MicroRNAs miR-18a and miR-452 regulate the replication of enterovirus 71 by targeting the gene encoding VP3.

MicroRNAs (miRNAs) are crucial in the process of host-pathogen interaction. In this study, we established a screening system for miRNAs of target genes to detect the effect of miRNAs on Enterovirus 71 (EV71) replication in rhabdomyosarcoma (RD) cells. A 3'-untranslated region (UTR) dual-luciferase assay was performed to confirm putative miRNA targets in EV71 genome. Firstly, 13 fragments of EV71 genome were inserted into the vector pMIR, and luciferase activities were analyzed to identify the putative miRNAs of target genes. The expression of the reporter protein was significantly downregulated in cells transfected with the vector containing gene VP3. Then we screened for miRNAs that might target to VP3 through online analysis software. In addition, Western blot, real-time PCR, virus titration, and morphological changes were considered to examine the effects of miRNAs on virus replication. The results suggested that miR-18a and miR-452 repress the reproduction of EV71 virus by binding to VP3. Moreover, EV71 infection also affected the expression of endogenous miR-18a and miR-452. In addition, no significant cytotoxic effects were observed. The results from this study suggest that the intracellular miRNAs may play vital roles in the host-virus interaction.

Computational predicting the human infectivity of H7N9 influenza viruses isolated from avian hosts.

The genome composition of a given avian influenza virus is the primary determinant of its potential for cross-species transmission from birds to humans. Here, we introduce a viral genome-based computational tool that can be used to evaluate the human infectivity of avian isolates of influenza A H7N9 viruses, which can enable prediction of the potential risk of these isolates infecting humans. This tool, which is based on a novel class weight-biased logistic regression (CWBLR) algorithm, uses the sequences of the eight genome segments of an H7N9 strain as the input and gives the probability of this strain infecting humans (reflecting its human infectivity). We examined the replication efficiency and the pathogenicity of several H7N9 avian isolates that were predicted to have very low or high human infectivity by the CWBLR model in cell culture and in mice, and found that the strains with high predicted human infectivity replicated more efficiently in mammalian cells and were more infective in mice than those that were predicted to have low human infectivity. These results demonstrate that our CWBLR model can serve as a powerful tool for predicting the human infectivity and cross-species transmission risks of H7N9 avian strains.

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.

Molecular Basis of a Protective/Neutralizing Monoclonal Antibody Targeting Envelope Proteins of both Tick-Borne Encephalitis Virus and Louping Ill Virus.

Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of the LIV envelope (E) protein and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either the LIV or TBEV E protein. The structures reveal that MAb 4.2 binds to the lateral ridge of domain III of the E protein (EDIII) of LIV or TBEV, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFV antiviral development.IMPORTANCE Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral countermeasure development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to low vaccination coverage. In this study, we solved the crystal structures of the LIV envelope protein (E) and a broadly neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. The structures of E-antibody complexes showed that MAb 4.2 targets the lateral ridge of both the TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab.

Monoclonal antibodies (mAbs) targeting the co-stimulatory molecule 4-1BB are of interest for tumor immunotherapy. We determined the complex structures of human 4-1BB with 4-1BB ligand (4-1BBL) or utomilumab to elucidate the structural basis of 4-1BB activation. The 4-1BB/4-1BBL complex displays a typical TNF/TNFR family binding mode. The structure of utomilumab/4-1BB complex shows that utomilumab binds to dimeric 4-1BB with a distinct but partially overlapping binding area with 4-1BBL. Competitive binding analysis demonstrates that utomilumab blocks the 4-1BB/4-1BBL interaction, indicating the interruption of ligand-mediated signaling. The binding profiles of 4-1BBL and utomilumab to monomeric or dimeric 4-1BB indicate limited cross-linking of 4-1BB molecules. These findings provide mechanistic insight into the binding of 4-1BB with its ligand and its agonist mAb, which may facilitate the future development of anti-4-1BB biologics for tumor immunotherapy.

Bunyavirales ribonucleoproteins: the viral replication and transcription machinery.

The Bunyavirales order is one of the largest groups of segmented negative-sense single-stranded RNA viruses, which includes many pathogenic strains that cause severe human diseases. The RNA segments of the bunyavirus genome are separately encapsidated by multiple copies of nucleoprotein (N), and both termini of each N-encapsidated genomic RNA segment bind to one copy of the viral L polymerase protein. The viral genomic RNA, N and L protein together form the ribonucleoprotein (RNP) complex that constitutes the molecular machinery for viral genome replication and transcription. Recently, breakthroughs have been achieved in understanding the architecture of bunyavirus RNPs with the determination of the atomic structures of the N and L proteins from various members of this order. In this review, we discuss the structures and functions of these bunyavirus RNP components, as well as viral genome replication and transcription mechanisms.

A New Discrete-Time Multi-Constrained $K$-Winner-Take-All Recurrent Network and Its Application to Prioritized Scheduling.

In this paper, we propose a novel discrete-time recurrent neural network aiming to resolve a new class of multi-constrained K-winner-take-all (K-WTA) problems. By facilitating specially designed asymmetric neuron weights, the proposed model is capable of operating in a fully parallel manner, thereby allowing true digital implementation. This paper also provides theorems that delineate the theoretical upper bound of the convergence latency, which is merely O(K). Importantly, via simulations, the average convergence time is close to O(1) in most general cases. Moreover, as the multi-constrained K-WTA problem degenerates to a traditional single-constrained problem, the upper bound becomes exactly two parallel iterations, which significantly outperforms the existing K-WTA models. By associating the neurons and neuron weights with routing paths and path priorities, respectively, we then apply the model to a prioritized flow scheduler for the data center networks. Through extensive simulations, we demonstrate that the proposed scheduler converges to the equilibrium state within near-constant time for different scales of networks while achieving maximal throughput, quality-of-service priority differentiation, and minimum energy consumption, subject to the flow contention-free constraints.

Inhibition of hepatitis B virus replication by activation of the cGAS-STING pathway.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) senses cytosolic DNA and catalyses synthesis of the second messenger cGAMP, which activates the downstream signalling adaptor protein STING, leading to the expression of type I interferons. Hepatitis B virus (HBV) is a small DNA virus, and the cGAS-STING pathway may inhibit HBV RNA synthesis and viral assembly in cell culture, but the exact roles of the cGAS pathway in the restriction of HBV replication in infection systems remain to be elucidated. In this study, replication of HBV was significantly inhibited both in cell culture and in vivo in a mouse model when the cGAS-STING pathway was activated by dsDNA or cGAMP. In contrast, the presence of enzymatically inactive cGAS mutant did not influence HBV replication. Moreover, knockdown of cGAS in human peripheral blood monocytes led to a higher level of intracellular HBV DNA. Collectively, our data indicate that the cGAS-STING pathway plays a role in the surveillance of HBV infection and may be exploited for development of novel anti-HBV strategies.

Resistance to Mutant Group 2 Influenza Virus Neuraminidases of an Oseltamivir-Zanamivir Hybrid Inhibitor.

Influenza virus neuraminidase (NA) drug resistance is one of the challenges to preparedness against epidemic and pandemic influenza virus infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug resistance of the influenza virus N1 NA is well characterized. Yet drug resistance of the N2 strain is not well understood. Here, we confirm that replacement of N2 E119 or I222 results in multidrug resistance, and when the replacements occur together, the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we showed that E119 replacement results in a loss of hydrogen bonding to oseltamivir and zanamivir, whereas I222 replacement results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we found that MS-257, a zanamivir-oseltamivir hybrid inhibitor, is less susceptible to drug resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug resistance. MS-257 serves as a next-generation anti-influenza virus drug candidate and serves also as a scaffold for further design of NAIs. IMPORTANCE: Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multidrug-resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multidrug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of the N2 E119V and I222L mutants and the dual mutant. Most importantly, we found that a novel oseltamivir-zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next-generation anti-influenza virus drug.

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.

Evidence for Within-Host Genetic Recombination among the Human Pegiviral Strains in HIV Infected Subjects.

The non-pathogenic Human Pegivirus (HPgV, formerly GBV-C/HGV), the most prevalent RNA virus worldwide, is known to be associated with reduced morbidity and mortality in HIV-infected individuals. Although previous studies documented its ubiquity and important role in HIV-infected individuals, little is known about the underlying genetic mechanisms that maintain high genetic diversity of HPgV within the HIV-infected individuals. To assess the within-host genetic diversity of HPgV and forces that maintain such diversity within the co-infected hosts, we performed phylogenetic analyses taking into account 229 HPgV partial E1-E2 clonal sequences representing 15 male and 8 female co-infected HIV patients from Hubei province of central China. Our results revealed the presence of eleven strongly supported clades. While nine clades belonged to genotype 3, two clades belonged to genotype 2. Additionally, four clades that belonged to genotype 3 exhibited inter-clade recombination events. The presence of clonal sequences representing multiple clades within the HIV-infected individual provided the evidence of co-circulation of HPgV strains across the region. Of the 23 patients, six patients (i.e., five males and one female) were detected to have HPgV recombinant sequences. Our results also revealed that while male patients shared the viral strains with other patients, viral strains from the female patients had restricted dispersal. Taken together, the present study revealed that multiple infections with divergent HPgV viral strains may have caused within-host genetic recombination, predominantly in male patients, and therefore, could be the major driver in shaping genetic diversity of HPgV.

Identification and Structure-Activity Relationships of Diarylhydrazides as Novel Potent and Selective Human Enterovirus Inhibitors.

Enterovirus 71 (EV71) plays an important role in hand-foot-and-mouth disease. In this study, a series of diarylhydrazide analogues was synthesized, and the systematic exploration of SAR led to potent enterovirus inhibitors, of which compound 15 exhibits significant improvements in inhibition potency with an EC50 value of 0.02 µM against EV71. It is very interesting that this class of diarylhydrazides exhibits activities against a series of human enteroviruses at the picomolar level, including EV71 and Coxsackieviruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5, and CVB6 (EC50 as low as 0.5 nM). Compared with the reference antienterovirus drug 1 (enviroxime) and known inhibitor 5 (WIN 51711), the four highly selective compounds 15, 27, 41 and 47 inhibited EV71 replication with EC50 values of 0.17-0.02 µM and SI values in a range of 978.4-12338. A preliminary mechanistic study indicated that VP1 might be the target site for this type of compound.

DYRK2 Negatively Regulates Type I Interferon Induction by Promoting TBK1 Degradation via Ser527 Phosphorylation.

Viral infection activates the transcription factors NF-κB and IRF3, which contribute to the induction of type I interferons (IFNs) and cellular antiviral responses. Protein kinases play a critical role in various signaling pathways by phosphorylating their substrates. Here, we identified dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) as a negative regulator of virus-triggered type I IFN induction. DYRK2 inhibited the virus-triggered induction of type I IFNs and promoted the K48-linked ubiquitination and degradation of TANK-binding kinase 1 (TBK1) in a kinase-activity-dependent manner. We further found that DYRK2 phosphorylated Ser527 of TBK1, which is essential for the recruitment of NLRP4 and for the E3 ubiquitin ligase DTX4 to degrade TBK1. These findings suggest that DYRK2 negatively regulates virus-triggered signaling by targeting TBK1 for phosphorylation and priming it for degradation, and these data provide new insights into the molecular mechanisms that dictate the cellular antiviral response.

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.

The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.

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.

Human respiratory syncytial virus infection is inhibited by IFN-induced transmembrane proteins.

The IFN immune system plays an essential role in protecting the host against most viral infections. In order to explore the interactions between the IFN pathway and human respiratory syncytial virus (RSV) infection, and to identify potential IFN-stimulated genes (ISGs) that may be involved in suppressing the replication of RSV, we utilized an IFN pathway-specific microarray to study the effects of RSV infection on the IFN pathway in HeLa cells. We showed that RSV infection enhanced the expression of a series of ISGs, including oligoadenylate synthetase 2, IFITM1 (IFN-induced transmembrane protein 1) and myxovirus resistance 2. Our results also showed that the IFITM proteins potently inhibited RSV infection mainly by interfering with both virus entry and the subsequent replication steps, but not the attachment process. The antiviral effect of IFITM3 was not affected by ubiquitination modification. Furthermore, knocking down the endogenous and IFN-induced expression of IFITM1 and 3 facilitated RSV infection. Expression of the IFITM proteins was found to delay the phosphorylation of IFN regulatory factor 3 through interfering with the detection of viral RNA by MDA5 (melanoma differentiation-associated gene 5) and RIG-I (retinoic acid-inducible gene I). These results demonstrated that the restriction of RSV infection by the IFITM proteins was achieved through the inhibition of virus entry and replication, and they provided further insight for exploring the mechanism of IFITM-protein-mediated virus restriction.

[MiR432* regulate the replication of coxsackievirus A16 in rhabdomyosarcoma cells].

OBJECTIVE: MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. In this study, we examined miRNAs' effects on the replication of Coxsackievirus A16 (CA16) in rhabdomyosarcoma cells. METHODS: We constructed target gene of miRNAs screening system. We used 3'untranslated region (UTR) dual luciferase reporter analysis to identify putative miRNA targets in the CA16 virus genome. First, 12 segments of CA16 virus genome were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated. Then, using online analysis programs we screened the miRNAs that may target to 5'-UTR. Furthermore, Western blot and real-time PCR test were used to study the effect of miRNAs on viral replication. RESULTS: The study showed that miR432 * could stimulate the replication of CA16 virus. On the contrary, miR432 * inhibitor could suppress CA16 virus replication. CONCLUSION: Cellular miRNAs could regulate the replication of CA16 virus in host cells. Our findings support the notion that the cellular miRNAs play an important role in the host and virus infection.