Hepatitis B virus RNAs co-opt ELAVL1 for stabilization and CRM1-dependent nuclear export.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.

Hepatitis B virus hijacks TSG101 to facilitate egress via multiple vesicle bodies.

Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.

HDAC6, A Novel Cargo for Autophagic Clearance of Stress Granules, Mediates the Repression of the Type I Interferon Response During Coxsackievirus A16 Infection.

Autophagic cargoes ensure selective autophagy for the recognition and removal of various cytosolic aggregated proteins, damaged organelles, or pathogens. Stress granules (SGs), as antiviral immune complexes, serve a positive role in the type I interferon (IFN) response and can be targeted by autophagy (termed granulophagy). However, the cargo of granulophagy remains elusive, and it is still unknown whether granulophagy plays a role in viral infection. Here, we found that histone deacetylase 6 (HDAC6), a component of viral RNA-induced SGs, is a novel granulophagic cargo that is recognized by p62/Sequestosome 1 (SQSTM1) and mediates the degradation of SGs in coxsackievirus A16 (CA16)-infected cells. CA16 viral RNA activated the protein kinase RNA-activated (PKR)/eukaryotic translation initiation factor 2-alpha (eIF2α) pathway to promote SG assembly. The SGs were degraded by CA16-triggered autophagy via the interaction between the ubiquitin-associated (UBA) domain of p62 and the ubiquitin-binding domain (UBD) of HDAC6, which was bridged by a poly-ubiquitin chain. We also found that granulophagy repressed the type I interferon response and facilitated viral replication. These results suggest that HDAC6 might be the first identified granulophagic cargo and granulophagy could be a strategy that viruses apply to repress the antiviral immune response.

The Late Domain of Prototype Foamy Virus Gag Facilitates Autophagic Clearance of Stress Granules by Promoting Amphisome Formation.

Prototype foamy virus (PFV), a complex retrovirus belonging to Spumaretrovirinae, maintains lifelong latent infection. The maintenance of lifelong latent infection by viruses relies on the repression of the type I interferon (IFN) response. However, the mechanism involving PFV latency, especially regarding the suppression of the IFN response, is poorly understood. Our previous study showed that PFV promotes autophagic flux. However, the underlying mechanism and the role of PFV-induced autophagy in latent infection have not been clarified. Here, we report that the PFV viral structural protein Gag induced amphisome formation and triggered autophagic clearance of stress granules (SGs) to attenuate type I IFN production. Moreover, the late domain (L-domain) of Gag played a central role in Alix recruitment, which promoted endosomal sorting complex required for transport I (ESCRT-I) formation and amphisome accumulation by facilitating late endosome formation. Our data suggest that PFV Gag represses the host IFN response through autophagic clearance of SGs by activating the endosome-autophagy pathway. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.IMPORTANCE Maintenance of lifelong latent infection for viruses relies on repression of the type I IFN response. Autophagy plays a double-edged sword in antiviral immunity. However, the role of autophagy in the regulation of the type I IFN response and the mechanism involving virus-promoted autophagy have not been fully elucidated. SGs are an immune complex associated with the antiviral immune response and are critical for type I IFN production. Autophagic clearance of SGs is one means of degradation of SGs and is associated with regulation of immunity, but the detailed mechanism remains unclear. In this article, we demonstrate that PFV Gag recruits ESCRT-I to facilitate amphisome formation. Our data also suggest that amphisome formation is a critical event for autophagic clearance of SGs and repression of the type I IFN response. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.

Analysis of potential functional significance of microRNA­3613­3p in human umbilical vein endothelial cells affected by heat stress.

Dysregulation of microRNA­3613­3p (miR­3613­3p) was previously reported in endothelial cells (ECs) during heat stress. The aim of the present study was to investigate the precise role of miR­3613­3p in heat stress. In the present study, potential gene targets of miR­3613­3p in heat­treated ECs were assessed, and the potential effects of miR­3613­3p were determined using Gene Ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to identify signaling pathways that may be affected by miR­3613­3p in heat­treated cells. Reverse transcription­quantitative PCR, western blotting and annexin V­FITC/propidium iodide staining were performed to detect miRNA expression, protein expression and apoptosis, respectively. Luciferase gene reporter assay was performed to evaluate the association between miR­3613­3p and mitogen­activated protein kinase kinase kinase 2 (MAP3K2). Bioinformatics analysis revealed 865 potential gene targets for miR­3613­3p and a series of functions and pathways in heat­treated ECs. 'Negative regulation of apoptotic process' was identified as a potential function of miR­3613­3p. In addition, functional analysis confirmed the downregulated expression levels of miR­3613­3p in ECs during heat stress, which was accompanied by an increase in apoptosis; restoration of miR­3613­3p expression inhibited apoptosis. MAP3K2 protein was demonstrated to be upregulated in heat­treated ECs, and overexpression of miR­3613­3p reduced MAP3K2 expression levels. Additionally, MAP3K2 was targeted by miR­3613­3p. These results indicated that miR­3613­3p may have complicated roles in ECs under heat stress. miR­3613­3p may serve an important role in the apoptosis of heat­treated ECs, and this effect may be partly achieved by targeting MAP3K2.

HMGB1 suppresses colon carcinoma cell apoptosis triggered by co­culture with dendritic cells via an ER stress­associated autophagy pathway.

High mobility group box protein 1 (HMGB1) is a versatile molecule that affects the immune system in various ways; however, its role in cancer immunity has not yet been completely elucidated. In the current study, bone marrow­derived dendritic cells from BALB/c mice and undifferentiated murine colon carcinoma CT26.WT cells were used as a cellular model to study the primary role of HMGB1 in colon cancer immunity. Annexin V and acridine orange/ethidium bromide staining was used to assess cellular apoptosis, Cell Counting kit 8 and lactate dehydrogenase assays were performed to evaluate cell viability and a monodansylcadaverine assay was used to detect autophagy. Western blot analysis was performed to detect the expression levels of proteins of interest. Endoplasmic reticulum (ER) stress and c­Jun N­terminal kinase phosphorylation were also investigated in CT26.WT cells exposed to dendritic cells. The present results demonstrated that the CT26.WT cells underwent apoptotic cell death following co­culturing with dendritic cells. However, pretreatment with HMGB1 resulted in a significant increase in viability of the CT26.WT cells exposed to dendritic cells. Furthermore, HMGB1 promoted ER stress­induced autophagy through the activation of JNK, which inhibited the apoptosis triggered by the dendritic cells, suggesting that HMGB1 has a role in immune evasion by colon cancer cells.

Analysis of miRNA expression profiling in human umbilical vein endothelial cells affected by heat stress.

To investigate the regulation of endothelial cell (EC) microRNAs (miRNAs) altered by heat stress, miRNA microarrays and bioinformatics methods were used to determine changes in miRNA profiles and the pathophysiological characteristics of differentially expressed miRNAs. A total of 31 differentially expressed miRNAs were identified, including 20 downregulated and 11 upregulated miRNAs. Gene Ontology (GO) enrichment analysis revealed that the validated targets of the differentially expressed miRNAs were significantly enriched in gene transcription regulation. The pathways were also significantly enriched in the Kyoto Encyclopedia of Genes and Genomes analysis, and most were cancer-related, including the mitogen-activated protein kinase signaling pathway, pathways involved in cancer, the Wnt signaling pathway, the Hippo signaling pathway, proteoglycans involved in cancer and axon guidance. The miRNA-gene and miRNA­GO network analyses revealed several hub miRNAs, genes and functions. Notably, miR­3613-3p played a dominant role in both networks. MAP3K2, MGAT4A, TGFBR1, UBE2R2 and SMAD4 were most likely to be controlled by the altered miRNAs in the miRNA-gene network. The miRNA­GO network analysis revealed significantly complicated associations between miRNAs and different functions, and that the significantly enriched functions targeted by the differentially expressed miRNAs were mostly involved in regulating gene transcription. The present study demonstrated that miRNAs are involved in the pathophysiology of heat-treated ECs. Understanding the functions of miRNAs may provide novel insights into the molecular mechanisms underlying the heat­induced pathophysiology of ECs.

JNK signaling is required for the MIP­1α­associated regulation of Kupffer cells in the heat stroke response.

Severe heat stroke (HS) consists of extreme hyperthermia with thermoregulatory failure, leading to high morbidity and mortality. Liver injury is a complication of HS that is associated with inflammatory responses and Kupffer cells (KCs), which are resident macrophages in the liver that serve as a major source of inflammatory cytokines; however, the association and the underlying mechanisms of KC functions in HS­induced endotoxemia and inflammation require an improved understanding. The important chemokine macrophage inflammatory protein­1α (MIP­1α) increases inflammatory responses and the secretion of inflammatory molecules from KCs, including tumor necrosis factor­α, interleukin (IL)­1ß and IL­6. In addition, the activation of c­Jun N­terminal kinase (JNK) signaling is responsible for the development of liver inflammation. Therefore, HS animal and cell models were constructed in order to investigate the pathways involved in the HS­induced dysfunction of KCs. The results of the present study suggest that JNK may be involved in the MIP­1α­associated pathogenesis of KCs in HS injury.

Sodium tanshinone IIA sulfonate improves inflammation, aortic endothelial cell apoptosis, disseminated intravascular coagulation and multiple organ damage in a rat heat stroke model.

The aim of the present study was to investigate the effects of sodium tanshinone IIA sulfonate (STS) on inflammatory responses, aortic endothelial cell apoptosis, disseminated intravascular coagulation (DIC) and multiple organ damage in an animal model of classic heat stroke (CHS). The rats in the heat stroke (HS) and STS­treated heat stroke (STS­HS) groups were placed into a pre­warmed animal temperature controller (ATC) at 35˚C. The moment at which the rectal temperature reached 43.5˚C was considered as the time of onset of HS. In the HS groups, the rats were removed from the ATC and allowed to recover at 26˚C for 0, 2, 6 or 12 h. In the STS­HS groups, the rats received femoral vein injections of 5­40 mg/kg STS immediately following the onset of HS and were subsequently placed at a temperature of 26˚C to recover for 6 h. In the present study, the serum levels of tumor necrosis factor (TNF)­α, interleukin (IL)­1ß and IL­6 were assessed using ELISA, and the numbers of apoptotic aortic endothelial cells were investigated using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick­end labeling combined with immunofluorescence. In the HS groups, the serum levels of TNF­α, IL­1ß and IL­6, as well as the numbers of apoptotic aortic endothelial cells were increased compared with the normothermic control group. Additionally, the plasma prothrombin time, activated partial thromboplastin time and D­dimer level were significantly increased in the HS group compared with the normothermic control group following recovery for 6 h. By contrast, the platelet count was decreased in the HS group compared with the normothermic control group. The serum levels of creatinine, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase were increased and histopathological damage to multiple organs was observed in the HS group following recovery for 6 h. In the STS­HS groups, cytokine levels and apoptotic aortic endothelial cell numbers were reduced compared with the HS group after 6 h recovery. STS (40 mg/kg) treatment additionally improved the serum levels of organ injury indicators and plasma indicators of coagulopathy, and prevented histopathological damage to multiple organs. These findings demonstrated that STS treatment may ameliorate multiple organ damage by attenuating inflammatory responses, aortic endothelial cell apoptosis and DIC in CHS. These results suggested that STS may hold potential as an alternative therapeutic strategy for the treatment of patients with HS.

Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

Coxsackievirus A16 infection triggers apoptosis in RD cells by inducing ER stress.

Coxsackievirus A16 (CA16) infection, which is responsible for hand, foot and mouth disease (HFMD), has become a common health problem in Asia due to the prevalence of the virus. Thus, it is important to understand the pathogenesis of CA16 infection. Viruses that induce endoplasmic reticulum (ER) stress are confronted with the unfolded protein response (UPR), which may lead to apoptotic cell death and influence viral replication. In this study, we found that CA16 infection could induce apoptosis and ER stress in RD cells. Interestingly, apoptosis via the activation of caspase-3, -8 and -9 in the extrinsic or intrinsic apoptotic pathways in RD cells was inhibited by 4-phenyl butyric acid (4PBA), a chemical chaperone that reduces ER stress. These results suggest that CA16 infection leads to ER stress, which in turn results in prolonged ER stress-induced apoptosis. This study provides a new basis for understanding CA16 infection and host responses.

Anti-CCL25 antibody prolongs skin allograft survival by blocking CCR9 expression and impairing splenic T-cell function.

Chemokines, by virtue of their ability to recruit immune cells into allografts, play critical roles in acute transplantation rejection. CCR9 and its ligand, CCL25, is one of the key regulators of thymocyte migration and maturation in normal and inflammatory conditions. Moreover, several studies have revealed that high expression of CCR9 and CCL25 participated in many kinds of diseases. However, the role of CCR9 in allograft rejection is still unclear. In this study, we established a murine skin transplantation model of acute rejection. Our findings showed that the proportion of CCR9-expressing T cells was significantly increased in the spleen of allotransplanted mice compared with syngeneic transplantation. Furthermore, expression of CCL25 in allograft was similarly increased. Neutralization of CCL25 by intravenous injection of anti-CCL25 monoclonal antibody significantly prolonged skin allograft survival, decreased the number of infiltrating cells, and simultaneously suppressed the chemotactic ability and the proliferation of the splenic T cells in response to allogeneic antigens. Finally, blockade of CCL25 also diminished the secretion of IFN-γ by splenic T cells. These studies indicated that CCR9/CCL25 was involved in acute transplantation rejection and anti-CCL25 strategies might be useful in preventing acute rejection.

High dose lipopolysaccharide triggers polarization of mouse thymic Th17 cells in vitro in the presence of mature dendritic cells.

Lipopolysaccharide (LPS) plays an important role in the activation of innate immune cells, leading to secretion of proinflammatory factors and bridging the adaptive immune system. Exposing total mouse thymic cells culture to LPS induced a unique expression profile of cytokines (IL-17A, IL-17F, and IL-22) and the essential ROR-γt master transcription factor, which suggested a preferential differentiation of thymocytes towards the Th17 cell phenotype. Th17-polarizing molecules (IL-23, IL-23R, IL-6, and TGF-ß) and IL-17A(+)CD4(+) thymocytes were also specifically produced by the in vitro LPS-stimulation of thymic cells. Furthermore, both the expression of Th17 differentiation-related molecules and the frequency of Th17 cells were significantly up-regulated with increasing doses of LPS, as evidenced by quantitative RT-PCR and flow cytometric analysis, respectively. The expressions and frequency reached maximum levels when LPS exposure had been maintained at an extremely high concentration (100 µg/mL) for 48 h. On the other hand, depletion of thymic dendritic cells (DCs) blocked the LPS-induced polarization of thymus-derived Th17 cell lineage. Addition of bone marrow-derived DCs (BMDCs) to the purified immature CD4(+) CD62L(low) thymocytes culture recovered the switch towards Th17 cells, which synergistically prompted the cytotoxic activity of CD8(+) T cells. Taken together, our data indicates that high doses of LPS can promote the differentiation of mouse thymus-derived Th17 cells by a mechanism involving components associated with mature DCs.

The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells.

The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their 'fresh' host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its 'fresh' host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA.

Foamy virus: an available vector for gene transfer in neural cells and other nondividing cells.

Foamy viruses (FVs) are classified to a subfamily of retrovirus presented in a wide range of hosts. None of FVs has been found to associate with any diseases in their native hosts. Such a unique biological character of FVs makes it suitable for the development of vectors for gene transfer. However, it is still controversial whether foamy virus vectors (FV vectors) can be applied to the central nervous system (CNS). In this review, we summarize the studies of FV vectors, which have been used for transduction of neural cells and other nondividing cells. We further discuss the potential mechanisms underlying the infection and propose that FVs can be used to develop transfer vectors for gene therapy in neurological disorders.