSUN2, along with m5C modifications, played multiple roles during the EV71 life cycle. Functional m5C modified nucleotides increased the translational efficiency and stability of EV71 RNAs. Additionally, NSUN2 was found to target the viral protein VP1 for binding and promote its stability by inhibiting the ubiquitination. Furthermore, both viral replication and pathogenicity in mice were largely attenuated when functional m5C residues were mutated. Taken together, this study characterizes distinct pathways mediated by NSUN2 in regulating EV71 replication, and highlights the importance of its catalyzed m5C modifications on EV71 RNAs for the viral replication and pathogenicity.

Study of the antivirus function mediated by STING in Micropterus salmoides.

Stimulator of interferon genes (STING) has been demonstrated as a critical mediator in the innate immune response to cytosolic DNA and RNA derived from different pathogens. While the role of Micropterus salmoides STING (MsSTING) in largemouth bass virus is still unknown. In this study, RT-qPCR assay and Western-blot assay showed that the expression levels of MsSTING and its downstream genes were up-regulated after LMBV infection. Pull down experiment proved that a small peptide called Fusion peptide (FP) that previously reported to target to marine and human STING as a selective inhibitor also interacted with MsSTING in vitro. Comparing with the RNA-seq of Largemouth bass infected with LMBV singly, 326 genes were significantly up-regulated and 379 genes were significantly down-regulated in the FP plus LMBV group in which Largemouth bass was treatment with FP before LMBV-challenged. KEGG analysis indicated that the differentially expressed genes (DEGs) were mainly related to signaling transduction, infectious disease viral, immune system and endocrine system. Besides, the survival rate of LMBV-infected largemouth bass was highly decreased following FP treatment. Taken together, our study showed that MsSTING played an important role in immune response against LMBV infection.

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

Epigenetic addition of m5C to HBV transcripts promotes viral replication and evasion of innate antiviral responses.

Eukaryotic five-methylcytosine (m5C) is an important regulator of viral RNA splicing, stability, and translation. However, its role in HBV replication remains largely unknown. In this study, functional m5C sites are identified in hepatitis B virus (HBV) mRNA. The m5C modification at nt 1291 is not only indispensable for Aly/REF export factor (ALYREF) recognition to promote viral mRNA export and HBx translation but also for the inhibition of RIG-I binding to suppress interferon-ß (IFN-ß) production. Moreover, NOP2/Sun RNA methyltransferase 2 (NSUN2) catalyzes the addition of m5C to HBV mRNA and is transcriptionally downregulated by the viral protein HBx, which suppresses the binding of EGR1 to the NSUN2 promoter. Additionally, NSUN2 expression correlates with m5C modification of type I IFN mRNA in host cells, thus, positively regulating IFN expression. Hence, the delicate regulation of NSUN2 expression induces m5C modification of HBV mRNA while decreasing the levels of m5C in host IFN mRNA, making it a vital component of the HBV life cycle. These findings provide new molecular insights into the mechanism of HBV-mediated IFN inhibition and may inform the development of new IFN-α based therapies.

N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71.

Chemical modifications are important for RNA function and metabolism. N4-acetylcytidine (ac4C) is critical for the translation and stability of mRNA. Although ac4C is found in RNA viruses, the detailed mechanisms through which ac4C affects viral replication are unclear. Here, we reported that the 5' untranslated region of the enterovirus 71 (EV71) genome was ac4C modified by the host acetyltransferase NAT10. Inhibition of NAT10 and mutation of the ac4C sites within the internal ribosomal entry site (IRES) suppressed EV71 replication. ac4C enhanced viral RNA translation via selective recruitment of PCBP2 to the IRES and boosted RNA stability. Additionally, ac4C increased the binding of RNA-dependent RNA polymerase (3D) to viral RNA. Notably, ac4C-deficient mutant EV71 showed reduced pathogenicity in vivo. Our findings highlighted the essential role of ac4C in EV71 infection and provided insights into potential antiviral treatments.

MIR22HG Aggravates Oxygen-Glucose Deprivation and Reoxygenation-Induced Cardiomyocyte Injury through the miR-9-3p/SH2B3 Axis.

Reperfusion therapy, the standard treatment for acute myocardial infarction (MI), can trigger necrotic death of cardiomyocytes and provoke ischemia/reperfusion (I/R) injury. However, molecular mechanisms that regulate cardiomyocyte death remain largely unknown. The abnormal expression of lncRNA MIR22HG has been found in types of diseases. The current study was aimed at exploring the function and mechanism of MIR22HG in I/R injury. In this study, mouse myocardial cells (HL-1) treated with oxygen-glucose deprivation and reoxygenation (OGD/R) were used as the in vitro models, and myocardial ischemia reperfusion injury (MIRI) animal models in vivo were established in male C57BL/6 mice. Experiments including CCK-8, flow cytometry, TUNEL, HE staining, RT-qPCR, western blotting, and luciferase reporter assays were performed to explore the function and potential mechanism of MIR22HG in MIRI in vitro and in vivo. Bioinformatics analysis was performed to predict the binding site between miR-9-3p and MIR22HG (or SH2B3). Our results indicated that the MIR22HG level was upregulated in cardiomyocytes after OGD/R treatment. The knockdown of MIR22HG promoted cell viability and inhibited apoptosis and extracellular matrix (ECM) production in OGD/R-treated HL-1 cells. In mechanism, MIR22HG binds to miR-9-3p, and miR-9-3p targets the SH2B3 3' untranslated region (UTR). Moreover, SH2B3 expression was positively regulated by MIR22HG but negatively modulated by miR-9-3p. Rescue assays suggested that the suppressive effect of MIR22HG knockdown on cell viability, apoptosis, and ECM accumulation was reversed by the overexpression of SH2B3. The in vivo experiments demonstrated that MIR22HG knockdown alleviated cardiomyocyte apoptosis and reduced myocardial infarct size in MIRI mice. In summary, MIR22HG knockdown alleviates myocardial injury through the miR-9-3p/SH2B3 axis.

Raised SPINK1 levels play a role in angiogenesis and the transendothelial migration of ALL cells.

The present study was designed to assess whether raised Serine protease inhibitor Kazal type 1 (SPINK1) expressions modulates angiogenesis. Human umbilical vein endothelial cells (HUVECs) exposed to SPINK1 were noted to exhibit raised expressions of interleukin-8 (IL-8) as well as VCAM-1 and ICAM-1 cell adhesion molecules in a dose-dependent manner. In co-culture system of HUVECs and Acute lymphoblastic leukemia (ALL) cells, SPINK1 exposure also resulted in enhanced endothelial cell motility and ALL cells trans-endothelial migration. High concentrations of SPINK1 caused in vitro cellular reorganization into tubes in Matrigel-cultured HUVECs and induced in vivo vascularization and brain infiltration of NOD/SCID ALL model mice. The further transcriptomic analysis indicated that SPINK1 treatment altered several biological processes of endothelial cells and led to activation of the MAPK pathway. This study is the first to determine the neovascularization effects of raised SPINK1.

Yes-Associated Protein Promotes the Development of Condyloma Acuminatum through EGFR Pathway Activation.

OBJECTIVE: Investigate the role of Yes-associated protein (YAP1) in the development of condyloma acuminatum (CA). METHODS: We enrolled 30 male patients with CA and 20 healthy individuals as a control group, to compare the YAP1 expression in their tissue samples. Following this, we overexpressed and downregulated YAP1 expression in HaCaT cells to examine the migratory, proliferative, and apoptotic potential of HaCaT cells expressing different levels of YAP1. RESULTS: In the CA patient tissue samples, an increase in YAP1 expression can be observed. In vitro,the overexpression of YAP1 was shown to promote the growth and migration of HaCaT cells and to activate epidermal growth factor receptor (EGFR) pathway-associated proteins, while the downregulation of YAP1 inhibited cell growth and migration of these cells. CONCLUSIONS: YAP1 promotes the growth of keratinocytes in CA through the activation of the EGFR pathway, and it may mediate the development of human papilloma virus-associated diseases.

LncRNA SNHG5 promotes growth and invasion in melanoma by regulating the miR-26a-5p/TRPC3 pathway.

INTRODUCTION: Melanoma has been reported as the most common malignancy in skin cancer. The small nucleolar RNA host gene 5 (SNHG5), an lncRNA, has been proven as a vital regulator in several types of carcinoma. This study was designed to investigate the detailed roles and possible mechanisms of SNHG5 in melanoma progression. METHODS: Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of SNHG5, miR-26a-5p and transient receptor potential, canonical 3 (TRPC3) mRNA in melanoma tissues and cells. CCK-8 assay was used to measure the cell viability. Flow cytometry assays were performed to determine the cell cycle distribution and apoptosis. The invasive ability was assessed by a 24-well Transwell insert. Western blot analysis was employed to evaluate the protein expression of TRPC3. Dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay were applied to identify the interactions among SNHG5, miR-26a-5p and TRPC3. RESULTS: The results showed that SNHG5 expression was increased in melanoma tumor tissues and cell lines. Higher SNHG5 expression was correlated with advanced pathogenic status. Moreover, SNHG5 could serve as a molecular sponge of miR-26a-5p. SNHG5 downregulation repressed proliferation, promoted apoptosis, and decreased invasion in melanoma cells, while these effects were greatly counteracted by miR-26a-5p inhibitor. Furthermore, miR-26a-5p directly targeted TRPC3 to suppress its expression, and this effect was aggravated following SNHG5 downregulation. Also, TRPC3 depletion exerted similar tumor-suppressive functions as SNHG5 knockdown. CONCLUSION: SNHG5 promoted melanoma development by inhibiting miR-26a-5p and facilitating TRPC3 expression, highlighting the potential of SNHG5 as a novel target therapy for melanoma.

Treatment of latent or subclinical Genital HPV Infection with 5-aminolevulinic acid-based photodynamic therapy.

BACKGROUND: Genital HPV infections are widely prevalent. HPV can persist and be transmitted to partners even after warts are gone. The HPV genotype and viral load assay can reveal whether infections are persistent or latent, and it can serve as a predictor of infection clearance. Although numerous studies have demonstrated that photodynamic therapy (PDT) is effective against condyloma acuminata, there is no data on its effects on latent, persistent infections. METHODS: A total of 20 patients with latent or subclinical HPV infection were evaluated. At each patient visit, polymerase chain reaction was used to identify HPV genotypes and measure the viral loads, which reflect the status of HPV infection. We administered 5-aminolevulinic acid (ALA)-based PDT weekly for patients with active infections, while no treatment was administered to patients with regressing infections. RESULTS: PDT treatment can effectively eliminate HPV, significantly reducing viral loads after three rounds of treatment (p < 0.001). The rate of negative HPV DNA test results was higher in patients with latent or subclinical infections than in patients with genital warts after one round of PDT treatment. CONCLUSIONS: ALA-PDT can effectively eliminate latent or subclinical HPV infections. Additionally, our assay for identifying HPV genotypes and viral loads, which reflect the status of HPV infection, can accurately guide ALA-PDT treatment.

Development of podophyllotoxin­loaded nanostructured lipid carriers for the treatment of condyloma acuminatum.

Condyloma acuminatum (CA) is a common sexually transmitted disease caused by human papillomavirus (HPV). Podophyllotoxin (POD), a cytotoxic compound, is able to effectively treat HPV; however, the severe irritation side effects of POD restrict its use as a treatment for CA. The aim of the present study was to construct novel POD­loaded nanostructured nanolipid carriers (POD­NLCs) and evaluate their physicochemical characteristics and cytotoxicity. POD­NLCs (0.5%) were prepared using emulsion­evaporation and low temperature­solidification methods with optimized conditions and preparations. Subsequently, the POD­NLCs were physicochemically characterized and their in vitro and in vivo release efficiencies and in vitro cytotoxicity were studied. The prepared POD­NLCs had an average particle size, ζ potential, polydispersity index and encapsulation efficacy of 178.5±20 nm, ­27±0.5 mV, 0.18±0.01 and 82.9±2%, respectively. In vitro and in vivo release studies demonstrated that POD­NLCs are able to provide sustained drug delivery for 72 h in vitro and 10 h in the mucosa. Compared with a tincture formulation of POD (POD­T), POD­NLC induced less inflammatory cytokine production in the cervical mucous and led to a decreased histopathological score. In addition, a cytotoxicity assay demonstrated that inhibition of the POD­NLCs was 98.4% at 24 h and remained >98% up to 72 h. Furthermore, more cells were arrested in the G2/M phase of the cell cycle following POD­NLC treatment compared with the POD­T treatment. The present study provides evidence that POD­NLC is a promising delivery system for the treatment of CA.

[Acute and chronic toxicity of 0.5% podophyllotoxin-loaded nanostructured lipid carriers to vaginal mucosa in rabbits and rats].

OBJECTIVE: To test the acute and chronic toxicity of topical application of 0.5% podophyllotoxin-loaded nanostructured lipid carriers (POD-NLC) to the vaginal mucosa. METHODS: Twelve New Zealand rabbits were randomized into 3 groups and subjected to daily topical applications of normal saline (control group), 0.5% podophyllotoxin tincture (POD-T) or 0.5% POD-NLC on the vaginal mucosa for 10 consecutive days, and the pathological changes in the mucosa were graded using the Eckstein scoring system.The acute toxicity of POD-NLC was tested in 20 SD female rats, which received intravaginal administration of POD-NLC or vehicle for 3 times within 24 h; After 14 days of continuous observation, the rats were dissected for calculating the viscera coefficient.For testing the chronic toxicity of POD-NLC, 80 SD female rats were randomized into 4 groups and subjected to daily intravaginal administration of the vehicle or POD-NLC at low, moderate or high doses for 13 consecutive weeks.The rats were weighed once a week and at the end of the experiment, 2/3 of the rats from each group were sacrificed to collect blood samples, calculate the viscera coefficient, and examine the pathological changes in the liver.The remaining 1/3 rats were observed for another 2 weeks without further drug treatment and the same examinations were performed. RESULTS: In the rabbits, 0.5% POD-NLC elicited only mild irritation while POD-T caused moderate irritation of the vaginal mucosa.In the acute toxicity test, the organ coefficients were comparable between the rats treated with the vehicle and POD-NLC (P>0.05).Long-term intravaginal administration of POD-NLC did not produce significant changes in the behavior, activity, body weight, blood biochemical profiles or organ coefficient as compared with the vehicle control group (P>0.05). CONCLUSIONS: Intravaginal administration of 0.5% POD-NLC causes very mild irritation without obvious acute or chronic toxicity to the vaginal mucosa in rabbits and rats.

Dynamics of HPV viral loads reflect the treatment effect of photodynamic therapy in genital warts.

BACKGROUND: Photodynamic therapy (PDT) has demonstrated good clinical cure rates and low recurrence rates in the treatment of genital warts. Human papillomavirus (HPV) genotypes and viral load assays can reflect the status of persistent or latent infection and serve as a predictor of infection clearance. METHODS: Specimens from 41 patients with HPV infection were obtained, and the HPV genotypes and viral load were analyzed using real-time polymerase chain reaction (PCR) assays. Traditional treatment, such as radiofrequency, microwave, or surgical therapy, was used to remove the visible lesions, and then PDT treatment was performed every week. HPV DNA testing was performed at every patient visit and the frequency of PDT treatment was determined by changes in HPV viral loads. RESULTS: HPV viral loads decreased significantly after PDT treatment. There were significant differences in HPV viral loads between pretherapy and three or six rounds of PDT treatment. Significant differences were also observed between single and multiple type HPV infection after six rounds of PDT treatment. Patients with single type HPV infection had significantly higher rates of negative HPV DNA test results, as compared with patients with multiple infections after six rounds of PDT treatment; however, there was no difference in recurrence rates between the two groups. CONCLUSIONS: Dynamic monitoring of HPV genotypes and viral loads can be used to guide PDT treatment and indicate PDT treatment efficacy in eliminating HPV.