Targeting HNRNPA2B1 inhibits enterovirus 71 replication in SK-N-SH cells.

OBJECTIVE: To investigate the effect of heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) on the replication of enterovirus 71 (EV-71) in SK-N-SH cells. METHODS: The mRNA and protein expression of HNRNPA2B1 in SK-N-SH cells were detected by real-time quantitative PCR (qRT-PCR) and western blotting (WB), respectively. WB was used to detect HNRNPA2B1 protein expression in the nucleus and cytosol. The localization of HNRNPA2B1 protein in the nucleus and cytosol was detected by immunofluorescence (IF). The expression of HNRNPA2B1 was inhibited by small interfering RNA (si-HNRNPA2B1). Viral RNA, viral structural protein VP1, and viral titer were detected by qRT-PCR, WB, and viral dilution counting, respectively. RESULTS: EV-71 infection significantly upregulates the expression of HNRNPA2B1 in SK-N-SH cells. EV-71 infection promotes HNRNPA2B1 nucleus-cytoplasm redistribution. Down-regulation of HNRNPA2B1 expression significantly inhibited EV-71 replication. CONCLUSION: HNRNPA2B1 protein redistributed from nucleus to cytoplasm and is highly expressed in the cytoplasm during EV-71 infection. Inhibition of HNRNPA2B1 levels effectively inhibits EV-71 replication in SK-N-SH cells.

A Review of Labeling Approaches Used in Small Extracellular Vesicles Tracing and Imaging.

Small extracellular vesicles (sEVs), a subset of extracellular vesicles (EVs) originating from the endosomal compartment, are a kind of lipid bilayer vesicles released by almost all types of cells, serving as natural carriers of nucleic acids, proteins, and lipids for intercellular communication and transfer of bioactive molecules. The current findings suggest their vital role in physiological and pathological processes. Various sEVs labeling techniques have been developed for the more advanced study of the function, mode of action, bio-distribution, and related information of sEVs. In this review, we summarize the existing and emerging sEVs labeling techniques, including fluorescent labeling, radioisotope labeling, nanoparticle labeling, chemical contrast agents labeling, and label-free technique. These approaches will pave the way for an in-depth study of sEVs. We present a systematic and comprehensive review of the principles, advantages, disadvantages, and applications of these techniques, to help promote applications of these labeling approaches in future research on sEVs.

Roles and mechanisms of exosomal microRNAs in viral infections.

Exosomes are small extracellular vesicles with a diameter of 30-150 nm that originate from endosomes and fuse with the plasma membrane. They are secreted by almost all kinds of cells and can stably transfer different kinds of cargo from donor to recipient cells, thereby altering cellular functions for assisting cell-to-cell communication. Exosomes derived from virus-infected cells during viral infections are likely to contain different microRNAs (miRNAs) that can be transferred to recipient cells. Exosomes can either promote or suppress viral infections and therefore play a dual role in viral infection. In this review, we summarize the current knowledge about the role of exosomal miRNAs during infection by six important viruses (hepatitis C virus, enterovirus A71, Epstein-Barr virus, human immunodeficiency virus, severe acute respiratory syndrome coronavirus 2, and Zika virus), each of which causes a significant global public health problem. We describe how these exosomal miRNAs, including both donor-cell-derived and virus-encoded miRNAs, modulate the functions of the recipient cell. Lastly, we briefly discuss their potential value for the diagnosis and treatment of viral infections.

Enterovirus A71 Promotes Exosome Secretion by the Nonstructural Protein 3A Interacting with Rab27a.

Exosomes are small membrane-bound vesicles which are intraluminal vesicles (ILVs) secreted to the extracellular space after multivesicular bodies (MVBs) fuse with the plasma membrane. Although it is known that exosomes play a multitude of roles during viral infection, the mechanism that regulates their secretion during viral infection is unknown. Here, we found that enterovirus A71 (EV-A71) infection increased exosome secretion both in vivo and in vitro. Importantly, the expression of nonstructural protein 3A was sufficient to promote exosome secretion, while a mutation affecting the amino acid 18 position abrogated this effect, without changing the size of exosomes in vivo or in vitro. Transmission electron microscopy (TEM) analysis revealed that 3A decreases the number of MVBs and ILVs in vivo and in vitro, which suggested 3A may boost the fusion between MVBs and the plasma membrane. Furthermore, we demonstrated that an interaction between 3A and the small GTPase protein, Rab27a, protected Rab27a from ubiquitination, resulted in increasing exosome release. Data indicated a novel mechanism by which EV-A71 3A modifies exosome secretion during viral infection. IMPORTANCE Research has shown that viral infection impacts exosome secretion, but its regulation mechanisms remain poorly understood. Nonstructural protein 3A of EV-A71 interacts with many host factors and is involved in the remodeling of cellular membranes. In this investigation, we applied exogenous expression of 3A protein for exploring its regulation on exosome secretion and utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors. Our results demonstrate that 3A protein upregulates the release of the exosomes and that the 3A mutant strain of EV-A71 induce less exosome release compared with the EV-A71 wild type. Viral 3A protein interacts with the host factor Rab27a to prevent it from being ubiquitinated, which in turn improves exosome secretion both in vitro and in vivo. EV-A71 3A protein is a novel viral factor in the control of exosome production.

Enterovirus-71 utilizes small extracellular vesicles to cross the blood-brain barrier for infecting the central nervous system via transcytosis.

BACKGROUND: Central nervous system (CNS) infections caused by Enterovirus 71 (EV71) pose a serious threat to children, causing severe neurogenic complications and even fatality in some patients. However, the pathogenesis of EV71 infections in the CNS remains unclear. METHODS: An in vitro blood-brain barrier (BBB) model was constructed by coculturing brain microvascular endothelial cells (BMECs) and astrocytes in transwell inserts for simulating CNS infections. EV71 virions and small extracellular vesicles (sEVs) derived from EV71-infected cells (EV71-sEVs) were isolated from the cell culture supernatant by density gradient centrifugation. The BBB model was separately infected with EV71 virions and EV71-sEVs. The mechanism of crossing the BBB was determined by inhibiting the different endocytic modes. A murine model of EV71 infection was constructed for confirming the results of in vitro experiments. RESULTS: The EV71-sEVs containing viral components were endocytosed by BMECs and released on the abluminal side of the BBB model, where they infected the astrocytes without disrupting the BBB in the early stages of infection. The integrity of the tight junctions (TJs) between BMECs was breached via downregulation of PI3K/Akt signaling in the late stages of infection. CONCLUSIONS: EV71 utilized the circulating sEVs for infecting the CNS by crossing the BBB.

Dual roles and potential applications of exosomes in HCV infections.

The hepatitis C virus (HCV) causes severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma, which have high morbidity and mortality. Antibody targeting receptor-mediated HCV infections have limited therapeutic benefits, suggesting that the transmission of HCV infections is possibly mediated via receptor-independent mechanisms. Exosomes are membrane-enclosed vesicles with a diameter of 30-200 nm, which originate from the fusion of endosomal multivesicular bodies with the plasma membrane. Accumulating evidence suggests that exosomes have a pivotal role in HCV infections. Exosomes can transfer viral and cellular bioactive substances, including nucleic acids and proteins, to uninfected cells, thus spreading the infection by masking these materials from immunological recognition. In addition, exosomes originating from some cells can deliver antiviral molecules or prompt the immune response to inhibit HCV infection. Exosomes can be used for the diagnosis of HCV-related diseases, and are being presently evaluated as therapeutic tools for anti-HCV drug delivery. This review summarizes the current knowledge on the dual roles and potential clinical applications of exosomes in HCV infections.

Revisiting PD-1/PD-L pathway in T and B cell response: Beyond immunosuppression.

The regulation of T cell response depends on co-inhibitory pathways that serve to control immune-mediated tissue damage and resolve inflammation by modulating the magnitude and duration of immune response. In this process, the axis of T-cell-expressed programmed death-1 (PD-1) and its ligands (PD-L1 and PD-L2) play a key role. While the PD-1/PD-L pathway has received considerable attention for its role in the maintenance of T cell exhaustion in cancer and chronic infection, the PD-1/PD-L pathway also plays diverse roles in regulating host immunity beyond T cell exhaustion. In this review, we will discuss emerging concepts in co-stimulatory functions of PD-1/PD-L pathway on T cell- and B cell response and explore the potential underlying mechanisms. In addition, based on the elevated expression of PD-1 and its ligands in local inflamed tissues, we further discussed the role of PD-1/PD-L pathway in autoimmune diseases.

The functional roles of m6A modification in T lymphocyte responses and autoimmune diseases.

RNA N6-methyladenosine (m6A) modification is abundant in eukaryotes, bacteria and archaea. It is an RNA modification mainly existing in messenger RNA (mRNAs) and has a significant effect on the metabolism and function of mRNAs. m6A modification is controlled by three types of proteins, namely methyltransferase as the "writers", demethylase as the "erasers", and specific m6A recognized protein (YTHDF1-3) as the "readers". Recent studies have shown that m6A modification plays an important role in cancer, viral infection and autoimmune diseases. In this review, we will elaborate on the m6A modifications in the homeostasis and differentiation of T cells. Then we will further summarize the effects of m6A modification on the T cell responses and T cell-mediated autoimmune diseases. This will advance T cell epigenetics research and provide potential biomarkers and therapeutic targets for autoimmune diseases.

New method for emergency decision making with an integrated regret theory-EDAS method in 2-tuple spherical linguistic environment.

With the frequent occurrence of various emergency events, emergency decision making (EDM) has become an important research focus recently and many studies have been conducted to decrease the negative impact of emergencies. Normally, it is essential for decision makers to make satisfactory and reasonable emergency decisions in the shortest possible time as inappropriate decisions may result in enormous economic losses and serious social consequences. To ensure that an emergency response can be made efficiently, we propose a new EDM method by integrating regret theory and evaluation based on distance from average solution (EDAS) method within the 2-tuple spherical linguistic environment. First, the 2-tuple spherical linguistic term sets (TSLTSs) are employed by decision makers to express their uncertain and vague evaluation information on emergency alternatives. Then, an integrated EDM method based on regret theory and EDAS method is proposed to rank emergency alternatives and find out the optimal one. Besides, the criteria importance through inter-criteria correlation (CRITIC) method is used to determine criteria weights objectively in the EDM process. Finally, the proposed regret theory-EDAS method is applied to select the optimal response solution for a public health emergency in China. The superiority and practicality of the designed method are further justified through a comparative analysis with other EDM methods.

Decades on emergency decision-making: a bibliometric analysis and literature review.

When an emergency occurs, effective decisions should be made in a limited time to reduce the casualties and economic losses as much as possible. In the past decades, emergency decision-making (EDM) has become a research hotspot and a lot of studies have been conducted for better managing emergency events under tight time constraint. However, there is a lack of a comprehensive bibliometric analysis of the literature on this topic. The objective of this paper is to provide academic community with a complete bibliometric analysis of the EDM researches to generate a global picture of developments, focus areas, and trends in the field. A total of 303 journal publications published between 2010 and 2020 were identified and analyzed using the VOSviewer in regard to cooperation network, co-citation network, and keyword co-occurrence network. The findings indicate that the annual publications in this research field have increased rapidly since 2014. Based on the cooperation network and co-citation network analyses, the most productive and influential countries, institutions, researchers, and their cooperation networks were identified. Using the co-citation network analysis, the landmark articles and the core journals in the EDM area are found out. With the help of the keyword co-occurrence network analysis, research hotspots and development of the EDM domain are determined. According to current trends and blind spots in the literature, possible directions for further investigation are finally suggested for EDM. The literature review results provide valuable information and new insights for both scholars and practitioners to grasp the current situation, hotspots and future research agenda of the EDM field.

Mechanism of cargo sorting into small extracellular vesicles.

Extracellular vesicles (EVs) are special membranous structures released by almost every cell type that carry and protect some biomolecules from being degraded. They transport important signaling molecules involved in cell communication, migration, and numerous physiological processes. EVs can be categorized into two main types according to their size: i) small extracellular vesicles (sEVs), such as exosomes (30-150 nm), released from the fusion of multivesicular bodies (MVBs) with the plasma membrane, and ii) large EVs, such as microvesicles (100-1000 nm). These are no longer considered a waste product of cells, but regulators of intercellular communication, as they can transport specific repertoires of cargos, such as proteins, lipids, and nucleic acids to receptor cells to achieve cell-to-cell communication. This indicates the existence of different mechanisms, which controls the cargos sorting into EVs. This review mainly gives a description about the biological roles of the cargo and the sorting mechanisms of sEVs, especially exosomes.

DNA aptamer against EV-A71 VP1 protein: selection and application.

BACKGROUND: Enterovirus 71 (EV-A71) is a highly infectious pathogen associated with hand, foot and mouth disease, herpangina, and various neurological complications, so it is important for the early detection and treatment of EV-A71. An aptamer is a nucleotide sequence that screened in vitro by the technology named systematic evolution of ligands by exponential enrichment technology (SELEX). Similar to antibodies, aptamers can bind to the targets with high specificity and affinity. Besides, emerging aptamers have many advantages comparing with antibodies, such as ease of synthesis and modification, having a wide variety of target materials, low manufacturing cost and easy flexibility in amending. Therefore, aptamers are promising in virus detection and anti-virus therapy. METHODS: Aptamers were selected by SELEX. Specificity, affinity and second structure were used to characterize the selected aptamers. Chemiluminescence was adopted to build an aptamer-based detection method for EV-A71. Cytopathogenic effects trial, the level of intracellular EV-A71 RNA and protein expression were used to evaluate the antiviral effect of the selected aptamers. RESULTS: Three DNA aptamers with high specificity and affinity for EV-A71structual protein VP1 were screened out. A rapid chemiluminutesescence aptamer biosensor for EV-A71 detection was designed out. The selected aptamers could inhibit the RNA replication and protein expression of EV-A71 in RD cells and ameliorate the cytopathogenic effects. CONCLUSIONS: The aptamers against EV-A71 have the potentiality to be applied as attractive candidates used for EV-A71 detection and treatment in the future.

The Role of Viral Proteins in the Regulation of Exosomes Biogenesis.

Exosomes are membrane-bound vesicles of endocytic origin, secreted into the extracellular milieu, in which various biological components such as proteins, nucleic acids, and lipids reside. A variety of external stimuli can regulate the formation and secretion of exosomes, including viruses. Viruses have evolved clever strategies to establish effective infections by employing exosomes to cloak their viral genomes and gain entry into uninfected cells. While most recent exosomal studies have focused on clarifying the effect of these bioactive vesicles on viral infection, the mechanisms by which the virus regulates exosomes are still unclear and deserve further attention. This article is devoted to studying how viral components regulate exosomes biogenesis, composition, and secretion.

The role of host cell Rab GTPases in influenza A virus infections.

Influenza A virus (IAV) is a crucial cause of respiratory infections in humans worldwide. Therefore, studies should clarify adaptation mechanisms of IAV and critical factors of the viral pathogenesis in human hosts. GTPases of the Rab family are the largest branch of the Ras-like small GTPase superfamily, and they regulate almost every step during vesicle-mediated trafficking. Evidence has shown that Rab proteins participate in the lifecycle of IAV. In this mini-review, we outline the regulatory mechanisms of different Rab proteins in the lifecycle of IAV. Understanding the role of Rab proteins in IAV infections is important to develop broad-spectrum host-targeted antiviral strategies.

A Mouse Model for Infection with Enterovirus A71 in Small Extracellular Vesicles.

Enterovirus A71 (EV-A71) is the major pathogen of hand, foot, and mouth disease (HFMD); in some severe cases, it could develop into central nervous system (CNS) disease such as aseptic meningitis, encephalitis, and neurogenic pulmonary edema in children under 5 years. The EV-A71 pathogenesis which is involved with the CNS is unclear due to the lack of a simple and reliable mouse model thus far. Most clinical EV-A71 isolates could not effectively infect the neonatal mouse, which used to be an EV-A71 infection model. The small extracellular vesicles (sEVs) released from clinical EV-A71 isolate-infected cells were infectious in cell lines and could cause a high viral replication in mice. Neonatal ICR mice were injected intraperitoneally with these infectious sEVs and showed more weight loss and higher mortality than those mice injected with the clinical EV-A71 isolate. By using these sEVs, we provided a simple and effective method by which we can generate a stable and valuable animal model for the studies of EV-A71 pathogenesis and therapy.IMPORTANCE EV-A71 was supposed to infect the CNS through the neural pathway and the circulation of the blood in previous studies. Reverse axon transport had been confirmed as an important pathway for EV-A71 to infect the CNS; however, it is still unknown how EV-A71 infects the CNS through the circulation of the blood. Combined with the infectivity of sEVs secreted from EV-A71-infected cells and the characteristic that sEVs could cross the blood-brain barrier, we considered that sEVs may play a vital role in EV-A71 pathogenesis of the CNS.

LncRNA AK036396 Inhibits Maturation and Accelerates Immunosuppression of Polymorphonuclear Myeloid-Derived Suppressor Cells by Enhancing the Stability of Ficolin B.

Long noncoding RNAs (lncRNA) are emerging as crucial regulators of cell biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) remains unclear. Here, we identified that the lncRNA F730016J06Rik (AK036396) was highly expressed in PMN-MDSCs and that lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), the expression of which could be assessed as a surrogate for PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an ortholog of mouse Fcnb, was increased and positively correlated with arginase1 (ARG1) expression. This suppressive molecule is released by MDSCs, and its production is commonly used to represent the suppressive activity of MDSCs in patients with lung cancer, suggesting clinical relevance for these findings. These results indicate that lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability.

Roles and potential applications of lncRNAs in HIV infection.

Long non-coding RNAs (lncRNAs) are defined as a class of RNA molecules with a length of more than 200 nucleotides that are not translated into protein, and are known to participate in a variety of biological processes. They have recently been implicated as having roles in viral infections, and several research groups have identified that complex interactions exist between lncRNAs and the progression of human immunodeficiency virus (HIV) infection. lncRNAs derived from both the human host and HIV itself are emerging as key regulators of various cellular functions, playing crucial roles in virus-host interactions and viral pathogenesis. This review provides a brief discussion of the roles and associated mechanisms of lncRNAs in HIV infection. Moreover, due to the continued lack of effective HIV vaccines or treatments, we provide an insight into the complex interplay between lncRNAs and HIV and suggest innovative therapeutic strategies for HIV/acquired immunodeficiency syndrome (AIDS). The available data on lncRNAs that have been associated with HIV infection and their potential applications for the treatment of HIV are summarized for the first time, providing a new perspective for the future development of therapeutic strategies.

Exosomes cloak the virion to transmit Enterovirus 71 non-lytically.

Enterovirus 71 (EV71) is a non-enveloped virus and it can be released from host cells through a traditional cytolytic manner. Now, we showed EV71 could be spread non-lytically between cells during early viral infection. In order to explain this phenomenon, we separated supernatant fluids of rhabdomyosarcoma (RD) cells cultures infected with EV71 by isopycnic gradient centrifugation. Two populations of virus particles were morphology indistinguishable by transmission electron microscope (TEM). It showed that some EV71 particles were wrapped inside extracellular vesicles which were verified to be exosomes by immunoassay and morphologic analysis. In addition, exosomes containing viral RNA were shed in plasma of EV71-infected encephalitis in children. Our findings indicate that the "non-enveloped" EV71 virions could be wrapped within exosomes which promote their spread in the absence of cell lysis.Abbreviation: EV71: enterovirus 71; EXO: exosome; RD: rhabdomyosarcoma; TEM: transmission electron microscope; HFMD: hand, foot, and mouth disease; HIV: immunodeficiency virus; HCV: hepatitis C virus; HTLV: Human T-cell lymphotropic virus; HAV: hepatitis A virus; MOI: multiplicity of infection; EVs: extracellular vesicles; VP1: viral capsid protein 1; NTA: nanoparticle tracking analysis; CNS: central nervous system.

CD4+ T Cell-Released Extracellular Vesicles Potentiate the Efficacy of the HBsAg Vaccine by Enhancing B Cell Responses.

T cells secrete bioactive extracellular vesicles (EVs), but the potential biological effects of CD4+ T cell EVs are not clear. The main purpose of this study is to investigate the effects of CD4+ T cell-derived EVs on B cell responses and examine their role in antigen-mediated humoral immune responses. In this study, CD4+ T cell EVs are purified from activated CD4+ T cells in vitro. After immunization with the Hepatitis B surface antigen (HBsAg) vaccine, CD4+ T cell EVs-treated mice show stronger humoral immune responses, which is indicated by a greater Hepatitis B surface antibody (HBsAb) level in serum and a greater proportion of plasma cells in bone marrow. In addition, it is found that EVs released from activated CD4+ T cells play an important role in B cell responses in vitro, which significantly promote B cell activation, proliferation, and antibody production. Interestingly, antigen-specific CD4+ T cell EVs are found to be more efficient than control EVs in enhancing B cell responses. Furthermore, it is shown that CD40 ligand (CD40L) is involved in CD4+ T cell EVs-mediated B cell responses. Overall, the results have demonstrated that CD4+ T cell EVs enhance B cell responses and serve as a novel immunomodulator to promote antigen-specific humoral immune responses.