Cyclin-dependent kinase 5 negatively regulates antiviral immune response by disrupting myeloid differentiation primary response protein 88 self-association.

As a member of the pattern recognition receptors (PRRs) involving in the innate immune system, Toll-like receptors (TLRs) can sense a wide range of microbial pathogens and combat infections by producing antimicrobial products, inflammatory cytokines, and chemokines. All TLRs, with the exception of TLR3, activate a signalling cascade via the myeloid differentiation primary response gene 88 (MyD88). Therefore, the activation of MyD88-dependent signalling pathway must be finely controlled. Herein, we identified that cyclin-dependent kinase 5 (CDK5) negatively regulated TLR-MyD88 signalling pathway by targeting MyD88. Overexpression of CDK5 reduced the production of interferons (IFNs), while a deficiency in CDK5 increased the expression of IFNs in response to vesicular stomatitis virus (VSV) infection. Mechanistically, CDK5 suppressed the formation of MyD88 homodimers, resulting in the attenuated production of IFNs induced by VSV infection. Surprisingly, its kinase activity does not play a role in this process. Therefore, CDK5 can act as an internal regulator to prevent excessive production of IFNs by restricting TLR-MyD88-induced activation of antiviral innate immunity in A549 cells.

Molecular weight determination of low molecular weight hyaluronic acid and chondroitin sulfate by gel permeation chromatography.

Low molecular weight (LWM) hyaluronic acid (HA) and chondroitin sulfate (CS) have a wide range of applications. To determine their molecular weight (MW), we developed a gel permeation chromatography (GPC) method, which is calibrated based on serrated peaks in the chromatograms. MW calibrants were obtained from the enzymolysis of HA and CS using hyaluronidase. The identical structure of calibrants and samples ensured the soundness of the method. The highest confidence MWs were up to 14,454 and 14,605 for HA and CS, respectively, and the standard curves showed very high correlation coefficients. Thanks to the changeless relationship between MW and its contribution to the GPC integral, the second calibration curves could be derived via one GPC column, also embodied correlation coefficients of >0.9999. The discrepancies of MW values were minuscule, and the measurement of a sample could be conducted in <30 min. The accuracy of the method was verified using LWM heparins, and the measured Mw values showed a 1.2 %-2.0 % error relative to pharmacopeia results. The MW results obtained for LWM-HA and LWM-CS samples were also consistent with the results obtained by multiangle laser light scattering. The method was also verified be able to measure the very low MWs.

Portable rapid detection of maize chlorotic mottle virus using RT-RAA/CRISPR-Cas12a based lateral flow assay.

Introduction: Maize lethal necrosis seriously threatens maize production worldwide, which was caused by coinfection by maize chlorotic mottle virus (MCMV) and a potyvirid. To effectively control maize lethal necrosis, it is vital to develop a rapid, sensitive, and specific detection method for the early diagnosis of MCMV in host plant tissues. Methods: We established a rapid detection procedure by combining the one-step reverse-transcription recombinase-aided amplification (one-step RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one tube (one-tube one-step RT-RAA/CRISPR-Cas12a), which can be implemented on a portable metal incubator at 37~42°C. Furthermore, the crude extract of total RNA from plant materials using alkaline-PEG buffer can be directly used as the template for one-step RT-RAA. Results: The developed one-tube one-step RT-RAA/CRISPR-Cas12a lateral flow assay can detect as low as 2.5 copies of the coat protein (CP) gene of MCMV and 0.96 pg of the total RNA extracted from MCMV infected maize leaves. Furthermore, the MCMV infected maize leaves at 5 dpi having no obvious symptoms was detected as weak positive. Discussion: The crude extraction method of total RNA from plant materials required no complicated device, and all the procedures could be implemented at room temperature and on a portable metal incubator, costing a total time of about 1h. The one-step RT-RAA reagents and CRISPR/Cas12a reagents can be lyophilized for easy storage and transportation of reagents, which makes this method more feasible for the filed detection. This method presents rapidness, robustness and on-site features in detecting viral RNA, and is a promising tool for the field application in minimally equipped laboratories.

Zika virus infection induced apoptosis by modulating the recruitment and activation of pro-apoptotic protein Bax.

Zika virus (ZIKV) infection is associated with microcephaly in newborns and serious neurological complications in adults. Apoptosis of neural progenitor cells induced by ZIKV infection is believed to be a main reason for ZIKV infection-related microcephaly. However, the detailed mechanism of ZIKV infection-induced apoptosis remains to be elucidated. In this report, ZIKV infection induced the conformational activation of the pro-apoptotic protein Bax, with subsequent formation of oligomers of Bax in the mitochondria. Cell apoptosis was reduced significantly in SY5Y cells subjected to Bax knockdown. Additionally, while decreasing Bax expression inhibited the release of Cyt c from the mitochondria and reduced the rate of loss of mitochondrial membrane potential induced by ZIKV infection, silencing Bak, caspase-8, and/or caspase-10 expression did not. Mitochondria isolated from the untreated ZIKV-infected cells displayed Bax-binding ability and the subsequent release of Cyt c. This study also indicated that the NS4B protein of ZIKV recruited Bax to the mitochondria and induced Bax conformational activation. The overexpressed NS4B was localized to the mitochondria and induced cell apoptosis by activating the pro-apoptotic protein Bax. All the above results indicated that ZIKV infection directly impacted the mitochondrial apoptotic pathway by modulating the recruitment and activation of Bax.Importance: Since the large outbreaks that occurred in the Pacific Islands and Latin America in 2013, Zika virus has been confirmed a neuroteratogenic pathogen and causative agent of microcephaly and other developmental anomalies of the central nervous system in children born to infected mothers. As the widespread apoptosis throughout the whole brain, studies in animal models have reinforced the link between microcephaly caused by ZIKV infection and NPC apoptosis. Currently, the detailed mechanism of ZIKV infection-induced apoptosis still remains to be elucidated. Here, we firstly demonstrate that ZIKV infection activated the classic signs of mitochondrial apoptotic pathway by modulating the recruitment and activation of Bax. ZIKV NS4B represents a novel viral apoptotic protein that can modulate the recruitment and activation of Bax and trigger the apoptotic program. This is a new insight into understanding the interplay between apoptosis and ZIKV infection.

Mitochondria Redistribution in Enterovirus A71 Infected Cells and Its Effect on Virus Replication.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD) and it also causes severe neurologic complications in infected children. The interactions between some viruses and the host mitochondria are crucial for virus replication and pathogenicity. In this study, it was observed that EV-A71 infection resulted in a perinuclear redistribution of the mitochondria. The mitochondria rearrangement was found to require the microtubule network, the dynein complex and a low cytosolic calcium concentration. Subsequently, the EV-A71 non-structural protein 2BC was identified as the viral protein capable of inducing mitochondria clustering. The protein was found localized on mitochondria and interacted with the mitochondrial Rho GTPase 1 (RHOT1) that is a key protein required for attachment between the mitochondria and the motor proteins, which are responsible for the control of mitochondria movement. Additionally, suppressing mitochondria clustering by treating cells with nocodazole, EHNA, thapsigargin or A23187 consistently inhibited EV-A71 replication, indicating that mitochondria recruitment played a crucial role in the EV-A71 life cycle. This study identified a novel function of the EV-A71 2BC protein and provided a potential model for the regulation of mitochondrial motility in EV-A71 infection.

Enterovirus 71 induces apoptosis by directly modulating the conformational activation of pro-apoptotic protein Bax.

Enterovirus 71 (EVA71), a virus of the genus Enterovirus in the family Picornaviridae, is one of the main causative agents of hand, foot and mouth disease in infected infants and young children. In this study, we report that cells with EVA71 infection exhibit increased levels of cytochrome c release and caspase-3 activation. EVA71 infection induces the conformational activation of pro-apoptotic protein Bax and the subsequent formation of oligomers of Bax in mitochondria. Inhibitors that block caspase-8 activation cannot inhibit apoptosis induced by EVA71 infection. Importantly, cells with Bax but not Bak or caspase-8 knockdown show resistance to apoptosis induced by EVA71 infection. Mitochondria isolated from EVA71-infected cells display clear Bax-binding ability and the subsequent release of cytochrome c. Therefore, these results indicate that EVA71 infection directly impacts the mitochondrial apoptotic pathway by modulating the recruitment and activation of Bax.

Detection and differentiation of influenza viruses with glycan-functionalized gold nanoparticles.

Accurate diagnosis of influenza viruses is difficult and generally requires a complex process because of viral diversity and rapid mutability. In this study, we report a simple and rapid strategy for the detection and differentiation of influenza viruses using glycan-functionalized gold nanoparticles (gGNPs). This method is based on the aggregation of gGNP probes on the viral surface, which is mediated by the specific binding of the virus to the glycans. Using a set of gGNPs bearing different glycan structures, fourteen influenza virus strains, including the major subtypes currently circulating in human and avian populations, were readily differentiated from each other and from a human respiratory syncytial virus in a single-step colorimetric procedure. The results presented here demonstrate the potential of this gGNP-based system in the development of convenient and portable sensors for the clinical diagnosis and surveillance of influenza viruses.

Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax.

To survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochrome c (Cyt c) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XL abrogates 2B-induced release of Cyt c and caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax. IMPORTANCE: EV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. This study provides evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cyt c release and mitochondrial permeabilization during EV71 infection.

Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

UNLABELLED: Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE: The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

Study on nanocomposite construction based on the multi-functional biotemplate self-assembled by the recombinant TMGMV coat protein for potential biomedical applications.

Nowadays there is a growing interest in bio-scaffolded nanoarchitectures. Rapid progress in nanobiotechnology and molecular biology has allowed the engineering of inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs) into self-assembling biological structures to facilitate the design of novel biomedical or bioimaging devices. Here we introduce a novel nanocomposite comprising a self-assembled protein scaffold based on a recombinant tobacco mild green mosaic tobamovirus (TMGMV) coat protein (CP) and the photocatalytic TiO2 nanoparticles attached to it, which may provide a generic method for materials engineering. A template containing a modified TMGMV CP (mCP) gene, with the first six C-terminal amino acid residues deleted to accommodate more foreign peptides and expressing a site-directed mutation of A123C for bioconjugation utility, and two genetically engineered mutants, Escherichia coli-based P-mCP-Ti7 containing a C-terminal TiO2 GEPI sequence of seven peptides (Ti7) and Hi5 insect cells-derived E-CP-Ti7-His6 C-terminally fused with Ti7+His6 tag were created. Expression vectors and protocols for enriching of the two CP variants were established and the resultant proteins were identified by western blot analysis. Their RNA-free self-assembling structures were analyzed by transmission electron microscopy (TEM) and immuno-gold labeling TEM analysis. Adherence of nanoparticles to the P-mCP-Ti7 induced protein scaffold was visualized by TEM analysis. Also discussed is the Cysteine thiol reactivity in bioconjugation reactions with the maleimide-functionalized porphyrin photosensitizers which can function as clinical photodynamic therapy agents. This study introduced a novel approach to producing an assembly-competent recombinant TMGMV CP, examined its ability to serve as a novel platform for the multivalent display of surface ligands and demonstrated an alternative method for nanodevice synthesis for nanobiotechnological applications by combining GEPIs-mediated immobilization with the controllability of self-assembling recombinant TMGMV CP.

Self-assembled bionanoparticles based on the Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) coat protein as a prospective bioscaffold for nanotechnological applications.

Biomolecule-nanoparticle hybrid bioconjugates based on bioscaffolds such as protein cages and virus capsules have been widely studied. Highly stable and durable biotemplates are a vital pillar in constructing bio-inorganic functional hybrid composites. Here, we introduce a highly heat-resistant coat protein (CP) of Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) isolated from the hyperthermophilic archaeon as a prospective biological matrix. Our experiments showed that STSV1 CP was successfully cloned and solubly expressed in the Escherichia coli Rosetta-(DE3) host strain. Protein expression was verified by SDS-PAGE and western blot analysis of the reference C-terminally six-histidine (His6) tagged STSV1 CP (HT-CP). Thermal stability experiments showed that the STSV1 coat protein remained fairly stable at 80 °C. The proteins can be purified facilely by heat treatment followed by size exclusion chromatography (SEC). Transmission electron microscopy (TEM) analysis of the purified STSV1 CP protein aggregates demonstrated that the protein could self-assemble into rotavirus-like nanostructures devoid of genetic materials under our experimental conditions. Similar results were obtained for the HT-CP purified by heat treatment followed by Ni-NTA and SEC, indicating that moderately engineered STSV1 CP can retain its self-assembly property. In addition, the STSV1 CP has a high binding affinity for TiO2 nanoparticles. This illustrates that the STSV1 CP can be used as a bioscaffold in nanobiotechnological applications.

Cell surface vimentin is an attachment receptor for enterovirus 71.

UNLABELLED: Enterovirus 71 (EV71) is a highly transmissible pathogenic agent that causes severe central nervous system diseases in infected infants and young children. Here, we reported that EV71 VP1 protein could bind to vimentin intermediate filaments expressed on the host cell surface. Soluble vimentin or an antibody against vimentin could inhibit the binding of EV71 to host cells. Accompanied with the reduction of vimentin expression on the cell surface, the binding of EV71 to cells was remarkably decreased. Further evidence showed that the N terminus of vimentin is responsible for the interaction between EV71 and vimentin. These results indicated that vimentin on the host cell surface may serve as an attachment site that mediated the initial binding and subsequently increased the infectivity of EV71. IMPORTANCE: This study delivers important findings on the roles of vimentin filaments in relation to EV71 infection and provides information that not only improves our understanding of EV71 pathogenesis but also presents us with potentially new strategies for the treatment of diseases caused by EV71 infections.

The nuclear protein Sam68 is redistributed to the cytoplasm and is involved in PI3K/Akt activation during EV71 infection.

Nuclear proteins can be triggered to be redistributed to the cytoplasm to assist with EV71 virus replication. This process is frequently involved in cellular signal transduction upon virus infection. In this study, we have demonstrated that a new nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), was translocated to the cytoplasm and was co-localized with EV71 during virus infection. Confocal microscopy and subcellular fractionation assay confirmed that virus 3C protease triggered the redistribution of Sam68 to the cytoplasm. Knockdown of Sam68 expression using ShRNA significantly inhibited virus replication, suggesting that Sam68 may be a host factor involved in EV71 life cycle. In addition, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Sam68 is known to be an upstream regulator of PI3K and our immunoprecipitation studies confirmed that Sam68 interacted directly with the p85 regulatory subunit of PI3K and mediated PI3K/Akt activation during EV71 infection. On the contrary, silencing of Sam68 dramatically abrogated Akt phosphorylation. These data, plus the fact that Sam68 is known to be a signaling adaptor protein, indicated that Sam68 is a signal molecule with a functional role in the PI3K/Akt signal pathway during EV71 infection.

Identification of the myelin oligodendrocyte glycoprotein as a cellular receptor for rubella virus.

Rubella virus (RV) is a highly transmissible pathogenic agent that causes the disease rubella. Maternal RV infection during early pregnancy causes the death of the fetus or congenital rubella syndrome in infants. However, the cellular receptor for RV has not yet been identified. In this study, we found that the myelin oligodendrocyte glycoprotein (MOG) specifically bound to the E1 envelope glycoprotein of RV, and an antibody against MOG could block RV infection. Most importantly, we also showed that ectopic expression of MOG on the cell surface of 293T cells rendered this nonpermissive cell line permissive for RV entry and replication. Thus, this study has identified a cellular receptor for RV and suggests that blocking the MOG attachment site of RV may be a strategy for molecular intervention of RV infection.

Inhibition of atrogin-1/MAFbx expression by adenovirus-delivered small hairpin RNAs attenuates muscle atrophy in fasting mice.

Atrogin-1 or muscle atrophy F-box (MAFbx) is a major atrophy-related E3 ubiquitin ligase highly expressed in skeletal muscle during muscle atrophy and other disease states such as sepsis, cancer cachexia, and fasting. In this paper, we report experiments inhibiting MAFbx activity in fasting mice and in the skeletal myoblast cell line C2C12 via an adenovirus-mediated small hairpin RNA (shRNA) expression system in order to assess its suitability as a therapeutic target. Our results demonstrated that downregulation of MAFbx by shRNAs attenuated muscle loss induced by fasting in mice. Furthermore, we showed that when MAFbx expression was blocked both in cells and in fasting mice, the level of a myogenic factor, MyoD, was upregulated; whereas a muscle negative regulator, growth differentiation factor (GDF)-8 (myostatin), was suppressed. Our results also suggested that lower levels of MAFbx could also enhance muscle cell differentiation that corresponded to the reduced expression of GDF-8 and the increased level of MyoD. Taken together, the present study showed that MAFbx could be a potential molecular target for treating muscle atrophy.

A retrovirus-based system to stably silence GDF-8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cells.

BACKGROUND: Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-beta superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation. METHODS: A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. RESULTS: In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. CONCLUSIONS: The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas.