Glucocorticoids Prevent Enterovirus 71 Capsid Protein VP1 Induced Calreticulin Surface Exposure by Alleviating Neuronal ER Stress.

Severe hand-foot-and-mouth disease (HFMD) caused by Enterovirus 71 (EV71) always accompanies with inflammation and neuronal damage in the central nervous system (CNS). During neuronal injuries, cell surface-exposed calreticulin (Ecto-CRT) is an important mediator for primary phagocytosis of viable neurons by microglia. Our data confirmed that brainstem neurons underwent neuronophagia by glia in EV71-induced death cases of HFMD. EV71 capsid proteins VP1, VP2, VP3, or VP4 did not induce apoptosis of brainstem neurons. Interestingly, we found VP1-activated endoplasmic reticulum (ER) stress and autophagy could promote Ecto-CRT upregulation, but ER stress or autophagy alone was not sufficient to induce CRT exposure. Furthermore, we demonstrated that VP1-induced autophagy activation was mediated by ER stress. Meaningfully, we found dexamethasone treatment could attenuate Ecto-CRT upregulation by alleviating VP1-induced ER stress. Altogether, these findings identify VP1-promoted Ecto-CRT upregulation as a novel mechanism of EV71-induced neuronal cell damage and highlight the potential of the use of glucocorticoids to treat severe HFMD patients with CNS complications.

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo.

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction. IMPORTANCE: Acute gastroenteritis, with its sequela diarrhea, is one of the most important causes of childhood morbidity and mortality worldwide. A variety of infectious agents cause gastroenteritis, and in many cases, an enterotoxin produced by the agent is involved in disease manifestations. Although we commonly think of bacteria as a source of toxins, at least one enteric virus, rotavirus, produces a protein with enterotoxigenic activity during viral replication. In these studies, we demonstrate that oral administration of the turkey astrovirus 2 (TAstV-2) structural (capsid) protein induces acute diarrhea, increases barrier permeability, and causes relocalization of NHE3 in the small intestine, suggesting that rotavirus may not be alone in possessing enterotoxigenic activity.

[Development of a novel adenovirus vector exhibiting microRNA-mediated suppression of the leaky expression of adenovirus genes].

Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.

Modeling of molecular interaction between apoptin, BCR-Abl and CrkL--an alternative approach to conventional rational drug design.

In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptin's action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency.

Chimeric capsid protein as a nanocarrier for siRNA delivery: stability and cellular uptake of encapsulated siRNA.

For the efficient cytoplasmic delivery of siRNA, we designed a chimeric capsid protein composed of a capsid shell, integrin targeting peptide, and p19 RNA binding protein. This recombinant protein assembled into a macromolecular container-like structure with capsid shell and provided a nanocarrier for siRNA delivery. Our capsid nanocarriers had dual affinity both for siRNA within the interior and integin receptors on the exterior, and the capsid shell structure allowed the encapsulated siRNAs to be protected from the external nucleases, leading to the enhanced stability of siRNA in serum conditions. The capsid nanocarriers could complex with siRNA in a size-dependent and sequence-independent manner and showed the pH-dependent complexing/dissocation behaviors with siRNA. Moreover, RGD peptides on the exterior surface of the capsid shell enabled the capsid nanocarriers to deliver siRNA into the cytosol of the target cells. Here, we demonstrated the superior efficiency of our siRNA/capsid nanocarrier complexes in RFP gene silencing, compared to untreated cells. These results provide an alternative approach to enhancing the stability of siRNA as well as to achieving targeted siRNA delivery.

Unscheduled Akt-triggered activation of cyclin-dependent kinase 2 as a key effector mechanism of apoptin's anticancer toxicity.

Apoptin, a protein from the chicken anemia virus, has attracted attention because it specifically kills tumor cells while leaving normal cells unharmed. The reason for this tumor selectivity is unclear and depends on subcellular localization, as apoptin resides in the cytoplasm of normal cells but in the nuclei of transformed cells. It was shown that nuclear localization and tumor-specific killing crucially require apoptin's phosphorylation by an as yet unknown kinase. Here we elucidate the pathway of apoptin-induced apoptosis and show that it essentially depends on abnormal phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation, resulting in the activation of the cyclin-dependent kinase CDK2. Inhibitors as well as dominant-negative mutants of PI3-kinase and Akt not only inhibited CDK2 activation but also protected cells from apoptin-induced cell death. Akt activated CDK2 by direct phosphorylation as well as by the phosphorylation-induced degradation of the inhibitor p27(Kip1). Importantly, we also identified CDK2 as the principal kinase that phosphorylates apoptin and is crucially required for apoptin-induced cell death. Immortalized CDK2-deficient fibroblasts and CDK2 knockdown cells were markedly protected against apoptin. Thus, our results not only decipher the pathway of apoptin-induced cell death but also provide mechanistic insights for the selective killing of tumor cells.

[The immune responses of the fusion protein consisted of two copies of T-cell and B-cell epitopes of food-and-mouth disease virus VP1 type O and LTB and STI enterotoxins of Escherichia coli].

A nucleotides fragment 2020VP1 related to T-cell epitope (aa21-40) and B-cell epitope (aa141-160) of VP1 of Food-and-mouth disease virus (FMDV)type O was synthesized, and the recombined expression vector r2020-LTB-2020-STI was correctly constructed, which was used to express the fusion protein consisted of two copies of aa21-40aa141-160 of FMDV VP1 and LTB and STI Enterotoxins of Escherichia coli. The fusion protein with molecular weight of about 45kD was successfully expressed in E.coli BL21(DE3) RIL at high level and confirmed by SDS-PAGE. The purified fusion protein could be specifically recognized by CTB antibody. The purified protein was then used to vaccinate guinea pigs, rabbits and Balb/c mice at 6-8 week age, and immune responses were finally observed. The fusion protein could induce proliferation of spleen T cells in vaccinated guinea pigs and elicit a high level of neutralizing antibody in rabbits. It could be concluded that the fusion protein could activate FMDV-specific cellular immune-response and humoral immune-response simultaneously. At the same time, the vaccinated mice could survive challenge of Enterotoxigenic Escherichia coli C83902, and the sera of vaccinated rabbits could neutralize the STI toxin. Moreover, the fusion protein had no STI toxicity, indicating that the fusion protein had LTB and STI immunogenicities. Therefore, it can be concluded that the fusion protein could be explored as a potentially efficient FMDV and ETEC vaccine.

Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway.

Apoptin, a small proline-rich protein derived from the chicken anaemia virus, induces cell death selectively in cancer cells. The signalling pathways of apoptin-induced, cancer cell-selective apoptosis are not well understood. Here, we demonstrate that apoptin triggers apoptosis by activating the mitochondrial/intrinsic pathway, and that it acts independently of the death receptor/extrinsic pathway. Jurkat cells deficient in either FADD or caspase-8 (which are both necessary for the extrinsic pathway) were equally as sensitive to apoptin as their parental clones. This demonstrates that apoptin is likely to act through the mitochondrial death pathway. Apoptin treatment causes a loss of mitochondrial membrane potential, and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Apoptin-induced cell death is counteracted by the anti-apoptotic Bcl-2 family members, Bcl-2 itself and Bcl-XL, as shown in Jurkat leukaemia cells. In addition, we describe the processing and activation of caspase-3. By contrast, cleavage of caspase-8, which is predominantly triggered by the death receptor pathway, is not observed. Furthermore, apoptin triggers the cytoplasmic translocation of Nur77, and the inhibition of Nur77 expression by siRNA significantly protects MCF7 cells from apoptin-triggered cell death. Thus, our data indicate that the apoptin death signal(s) ultimately converges at the mitochondria, and that it acts independently of the death receptor pathway.