Molecular engineering of a theranostic molecule that detects Aß plaques, inhibits Iowa and Dutch mutation Aß self-aggregation and promotes lysosomal biogenesis for Alzheimer's disease.

Extracellular clustering of amyloid-ß (Aß) and an impaired autophagy lysosomal pathway (ALP) are the hallmark features in the early stages of incurable Alzheimer's disease (AD). There is a pressing need to find or develop new small molecules for diagnostics and therapeutics for the early stages of AD. Herein, we report a small molecule, namely F-SLCOOH, which can bind and detect Aß1-42, Iowa mutation Aß, Dutch mutation Aß fibrils and oligomers exhibiting enhanced emission with high affinity. Importantly, F-SLCOOH can readily pass through the blood-brain barrier and shows highly selective binding toward the extracellular Aß aggregates in real-time in live animal imaging of a 5XFAD mice model. In addition, a high concentration of F-SLCOOH in both brain and plasma of wildtype mice after intraperitoneal administration was found. The ex vivo confocal imaging of hippocampal brain slices indicated excellent colocalization of F-SLCOOH with Aß positive NU1, 4G8, 6E10 A11 antibodies and THS staining dye, affirming its excellent Aß specificity and targetability. The molecular docking studies have provided insight into the unique and specific binding of F-SLCOOH with various Aß species. Importantly, F-SLCOOH exhibits remarkable anti-fibrillation properties against toxic Aß aggregate formation of Aß1-42, Iowa mutation Aß, and Dutch mutation Aß. F-SLCOOH treatment also exerts high neuroprotective functions and promotes autophagy lysosomal biogenesis in neuronal AD cell models. In summary, the present results suggest that F-SLCOOH is a highly promising theranostic agent for diagnosis and therapeutics of AD.

Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties.

Bovine parainfluenza virus type 3 infections induce ER stress-mediated autophagy to facilitate virus replication.

Bovine Parainfluenza Virus Type 3 (BPIV3) serves as a crucial pathogen in cattle, adept at triggering severe respiratory symptoms. This investigation explores the intricate interplay of endoplasmic reticulum stress (ER stress), unfolded protein response (UPR), and autophagy upon BPIV3 infection. In this study, we initially confirm a substantial increase in glucose regulatory protein 78 (GRP78) expression, accompanied by noticeable morphological changes and significant expansion of the ER lumen observed through transmission electron microscopy upon BPIV3 infection. Our findings indicate that ER Stress is induced during BPIV3 infection in vitro. Subsequently, we illustrate that BPIV3 triggers ER Stress to facilitate viral replication through heightened autophagy through treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) and utilizing small interfering RNA (siRNA) technology to knock down GRP78. Additionally, we observe that the activation of ER stress initiates the UPR via PERK and ATF6 pathways, with the IRE1 pathway not contributing to the regulation of ER stress-mediated autophagy. Moreover, intervention with the PERK inhibitor GSK2606414, ATF6 inhibitor Ceapin-A7, and siRNA technology successfully reverses BPIV3-induced autophagy. In summary, these findings propose that BPIV3 induces ER stress to enhance viral replication through increased autophagy, with the PERK and ATF6 pathways playing a significant role in ER stress-mediated autophagy.

Altered CD226/TIGIT expressions were associated with NK phenotypes in primary antiphospholipid syndrome and affected by IL-4/JAK pathway.

Natural killer (NK) cells were reported to be involved in the pathogenesis of primary antiphospholipid syndrome (pAPS). Immunosuppressive receptor T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and activating receptor cluster of differentiation 226 (CD226) are specifically expressed on NK cells with competitive functions. This study aims to investigate the expression diversities of CD226/TIGIT on NK subsets and their associations with NK subsets activation phenotypes and potential clinical significance, furthermore, to explore potential cause for CD226/TIGIT expression diversities in pAPS. We comparatively assessed the changes of CD56brightNK, CD56dimNK, and NK-like cells in 70 pAPS patients compared with control groups, including systemic lupus erythematosus, asymptomatic antiphospholipid antibodies carriers (asymp-aPLs carriers), and healthy controls and their expression diversities of CD226/TIGIT by flow cytometry. CD25, CD69, CD107α expression, and interferon gamma (IFN-γ) secretion levels of NK subsets were detected to determine the potential association of CD226/TIGIT expression with NK subsets phenotypes. CD226/TIGIT expression levels were compared among different subgroups divided by aPLs status. Moreover, in vitro cultures were conducted to explore the potential mechanisms of CD226/TIGIT expression imbalance. CD56brightNK and CD3+CD56+NK-like cells were significantly increased while CD56dimNK cells were obviously decreased in pAPS, and CD56brightNK and NK-like cells exhibited significantly higher CD226 but lower TIGIT expressions. CD226+CD56brightNK and TIGIT-CD56brightNK cells show higher CD69 expression and IFN-γ secretion capacity, and CD226+NK-like and TIGIT-NK-like cells showed higher expressions of CD25 and CD69 but lower apoptosis rate than CD226- and TIGIT+CD56brightNK/NK-like cells, respectively. The imbalanced CD226/TIGIT expressions were most significant in aPLs triple-positive group. Imbalanced expressions of CD226/TIGIT on CD56brightNK and NK-like cells were aggravated after interleukin-4 (IL-4) stimulation and recovered after tofacitinib blocking. Our data revealed significant imbalanced CD226/TIGIT expressions on NK subsets in pAPS, which closely associated with NK subsets phenotypes and more complicated autoantibody status. CD226/TIGIT imbalanced may be affected by IL-4/Janus Kinase (JAK) pathway activation.

Corynoxine promotes TFEB/TFE3-mediated autophagy and alleviates Aß pathology in Alzheimer's disease models.

Autophagy impairment is a key factor in Alzheimer's disease (AD) pathogenesis. TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) are nuclear transcription factors that regulate autophagy and lysosomal biogenesis. We previously showed that corynoxine (Cory), a Chinese medicine compound, protects neurons from Parkinson's disease (PD) by activating autophagy. In this study, we investigated the effect of Cory on AD models in vivo and in vitro. We found that Cory improved learning and memory function, increased neuronal autophagy and lysosomal biogenesis, and reduced pathogenic APP-CTFs levels in 5xFAD mice model. Cory activated TFEB/TFE3 by inhibiting AKT/mTOR signaling and stimulating lysosomal calcium release via transient receptor potential mucolipin 1 (TRPML1). Moreover, we demonstrated that TFEB/TFE3 knockdown abolished Cory-induced APP-CTFs degradation in N2aSwedAPP cells. Our findings suggest that Cory promotes TFEB/TFE3-mediated autophagy and alleviates Aß pathology in AD models.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

Fe65-engineered neuronal exosomes encapsulating corynoxine-B ameliorate cognition and pathology of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the predominant impairment of neurons in the hippocampus and the formation of amyloid plaques, hyperphosphorylated tau protein, and neurofibrillary tangles in the brain. The overexpression of amyloid-ß precursor protein (APP) in an AD brain results in the binding of APP intracellular domain (AICD) to Fe65 protein via the C-terminal Fe65-PTB2 interaction, which then triggers the secretion of amyloid-ß and the consequent pathogenesis of AD. Apparently, targeting the interaction between APP and Fe65 can offer a promising therapeutic approach for AD. Recently, exosome, a type of extracellular vesicle with diameter around 30-200 nm, has gained much attention as a potential delivery tool for brain diseases, including AD, due to their ability to cross the blood-brain barrier, their efficient uptake by autologous cells, and their ability to be surface-modified with target-specific receptor ligands. Here, the engineering of hippocampus neuron cell-derived exosomes to overexpress Fe65, enabled the development of a novel exosome-based targeted drug delivery system, which carried Corynoxine-B (Cory-B, an autophagy inducer) to the APP overexpressed-neuron cells in the brain of AD mice. The Fe65-engineered HT22 hippocampus neuron cell-derived exosomes (Fe65-EXO) loaded with Cory-B (Fe65-EXO-Cory-B) hijacked the signaling and blocked the natural interaction between Fe65 and APP, enabling APP-targeted delivery of Cory-B. Notably, Fe65-EXO-Cory-B induced autophagy in APP-expressing neuronal cells, leading to amelioration of the cognitive decline and pathogenesis in AD mice, demonstrating the potential of Fe65-EXO-Cory-B as an effective therapeutic intervention for AD.

Antigenic variation in hemagglutinin-neuraminidase of Newcastle disease virus isolated from Tibet, China.

Vaccines are widely used to prevent Newcastle disease virus (NDV). Under the pressure of immunization, NDVs with mutations among epitopes of F and HN protein were isolated, which indicates that the efficiency of vaccine may decrease in terms of preventing emerged NDV. However, the lack of evidences to support whether these mutations contribute to antigenic mutation and immune escape in NDV leading to the controversy that the matched vaccine is more effective than the mismatched vaccine. In this study, a genotype VII velogenic NDV strain (C22) was isolated from a vaccinated farm in Tibet, China. We found that this strain was close to NDV from east China, but it had a specific mutation (K138R) in one epitope (131DYIGGIGKE139) of HN protein. This mutation might change the interaction between amino acids in stalk-head link region of HN protein and then induce the specific antibody to worse recognize the C22 strain, but it did not alter viral virulence and growth ability. Then, the C22 strain was attenuated via modification of the F protein cleavage site to generate a matched vaccine. Comparing to a mismatched vaccine (LaSota), this matched vaccine showed advantages in inhibiting viral shedding and tissue damage. However, both vaccines induced chicken to generate similar level of neutralizing antibodies against C22, C22mut (R138K) and LaSota. These results suggest that the epitope mutation is insufficient to help NDV escaping neutralizing antibodies of vaccinated chicken, supporting that the merits of NDV matched vaccine are not totally related to humoral immunity.

PPARɑ Ligand Caudatin Improves Cognitive Functions and Mitigates Alzheimer's Disease Defects By Inducing Autophagy in Mice Models.

The autophagy-lysosomal pathway (ALP) is a major cellular machinery involved in the clearance of aggregated proteins in Alzheimer disease (AD). However, ALP is dramatically impaired during AD pathogenesis via accumulation of toxic amyloid beta (Aß) and phosphorylated-Tau (phospho-Tau) proteins in the brain. Therefore, activation of ALP may prevent the increased production of Aß and phospho-Tau in AD. Peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor that can activate autophagy, and transcriptionally regulate transcription factor EB (TFEB) which is a key regulator of ALP. This suggests that targeting PPARα, to reduce ALP impairment, could be a viable strategy for AD therapy. In this study, we investigated the anti-AD activity of Caudatin, an active constituent of Cynanchum otophyllum (a traditional Chinese medicinal herb, Qing Yang Shen; QYS). We found that Caudatin can bind to PPARα as a ligand and augment the expression of ALP in microglial cells and in the brain of 3XTg-AD mice model. Moreover, Caudatin could activate PPARα and transcriptionally regulates TFEB-augmented lysosomal degradation of Aß and phosphor-Tau aggregates in AD cell models. Oral administration of Caudatin decreased AD pathogenesis and ameliorated the cognitive dysfunction in 3XTg-AD mouse model. Conclusively, Caudatin can be a potential AD therapeutic agent via activation of PPARα-dependent ALP.

Impact and Advances in the Role of Bacterial Extracellular Vesicles in Neurodegenerative Disease and Its Therapeutics.

Bacterial Extracellular Vesicles (BEVs) possess the capability of intracellular interactions with other cells, and, hence, can be utilized as an efficient cargo for worldwide delivery of therapeutic substances such as monoclonal antibodies, proteins, plasmids, siRNA, and small molecules for the treatment of neurodegenerative diseases (NDs). BEVs additionally possess a remarkable capacity for delivering these therapeutics across the blood-brain barrier to treat Alzheimer's disease (AD). This review summarizes the role and advancement of BEVs for NDs, AD, and their treatment. Additionally, it investigates the critical BEV networks in the microbiome-gut-brain axis, their defensive and offensive roles in NDs, and their interaction with NDs. Furthermore, the part of BEVs in the neuroimmune system and their interference with ND, as well as the risk factors made by BEVs in the autophagy-lysosomal pathway and their potential outcomes on ND, are all discussed. To conclude, this review aims to gain a better understanding of the credentials of BEVs in NDs and possibly discover new therapeutic strategies.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer.

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-ß, IFN-γ, interleukin-1ß (IL-1ß), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

Chicken ISG12(2) attenuates Newcastle disease virus and enhances the efficiency of Newcastle disease vaccine via activating immune pathways.

Low virulence and strong immunogenicity are quite important for Newcastle disease virus (NDV) producing Newcastle disease (ND) living-attenuated vaccine. However, immunogenicity of NDV positively correlates to its virulence. Usually, the velogenic NDV induces stronger immune responses of poultry than the lentogenic strain, but virulent NDV poses a risk for chicken. In this study, we identified the chicken interferon (IFN)-stimulated gene 12-2 (ISG12(2)) not only attenuated NDV, but also increased immunogenicity of ND vaccine strain. First, we found that NDV infection or IFNs stimulation induced expression of chicken ISG12(2) that reinforced expression of IFNs. Over-expression or knock-down proved that chicken ISG12(2) inhibited NDV replication. Then, recombinant NDV LaSota strains (rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2)), expressing ISG12(2), were rescued. Pathogenicity tests showed that ISG12(2) expression attenuated NDV. RNA-seq or RT-qPCR demonstrated that, comparing to rLaSota/Fmut and rLaSota, rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2) induced hosts to produce cytokines enriching in innate and adaptive immune pathways in vitro and in vivo. Finally, we showed that rLaSota/ISG12(2) vaccination improved immune condition of chicken to quickly respond NDV infection and then enhance protection. These results suggest that chicken ISG12(2) is a potential novel molecular adjuvant to regulate immune responses, which decrease virulence and increase immunogenicity of NDV. The chicken ISG12(2) may contribute to the development of high efficient poultry vaccine.

Musashi1 inhibit the release of Newcastle disease viruses through preventing apoptosis of DF-1 cells.

The efficient proliferation of Newcastle disease virus (NDV) depends on its inhibition of host cell innate immunity. V protein acts as a nonstructural protein which plays a significant role in virus replication, whereas its function remains to be further explored. In this study, Musashi RNA binding protein 1 (MSI1) was selected and its interaction with V protein was further verified by Co-immunoprecipitation (Co-IP) and Immuno-colocalization test. Through the transfection of pCMV-HA-MSI1 in DF-1 cells, the overexpression of MSI1 reduced virus particles in the cell supernatant but not reduced mRNA and virus protein in cells pellet, which suggests that MSI1may act as a new antiviral molecule by inhibiting viral release. Cell early apoptosis was detected by flow cytometry (FCM), the result shows that overexpression of MSI1 inhibit cell apoptosis, implying MSI1 Inhibit virus release may through this way. Taken together, MSI1 and NDV V protein has a detectable interaction, and may block apoptosis to inhibit the release of NDV. However, this is the first report about the interaction between MSI1 and V protein of NDV that can inhibit the NDV replicated.

Electroacupuncture ameliorates beta-amyloid pathology and cognitive impairment in Alzheimer disease via a novel mechanism involving activation of TFEB (transcription factor EB).

Alzheimer disease (AD) is the most prevalent neurodegenerative disorder leading to dementia in the elderly. Unfortunately, no cure for AD is available to date. Increasing evidence has proved the roles of misfolded protein aggregation due to impairment of the macroautophagy/autophagy-lysosomal pathway (ALP) in the pathogenesis of AD, and thus making TFEB (transcription factor EB), which orchestrates ALP, as a promising target for treating AD. As a complementary therapy, acupuncture or electroacupuncture (EA) has been commonly used for treating human diseases. Although the beneficial effects of acupuncture for AD have been primarily studied both pre-clinically and clinically, the real efficacy of acupuncture on AD remains inconclusive and the underlying mechanisms are largely unexplored. In this study, we demonstrated the cognitive-enhancing effect of three-needle EA (TNEA) in an animal model of AD with beta-amyloid (Aß) pathology (5xFAD). TNEA reduced APP (amyloid beta (A4) precursor protein), C-terminal fragments (CTFs) of APP and Aß load, and inhibited glial cell activation in the prefrontal cortex and hippocampus of 5xFAD. Mechanistically, TNEA activated TFEB via inhibiting the AKT-MAPK1-MTORC1 pathway, thus promoting ALP in the brains. Therefore, TNEA represents a promising acupuncture therapy for AD, via a novel mechanism involving TFEB activation.Abbreviations Aß: ß-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; AKT1: thymoma viral proto-oncogene 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta (A4) precursor protein; BACE1: beta-site APP cleaving enzyme 1; CQ: chloroquine; CTFs: C-terminal fragments; CTSD: cathepsin D; EA: electroacupuncture; FC: fear conditioning; GFAP: glial fibrillary acidic protein; HI: hippocampus; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPT: microtubule-associated protein tau; MTORC1: mechanistic target of rapamycin kinase complex 1; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TNEA: three-needle electroacupuncture.

Newcastle disease virus selectively infects dividing cells and promotes viral proliferation.

Newcastle disease virus (NDV) can select cells to infect, but the mechanism of its cell selectivity has not been comprehensively investigated. Here, we use HeLa cells to establish that NDV can selectively infect cells at the single-cell level. We labeled proliferating cells with 5'-bromo-2-deoxyuridine (BrdU) and examined the colocalization of BrdU with NDV in cells to clarify the relationships between NDV infection and cell proliferation. Receptors at the plasma membrane mediate NDV entry into host cells. We labeled sialic acid receptor isoforms, compared their densities between different cell types and measured the sialic acid receptor densities in different cell phases. Our results suggest that NDV displays host tropism to HeLa cells compared to BHK cells and that the differences in the receptor isoform expression patterns between cell types contribute to the selection of HeLa by NDV. At the single-cell level, the dynamics of receptor expression changes during different cell phases contributing to the selection of cells in S/G2 phase for NDV infection. Furthermore, cell proliferation benefits viral replication, and enhanced virus replication leads to increased damage to cells. The elucidation of the mechanisms underlying host cell selection by NDV may help in the screening and characterizing of additional candidate oncolytic virus strains.

Newcastle disease virus V protein inhibits apoptosis in DF-1 cells by downregulating TXNL1.

Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.

Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP.

Newcastle disease virus (NDV) has been classified by the World Organization for Animal Health (OIE) as a notable disease-causing virus, and this virus has the ability to infect a wide range of birds. V protein is a non-structural protein of NDV. V protein has been reported to inhibit cell apoptosis (Park et al., 2003a) and promote viral replication (Huang et al., 2003), however, the mechanisms of action of V protein have not been elucidated. In the present study, a yeast two-hybrid screen was performed, and V protein was found to interact with the CacyBP/SIP protein. The results of co-immunoprecipitation and immuno-colocalization assays confirmed the interaction between V protein and CacyBP/SIP. The results of quantitative-PCR and viral plaque assays showed that overexpression of CacyBP/SIP inhibited viral replication in DF-1 cells. Overexpression of CacyBP/SIP in DF-1 cells induced caspase3-dependent apoptosis. The effect of knocking down CacyBP/SIP by siRNA was the opposite of that observed upon overexpression. Moreover, it is known that NDV induces cell apoptosis via multiple caspase-dependent pathways. Furthermore, V protein inhibited cell apoptosis and downregulated CacyBP/SIP expression in DF-1 cells. Taken together, the findings of the current study indicate that V protein interacts with CacyBP/SIP, thereby regulating cell apoptosis and viral replication.

Newcastle Disease Virus V Protein Promotes Viral Replication in HeLa Cells through the Activation of MEK/ERK Signaling.

Newcastle disease virus (NDV) can infect a wide range of domestic and wild bird species. The non-structural V protein of NDV plays an important role in antagonizing innate host defenses to facilitate viral replication. However, there is a lack of knowledge related to the mechanisms through which the V protein regulates viral replication. The extracellular signal-regulated kinase (ERK) signaling pathway in the host is involved in a variety of functions and is activated by several stimuli, including viral replication. In this study, we show that both the lentogenic strain, La Sota, and the velogenic strain, F48E9, of NDV activate the mitogen-activated protein kinase (MEK)/ERK signaling pathway. The pharmacological inhibition of ERK1/2 phosphorylation using the highly selective inhibitors U0126 and SCH772984 resulted in the reduced levels of NDV RNA in cells and virus titers in the cell supernatant, which established an important role for the MEK/ERK signaling pathway in NDV replication. Moreover, the overexpression of the V protein in HeLa cells increased the phosphorylation of ERK1/2 and induced the transcriptional changes in the genes downstream of the MEK/ERK signaling pathway. Taken together, our results demonstrate that the V protein is involved in the ERK signaling pathway-mediated promotion of NDV replication and thus, can be investigated as a potential antiviral target.