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changes in cellular mrna stability, splicing, and polyadenylation through hur protein sequestration by a cytoplasmic rna virus.the impact of rna viruses on the posttranscriptional regulation of cellular gene expression is unclear. sindbis virus causes a dramatic relocalization of the cellular hur protein from the nucleus to the cytoplasm in infected cells. this is to the result of the expression of large amounts of viral rnas that contain high-affinity hur binding sites in their 3' utrs effectively serving as a sponge for the hur protein. sequestration of hur by sindbis virus is associated with destabilization of cellul ...201324210824
mayaro virus infection, amazon basin region, peru, 2010-2013.during 2010-2013, we recruited 16 persons with confirmed mayaro virus infection in the peruvian amazon to prospectively follow clinical symptoms and serologic response over a 12-month period. mayaro virus infection caused long-term arthralgia in more than half, similar to reports of other arthritogenic alphaviruses.024210165
engineered alphavirus replicon vaccines based on known attenuated viral mutants show limited effects on immunogenicity.the immunogenicity of alphavirus replicon vaccines is determined by many factors including the level of antigen expression and induction of innate immune responses. characterized attenuated alphavirus mutants contain changes to the genomic 5' utr and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (ifn) induction. in an attempt to create more potent replicon vaccines, we engineered a panel of venezuelan equine ...201324210122
identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.chikungunya virus (chikv) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. the re-emergence of chikv has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. unfortunately, no effective treatment is currently available. the present study reports the use of resazurin in a cell-based high-throughput assay, and an i ...201324205414
mice deficient in interferon-gamma or interferon-gamma receptor 1 have distinct inflammatory responses to acute viral encephalomyelitis.interferon (ifn)-gamma is an important component of the immune response to viral infections that can have a role both in controlling virus replication and inducing inflammatory damage. to determine the role of ifn-gamma in fatal alphavirus encephalitis, we have compared the responses of wild type c57bl/6 (wtb6) mice with mice deficient in either ifn-gamma (gko) or the alpha-chain of the ifn-gamma receptor (grko) after intranasal infection with a neuroadapted strain of sindbis virus. mortalities ...201324204622
inhibition of arenavirus by a3, a pyrimidine biosynthesis inhibitor.arenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. currently, there are no fda-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. the pyrimidine biosynthesis inhibitor a3, which was identified in a high-throughput screen ...201324198417
synaptic nmda receptor stimulation activates pp1 by inhibiting its phosphorylation by cdk5.the serine/threonine protein phosphatase protein phosphatase 1 (pp1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how pp1 activity is controlled in different forms of synaptic plasticity remains unknown. we find that synaptic n-methyl-d-aspartate (nmda) receptor stimulation in neurons leads to activation of pp1 through a mechanism involving inhibitory phosphorylation at thr320 by cdk5. synaptic stimulation led to p ...201324189275
human bocavirus in patients with encephalitis, sri lanka, 2009-2010.we identified human bocavirus (hbov) dna by pcr in cerebrospinal fluid from adults and children with encephalitis in sri lanka. hbov types 1, 2, and 3 were identified among these cases. phylogenetic analysis of hbov1 strain sequences found no subclustering with strains previously identified among encephalitis cases in bangladesh.024188380
biology of the tam receptors.the tam receptors--tyro3, axl, and mer--comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. the tams and their ligands--gas6 and protein s--are essenti ...201324186067
ampars and synaptic plasticity: the last 25 years.the study of synaptic plasticity and specifically ltp and ltd is one of the most active areas of research in neuroscience. in the last 25 years we have come a long way in our understanding of the mechanisms underlying synaptic plasticity. in 1988, ampa and nmda receptors were not even molecularly identified and we only had a simple model of the minimal requirements for the induction of plasticity. it is now clear that the modulation of the ampa receptor function and membrane trafficking is criti ...024183021
design, synthesis and biological evaluation of phosphorodiamidate prodrugs of antiviral and anticancer nucleosides.we herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. twenty-five symmetrical phosphorodiamidates were synthesized, bearing esterified l-alanine (and in one case d-alanine) in the prodrug moiety, each as single stereoisomer. the presence of an achiral phosphorus represents a potential advantage over the phosphoramidate protide approach, where diastereoisomeric mixtures are routinely ...201324177359
architecture and biogenesis of plus-strand rna virus replication factories.plus-strand rna virus replication occurs in tight association with cytoplasmic host cell membranes. both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. this compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral rna from cellular defense mechanisms. electron tomography studies conducted during the last couple of y ...024175228
neuronal network imaging in acute slices using ca2+ sensitive bioluminescent reporter.genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. imaging of ca(2+) indicators is widely used to record transient intracellular ca(2+) increases associated with bioelectrical activity. the natural bioluminescent ca(2+) sensor aequorin has been historically the first ca(2+) indicator used to address biological questions. aequorin imaging offers several advantages over fluorescent reporte ...201424166366
european surveillance for west nile virus in mosquito populations.a wide range of arthropod-borne viruses threaten both human and animal health either through their presence in europe or through risk of introduction. prominent among these is west nile virus (wnv), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. endemic outbreaks of west nile fever have been reported in italy, greece, france, romania, hungary, russia and spain, with further spread expected. most outbreaks in western europe have been due ...201324157510
optimization of novel indole-2-carboxamide inhibitors of neurotropic alphavirus replication.neurotropic alphaviruses, which include western equine encephalitis virus (weev) and fort morgan virus, are mosquito-borne pathogens that infect the central nervous system causing acute and potentially fatal encephalitis. we previously reported a novel series of indole-2-carboxamides as alphavirus replication inhibitors, one of which conferred protection against neuroadapted sindbis virus infection in mice. we describe here further development of this series, resulting in 10-fold improvement in ...201324151954
infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes.replication of positive-sense rna viruses is associated with the rearrangement of cellular membranes. previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (sars-cov) showed that they generate double-membrane vesicles (dmvs) and convoluted membranes as part of a reticular membrane network. here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus i ...201324149513
mosquitoes of western yunnan province, china: seasonal abundance, diversity, and arbovirus associations.the western borderland between yunnan province, china, and myanmar is characterized by a climate that facilitates year-round production of mosquitoes. numerous mosquito-transmitted viruses, including japanese encephalitis virus circulate in this area. this project was to describe seasonal patterns in mosquito species abundance and arbovirus activity in the mosquito populations.201324146951
isg15 regulates peritoneal macrophages functionality against viral infection.upon viral infection, the production of type i interferon (ifn) and the subsequent upregulation of ifn stimulated genes (isgs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. the ubiquitin-like protein (ubl) isg15 is a critical ifn-induced antiviral molecule that protects against several viral infections, but the mechanism by which isg15 exerts its antiviral function is not completely understood. here, we report that isg15 plays ...201324137104
non-specific dsrna-mediated antiviral response in the honey bee.honey bees are essential pollinators of numerous agricultural crops. since 2006, honey bee populations have suffered considerable annual losses that are partially attributed to colony collapse disorder (ccd). ccd is an unexplained phenomenon that correlates with elevated incidence of pathogens, including rna viruses. honey bees are eusocial insects that live in colonies of genetically related individuals that work in concert to gather and store nutrients. their social organization provides numer ...201324130869
the transmembrane domain and acidic lipid flip-flop regulates voltage-dependent fusion mediated by class ii and iii viral proteins.voltage dependence of fusion induced by class ii and class iii viral fusion proteins was investigated. class ii proteins from ross river and sindbus virus and a mutant class iii protein from epstein barr virus were found to induce cell-cell fusion that is voltage dependent. combined with previous studies, in all, four class ii and two class iii protein have now been shown to exhibit voltage-dependent fusion, demonstrating that this is probably a general phenomenon for these two classes of viral ...201324124539
insect antiviral innate immunity: pathways, effectors, and connections.insects are infected by a wide array of viruses some of which are insect restricted and pathogenic, and some of which are transmitted by biting insects to vertebrates. the medical and economic importance of these viruses heightens the need to understand the interaction between the infecting pathogen and the insect immune system in order to develop transmission interventions. the interaction of the virus with the insect host innate immune system plays a critical role in the outcome of infection. ...201324120681
control of the rescue and replication of semliki forest virus recombinants by the insertion of mirna target sequences.due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. these properties may, however, be potentially hazardous unless the virus infection is controlled. while the rna genome of alphaviruses precludes the standard control techniques, host mirnas can be used to down-regulate viral replication. in this study, target sites from ubiquitous mirnas and those of mirnas under-represented in cervical cancer cells were ...201324098728
antiviral mechanisms of human defensins.defensins are an effector component of the innate immune system with broad antimicrobial activity. humans express two types of defensins, α- and β-defensins, which have antiviral activity against both enveloped and non-enveloped viruses. the diversity of defensin-sensitive viral species reflects a multitude of antiviral mechanisms. these include direct defensin targeting of viral envelopes, glycoproteins, and capsids in addition to inhibition of viral fusion and post-entry neutralization. bindin ...201324095897
the antiviral activities of isg15.post-translational protein modification is an important strategy for the regulation of the cell proteome independent of the need for new gene expression. ubiquitin and ubiquitin-like modifiers mediate the regulation of protein levels, signaling pathways, vesicular trafficking, and many other cellular processes through their covalent conjugation to proteins. interferon stimulated gene 15 (isg15) is a ubiquitin-like modifier induced by type i interferon. in addition to conjugating to potentially h ...201324095857
evasion of superinfection exclusion and elimination of primary viral rna by an adapted strain of hepatitis c virus.cells that are productively infected by hepatitis c virus (hcv) are refractory to a second infection by hcv via a block in viral replication known as superinfection exclusion. the block occurs at a postentry step and likely involves translation or replication of the secondary viral rna, but the mechanism is largely unknown. to characterize hcv superinfection exclusion, we selected for an hcv variant that could overcome the block. we produced a high-titer hc-j6/jfh1 (jc1) viral genome with a fluo ...201324089557
highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.the continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. to analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. the reporter virus encodes the small and bright nanoluc luciferase whose activity serves as an extremely sensitive readout of viral infection. this virus stably maintains the reporter const ...201324089552
induction of cytopathogenicity in human glioblastoma cells by chikungunya virus.chikungunya virus (chikv), an arthritogenic old-world alphavirus, has been implicated in the central nervous system (cns) infection in infants and elderly patients. astrocytes are the major immune cells of the brain parenchyma that mediate inflammation. in the present study we found that a local isolate of chikv infect and activate u-87 mg cells, a glioblastoma cell line of human astrocyte origin. the infection kinetics were similar in infected u-87 mg cells and the human embryo kidney (hek293) ...201324086645
panorama from the oncolytic virotherapy summit. 024081122
non-encapsidation activities of the capsid proteins of positive-strand rna viruses.viral capsid proteins (cps) are characterized by their role in forming protective shells around viral genomes. however, cps have additional and important roles in the virus infection cycles and in the cellular responses to infection. these activities involve cp binding to rnas in both sequence-specific and nonspecific manners as well as association with other proteins. this review focuses on cps of both plant and animal-infecting viruses with positive-strand rna genomes. we summarize the structu ...201324074574
molecular mechanisms involved in the pathogenesis of alphavirus-induced arthritis.arthritogenic alphaviruses, including ross river virus (rrv), chikungunya virus (chikv), sindbis virus (sinv), mayaro virus (mayv), o'nyong-nyong virus (onnv), and barmah forest virus (bfv), cause incapacitating and long lasting articular disease/myalgia. outbreaks of viral arthritis and the global distribution of these diseases point to the emergence of arthritogenic alphaviruses as an important public health problem. this review discusses the molecular mechanisms involved in alphavirus-induced ...201324069610
a fusion-inhibiting peptide against rift valley fever virus inhibits multiple, diverse viruses.for enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. this fusion process is mediated by at least three classes of fusion proteins (class i, ii, and iii) based on the protein sequence and structure. for rift valley fever virus (rvfv), the glycoprotein gc (class ii fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. here, we show that p ...201324069485
apoptosis induction influences reovirus replication and virulence in newborn mice.apoptosis is a type of controlled cell death that is essential for development and tissue homeostasis. it also serves as a robust host response against infection by many viruses. the capacity of neurotropic viruses to induce apoptosis strongly correlates with virulence. however, the precise function of apoptosis in viral infection is not well understood. reovirus is a neurotropic virus that induces apoptosis in a variety of cell types, including central nervous system neurons, leading to fatal e ...201324067960
hnrnp a1: the swiss army knife of gene expression.eukaryotic cells express a large variety of rna binding proteins (rbps), with diverse affinities and specificities towards target rnas. these proteins play a crucial role in almost every aspect of rna biogenesis, expression and function. the heterogeneous nuclear ribonucleoproteins (hnrnps) are a complex and diverse family of rna binding proteins. hnrnps display multiple functions in the processing of heterogeneous nuclear rnas into mature messenger rnas. hnrnp a1 is one of the most abundant and ...201324065100
development & evaluation of biotinylated dna probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & csf samples.the resurgence of chikungunya virus (chikv) in the indian ocean islands and india has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. the early confirmatory diagnosis of chikv is essential for management as well as control of unprecedented epidemics. the present study describes the development and evaluation of a highly sensitive and specific e1 structural gene specific biotinylated dna probe for detection of chikungunya viru ...024056565
an easy operating pathogen microarray (eopm) platform for rapid screening of vertebrate pathogens.infectious diseases emerge frequently in china, partly because of its large and highly mobile population. therefore, a rapid and cost-effective pathogen screening method with broad coverage is required for prevention and control of infectious diseases. the availability of a large number of microbial genome sequences generated by conventional sanger sequencing and next generation sequencing has enabled the development of a high-throughput high-density microarray platform for rapid large-scale scr ...201324053492
towards the elements of successful insect rnai.rna interference (rnai), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. the accumulating body of literature on insect rnai has revealed that the efficiency of rnai varies between different species, the mode of rnai delivery, and the genes being targeted. there is also variation in the duration of transcript suppression. at present, we have a limited ca ...201324041495
previous exposure to an rna virus does not protect against subsequent infection in drosophila melanogaster.immune priming has been shown to occur in a wide array of invertebrate taxa, with individuals exposed to a pathogen showing increased protection upon subsequent exposure. however, the mechanisms underlying immune priming are poorly understood. the antiviral rnai response in drosophila melanogaster is an ideal candidate for providing a specific and acquired response to subsequent infection. we exposed d. melanogaster to two challenges of a virus known to produce an antiviral rnai response, to exa ...201324040086
rig-i and mda-5 detection of viral rna-dependent rna polymerase activity restricts positive-strand rna virus replication.type i interferons (ifn) are important for antiviral responses. melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-induced gene i (rig-i) proteins detect cytosolic double-stranded rna (dsrna) or 5'-triphosphate (5'-ppp) rna and mediate ifn production. cytosolic 5'-ppp rna and dsrna are generated during viral rna replication and transcription by viral rna replicases [rna-dependent rna polymerases (rdrp)]. here, we show that the semliki forest virus (sfv) rna replicase can induce ...201324039580
systemic tumor-specific gene delivery.the objective of a systemically administered cancer gene therapy is to achieve gene expression that is isolated to the tumor tissue. unfortunately, viral systems have strong affinity for the liver, and delivery from non-viral cationic systems often results in high expression in the lungs. non-specific delivery to these organs must be overcome if tumors are to be aggressively treated with genes such as il-12 which activates a tumor immune response, and tnf-alpha which can induce tumor cell apopto ...201324035974
host restriction factor screening: let the virus do the work.in this issue of cell host & microbe, varble et al. (2013) engineer a library of rna viruses to express small interfering rnas and couple this with the power of virus evolution and selection to screen for host genes that when silenced resulted in greater viral infection in vivo.024034609
seroprevalence of sindbis virus and associated risk factors in northern sweden.mosquito-borne sindbis virus (sinv) cause disease characterized by rash, fever and arthritis which often leads to long-lasting arthralgia. to determine the seroprevalence of sinv and associated risk factors in northern sweden, a randomly selected population aged between 25 and 74 years were invited to join the monica study. serum from 1611 samples were analysed for specific igg antibodies. overall, 2·9% had igg against sinv. more men (3·7%) than women (2·0%) were sinv seropositive (p = 0·047) an ...201424029159
sindbis viral vectors transiently deliver tumor-associated antigens to lymph nodes and elicit diversified antitumor cd8+ t-cell immunity.tumors are theoretically capable of eliciting an antitumor immune response, but are often poorly immunogenic. oncolytic viruses (ovs) have recently emerged as a promising strategy for the immunogenic delivery of tumor-associated antigens (taas) to cancer patients. however, safe and effective ov/taa therapies have not yet been established. we have previously demonstrated that vectors based on sindbis virus (sv) can inhibit tumor growth and activate the innate immune system in mice. here, we demon ...201324025748
emerging roles for immunomodulatory functions of free isg15.type i interferons (ifns) exert their effects through the induction of hundreds of ifn-stimulated genes (isgs), many of which function by inhibiting viral replication and modulating immune responses. isg15, a di-ubiquitin-like protein, is one of the most abundantly induced isgs and is critical for control of certain viral and bacterial infections. like ubiquitin, isg15 is covalently conjugated to target proteins. in addition, free unconjugated isg15 is present both intra- and extracellularly. al ...201324010825
functional specialization of the small interfering rna pathway in response to virus infection.in drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded rna (dsrna) is processed into small interfering rnas (sirnas) by dicer-2 (dcr-2) in association with a dsrna-binding protein (dsrbp) cofactor called loquacious (loqs-pd). sirnas are then loaded onto argonaute-2 (ago2) by the action of dcr-2 with another dsrbp cofactor called r2d2. loaded ago2 executes the destruction of target rnas that have sequence complementarity to sirnas. although dcr-2, r ...201324009507
the amino-terminal domain of alphavirus capsid protein is dispensable for viral particle assembly but regulates rna encapsidation through cooperative functions of its subdomains.venezuelan equine encephalitis virus (veev) is a pathogenic alphavirus, which circulates in the central, south, and north americas, including the united states, and represents a significant public health threat. in recent years, strong progress has been made in understanding the structure of veev virions, but the mechanism of their formation has yet to be investigated. in this study, we analyzed the functions of different capsid-specific domains and its amino-terminal subdomains in viral particl ...201324006447
encapsidation of host-derived factors correlates with enhanced infectivity of sindbis virus.the genus alphavirus consists of a group of enveloped, single-stranded rna viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. here we report that sindbis virus (sinv) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. in contrast, mosquito-derived sinv consists of a homogeneous population of particles. our findings indicate that the denser particle subp ...201324006438
discovery of host-viral protein complexes during infection.viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. therefore, for their efficient replication and spread, viruses depend on dynamic and temporally regulated interactions with host proteins. the rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. the resulting discoveries often lead to unique and innovative hypotheses ...023996249
autophagy in the immune system.autophagy is an intracellular homeostatic mechanism important for the degradation of waste components from the cytoplasm in acidic lysosomal compartments. originally, surplus parts of the cytoplasm that acted as targets for autophagy were thought to comprise cellular organelles and proteins, but this has now extended to include a range of pathogens with particular emphasis on intracellular bacteria. the finding that autophagy can sequester intracellular bacteria and mediate their destruction has ...023991647
replication vesicles are load- and choke-points in the hepatitis c virus lifecycle.hepatitis c virus (hcv) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. to understand how the virus establishes its tightly controlled intracellular rna replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular hcv rna replication. we therefore quantitatively measured viral rna and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly ...201323990783
chikungunya virus capsid protein contains nuclear import and export signals.chikungunya virus (chikv) is an alphavirus of the togaviridae family. after autoproteolytic cleavage, the chikv capsid protein (cp) is involved in rna binding and assembly of the viral particle. the monomeric cp is approximately 30 kda in size and is small enough for passive transport through nuclear pores. some alphaviruses are found to harbor nuclear localization signals (nls) and transport of these proteins between cellular compartments was shown to be energy dependent. the active nuclear imp ...201323984714
erk signaling couples nutrient status to antiviral defense in the insect gut.a unique facet of arthropod-borne virus (arbovirus) infection is that the pathogens are orally acquired by an insect vector during the taking of a blood meal, which directly links nutrient acquisition and pathogen challenge. we show that the nutrient responsive erk pathway is both induced by and restricts disparate arboviruses in drosophila intestines, providing insight into the molecular determinants of the antiviral "midgut barrier." wild-type flies are refractory to oral infection by arboviru ...201323980175
multi-gene detection and identification of mosquito-borne rna viruses using an oligonucleotide microarray.arthropod-borne viruses are important emerging pathogens world-wide. viruses transmitted by mosquitoes, such as dengue, yellow fever, and japanese encephalitis viruses, infect hundreds of millions of people and animals each year. global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. here, we report an oligonucleotide dna microarray design, termed arbochip5.1, for multi-gene detection and identifica ...201323967358
coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.no therapeutics or vaccines currently exist for human coronaviruses (hcovs). the severe acute respiratory syndrome-associated coronavirus (sars-cov) epidemic in 2002-2003, and the recent emergence of middle east respiratory syndrome coronavirus (mers-cov) in april 2012, emphasize the high probability of future zoonotic hcov emergence causing severe and lethal human disease. additionally, the resistance of sars-cov to ribavirin (rbv) demonstrates the need to define new targets for inhibition of c ...201323966862
a compact viral processing proteinase/ubiquitin hydrolase from the otu family.turnip yellow mosaic virus (tymv)--a member of the alphavirus-like supergroup of viruses--serves as a model system for positive-stranded rna virus membrane-bound replication. tymv encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (pro). we recently reported that pro is actually a multifunctional enzyme with a specific ubiquitin hydrolase (dub) activity that contributes to viral infectivity. here, we report the ...201323966860
bed bugs and infectious disease: a case for the arboviruses. 201323966852
detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification.early and rapid detection of dengue virus (denv) infection during the febrile period is crucial for proper patient management and prevention of disease spread. an easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.201323964963
inhibition of hiv-1 replication by secondary metabolites from endophytic fungi of desert plants.most antiretroviral drugs currently in use to treat an hiv-1 infection are chemically synthesized and lead to the development of viral resistance, as well as cause severe toxicities. however, a largely unexplored source for hiv-1 drug discovery is endophytic fungi that live in a symbiotic relationship with plants. these fungi produce biologically active secondary metabolites, which are natural products that are beneficial to the host. we prepared several hundred extracts from endophytic fungi of ...201323961302
potential roles of microglial cell progranulin in hiv-associated cns pathologies and neurocognitive impairment.progranulin (pgrn) is a highly unusual molecule with both neuronal and microglial expression with two seemingly unrelated functions, i.e., as a neuronal growth factor and a modulator of neuroinflammation. haploinsufficiency due to loss of function mutations lead to a fatal presenile dementing illness (frontotemporal lobar degeneration), indicating that adequate expression of pgrn is essential for successful aging. pgrn might be a particularly relevant factor in the pathogenesis of hivencephaliti ...023959579
methotrexate treatment causes early onset of disease in a mouse model of ross river virus-induced inflammatory disease through increased monocyte production.part of the togaviridae family, alphaviruses, including chikungunya virus (chikv), sindbis virus (sinv) and ross river virus (rrv), are able to cause significant inflammatory pathologies ranging from arthritis to encephalitis. following symptomatic infection with arthritis-associated alphaviruses, patients often experience severe joint pain, affecting distal and small joints, which can last six months or longer. recently, methotrexate (mtx), a disease modifying anti-rheumatic drug (dmard), was u ...201323951095
virus infection, antiviral immunity, and autoimmunity.as a group of disorders, autoimmunity ranks as the third most prevalent cause of morbidity and mortality in the western world. however, the etiology of most autoimmune diseases remains unknown. although genetic linkage studies support a critical underlying role for genetics, the geographic distribution of these disorders as well as the low concordance rates in monozygotic twins suggest that a combination of other factors including environmental ones are involved. virus infection is a primary fac ...023947356
norovirus genome circularization and efficient replication are facilitated by binding of pcbp2 and hnrnp a1.sequences and structures within the terminal genomic regions of plus-strand rna viruses are targets for the binding of host proteins that modulate functions such as translation, rna replication, and encapsidation. using murine norovirus 1 (mnv-1), we describe the presence of long-range rna-rna interactions that were stabilized by cellular proteins. the proteins potentially responsible for the stabilization were selected based on their ability to bind the mnv-1 genome and/or having been reported ...201323946460
therapeutic targeting of autophagy in disease: biology and pharmacology.autophagy, a process of self-digestion of the cytoplasm and organelles through which cellular components are recycled for reuse or energy production, is an evolutionarily conserved response to metabolic stress found in eukaryotes from yeast to mammals. it is noteworthy that autophagy is also associated with various pathophysiologic conditions in which this cellular process plays either a cytoprotective or cytopathic role in response to a variety of stresses such as metabolic, inflammatory, neuro ...201323943849
a proteomic perspective of inbuilt viral protein regulation: pul46 tegument protein is targeted for degradation by icp0 during herpes simplex virus type 1 infection.much like the host cells they infect, viruses must also regulate their life cycles. herpes simples virus type 1 (hsv-1), a prominent human pathogen, uses a promoter-rich genome in conjunction with multiple viral trans-activating factors. following entry into host cells, the virion-associated outer tegument proteins pul46 and pul47 act to increase expression of viral immediate-early (α) genes, thereby helping initiate the infection life cycle. because pul46 has gone largely unstudied, we employed ...201323938468
isolation of midgut escape mutants of two american genotype dengue 2 viruses from aedes aegypti.several studies have shown that american genotype dengue 2 viruses (denv2) have reduced viral fitness in the mosquito vector, aedes aegypti, compared to other denv2 genotypes. diminished replication efficiency or inability to efficiently traverse membrane barriers encompassing organs such as the midgut or salivary glands are considered major factors negatively impacting viral fitness in the mosquito.201323937713
diagnosis of barmah forest virus infection by a nested real-time sybr green rt-pcr assay.barmah forest virus (bfv) is a mosquito borne (+) ssrna alphavirus found only in australia. it causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. we developed a real-time pcr assay to detect bfv in an effort to improve diagnosis early in the course of infection. the limit of detection was 16 genome equivalents with a specificity of 100%. fifty five serum samples from bfv-infected patients were tested by the pcr. 52 of 53 antibody-positive samples were pcr negat ...201323935816
prolonged polyarthralgia in a german traveller with mayaro virus infection without inflammatory correlates.mayaro virus is endemic in south america and sporadic outbreaks have been described. it causes a dengue-like febrile illness accompanied by severe and long-lasting polyarthralgias. outside endemic regions, however, the disease is not well known and can be misdiagnosed as dengue. international travellers are at risk to acquire mayaro virus and due to increased worldwide travel infectious disease specialists need to be aware of such rare clinical entities.201323927600
directed evolution of a virus exclusively utilizing human epidermal growth factor receptor as the entry receptor.rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. here we describe a directed-evolution strategy, named the tandem selection and enrichment system (tses), and its use in generating virus with exclusive specificity for a particular cellular receptor. in tses, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of ...201323926357
comprehensive mapping of common immunodominant epitopes in the eastern equine encephalitis virus e2 protein recognized by avian antibody responses.eastern equine encephalitis virus (eeev) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. eeev can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. the e2 protein of eeev and other alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. while many therapeutic and diagnostic applications of e2 protein-specific antibodies have been reported, the ...201323922704
fusion of mapple and venus fluorescent proteins to the sindbis virus e2 protein leads to different cell-binding properties.fluorescent proteins (fps) are widely used in real-time single virus particle studies to visualize, track and quantify the spatial and temporal parameters of viral pathways. however, potential functional differences between the wild type and the fp-tagged virus may specifically affect particular stages in the virus life-cycle. in this work, we genetically modified the e2 spike protein of sindbis virus (sinv) with two fps. we inserted mapple, a red fp, or venus, a yellow fp, at the n-terminus of ...201323916968
a prolyl-isomerase mediates dopamine-dependent plasticity and cocaine motor sensitization.synaptic plasticity induced by cocaine and other drugs underlies addiction. here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. in response to d1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mglur5) is phosphorylated by microtubule-associated protein kinase (mapk), which we show potentiates pin1-mediated prolyl-isomerization of m ...023911326
delivery systems for gene therapy.the structure of dna was unraveled by watson and crick in 1953, and two decades later arber, nathans and smith discovered dna restriction enzymes, which led to the rapid growth in the field of recombinant dna technology. from expressing cloned genes in bacteria to expressing foreign dna in transgenic animals, dna is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. gene therapy provides ...023901186
characterization of an early-stage fusion intermediate of sindbis virus using cryoelectron microscopy.the sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein e1 in conjunction with biochemical studies. however, the molecular structures of the hypothesized fusion intermediates have remained obscure due to difficulties inherent in the dynamic nature of the process. we developed an experimental system that uses liposomes as the target membrane to capture sindbis virus, a prototypical alp ...201323898184
the small gtpase arf1 modulates arp2/3-mediated actin polymerization via pick1 to regulate synaptic plasticity.inhibition of arp2/3-mediated actin polymerization by pick1 is a central mechanism to ampa receptor (ampar) internalization and long-term depression (ltd), although the signaling pathways that modulate this process in response to nmda receptor (nmdar) activation are unknown. here, we define a function for the gtpase arf1 in this process. we show that arf1-gtp binds pick1 to limit pick1-mediated inhibition of arp2/3 activity. expression of mutant arf1 that does not bind pick1 leads to reduced sur ...023889934
optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (cali).optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. in the current study, we engineered an optogenetic technique that directly inhibits neurotransmitter release. we used a genetically encoded singlet oxygen generator, minisog, to conduct chromophore assisted light inactivation (cali) of synaptic proteins. fusions of minisog to vamp2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. in c ...023889931
metabolism of amyloid β peptide and pathogenesis of alzheimer's disease.the conversion of what has been interpreted as "normal brain aging" to alzheimer's disease (ad) via transition states, i.e., preclinical ad and mild cognitive impairment, appears to be a continuous process caused primarily by aging-dependent accumulation of amyloid β peptide (aβ) in the brain. this notion however gives us a hope that, by manipulating the aβ levels in the brain, we may be able not only to prevent and cure the disease but also to partially control some very significant aspects of ...023883611
map1b-dependent rac activation is required for ampa receptor endocytosis during long-term depression.the microtubule-associated protein 1b (map1b) plays critical roles in neurite growth and synapse maturation during brain development. this protein is well expressed in the adult brain. however, its function in mature neurons remains unknown. we have used a genetically modified mouse model and shrna techniques to assess the role of map1b at established synapses, bypassing map1b functions during neuronal development. under these conditions, we found that map1b deficiency alters synaptic plasticity ...201323881099
translation of viral mrnas that do not require eif4e is blocked by the inhibitor 4egi-1.high throughput screening has rendered new inhibitors of eukaryotic protein synthesis. one such molecule, 4egi-1 has been reported to selectively block the initiation factor eif4e. we have investigated the action of this inhibitor on translation directed by several viral mrnas which, in principle, do not utilize eif4e. we found that 4egi-1 inhibits translation directed by poliovirus ires, in rabbit reticulocyte lysates, to a similar extent as capped mrna. moreover, 4egi-1 inhibits translation dr ...201323870416
magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of semliki forest virus.alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. during the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. in addition to viral components, these organelles harbor an unknown number of host proteins. in this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was ...201323864636
the c-terminal domain of chikungunya virus nsp2 independently governs viral rna replication, cytopathicity, and inhibition of interferon signaling.alphavirus nonstructural protein 2 (nsp2) has pivotal roles in viral rna replication, host cell shutoff, and inhibition of antiviral responses. mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsp2. results show that (i) nsp2 mutation p718s only in combination with kr649aa or adaptive mutation d711g allowed noncytopathic replicon rna replication, (ii) prohibiting nsp2 nuclear localization abrogates inhibition of antiviral interferon-ind ...201323864632
the role of e3 in ph protection during alphavirus assembly and exit.alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of e2/e1 glycoprotein heterodimers. during virus entry, the e2/e1 dimer dissociates within the acidic endosomal environment, freeing the e1 protein to mediate fusion of the viral and endosome membranes. e2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature e2 and a small peripheral glycoprotein, e3. the immature p62/e1 dimer is acid resistant, ...201323864626
presentation overrides specificity: probing the plasticity of alphaviral proteolytic activity through mutational analysis.semliki forest virus (genus alphavirus) is an important model for studying regulated nonstructural (ns) polyprotein processing. in this study, we evaluated the strictness of the previously outlined cleavage rules, accounting for the timing and outcome of each of three cleavages within the ns polyprotein p1234, and assessed the significance of residues p6 to p4 within the cleavage sites using an alanine scanning approach. the processing of the 1/2 and 3/4 sites was most strongly affected followin ...201323864614
statistical analysis of sizes and shapes of virus capsids and their resulting elastic properties.from the analysis of sizes of approximately 130 small icosahedral viruses we find that there is a typical structural capsid protein, having a mean diameter of 5 nm and a mean thickness of 3 nm, with more than two thirds of the analyzed capsid proteins having thicknesses between 2 nm and 4 nm. to investigate whether, in addition to the fairly conserved geometry, capsid proteins show similarities in the way they interact with one another, we examined the shapes of the capsids in detail. we classif ...201323860870
viral vectors for vaccine applications.traditional approach of inactivated or live-attenuated vaccine immunization has resulted in impressive success in the reduction and control of infectious disease outbreaks. however, many pathogens remain less amenable to deal with the traditional vaccine strategies, and more appropriate vaccine strategy is in need. recent discoveries that led to increased understanding of viral molecular biology and genetics has rendered the used of viruses as vaccine platforms and as potential anti-cancer agent ...201323858400
virus replicon particle based chikungunya virus neutralization assay using gaussia luciferase as readout.chikungunya virus (chikv) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. so far, no specific treatment or vaccine is available. as nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for chikv diagnosis and for determination of the immune status of a patient. furthermore, neutralization assays represent a useful tool to validate the efficacy of poten ...201323855906
inhibition of hepatitis b virus replication by the host zinc finger antiviral protein.the zinc finger antiviral protein (zap) is a mammalian host restriction factor that inhibits the replication of a variety of rna viruses, including retroviruses, alphaviruses and filoviruses, through interaction with the zap-responsive elements (zre) in viral rna, and recruiting the exosome to degrade rna substrate. hepatitis b virus (hbv) is a pararetrovirus that replicates its genomic dna via reverse transcription of a viral pregenomic (pg) rna precursor. here, we demonstrate that the two isof ...201323853601
viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways.early innate and cell-intrinsic responses are essential to protect host cells against pathogens. in turn, viruses have developed sophisticated mechanisms to establish productive infections by counteracting host innate immune responses. increasing evidence indicates that these antiviral factors may have a dual role by directly inhibiting viral replication as well as by sensing and transmitting signals to induce antiviral cytokines. recent studies have pointed at new, unappreciated mechanisms of v ...201323850008
recombinant varicella-zoster virus vaccines as platforms for expression of foreign antigens.varicella-zoster virus (vzv) vaccines induce immunity against childhood chickenpox and against shingles in older adults. the safety, efficacy, and widespread use of vzv vaccines suggest that they may also be effective as recombinant vaccines against other infectious diseases that affect the young and the elderly. the generation of recombinant vzv vaccines and their evaluation in animal models are reviewed. the potential advantages and limitations of recombinant vzv vaccines are addressed.201323843791
heterogeneous nuclear ribonucleoprotein k supports vesicular stomatitis virus replication by regulating cell survival and cellular gene expression.the heterogeneous nuclear ribonucleoprotein k (hnrnp k) is a member of the family of hnrnps and was recently shown in a genome-wide small interfering rna (sirna) screen to support vesicular stomatitis virus (vsv) growth. to decipher the role of hnrnp k in vsv infection, we conducted studies which suggest that the protein is required for vsv spreading. virus binding to cells, entry, and nucleocapsid uncoating steps were not adversely affected in the absence of hnrnp k, whereas viral genome transc ...201323843646
amalgamating oncolytic viruses to enhance their safety, consolidate their killing mechanisms, and accelerate their spread.oncolytic viruses are structurally and biologically diverse, spreading through tumors and killing them by various mechanisms and with different kinetics. here, we created a hybrid vesicular stomatitis/measles virus (vsv/mv) that harnesses the safety of oncolytic mv, the speed of vsv, and the tumor killing mechanisms of both viruses. oncolytic mv targets cd46 and kills by forcing infected cells to fuse with uninfected neighbors, but propagates slowly. vsv spreads rapidly, directly lysing tumor ce ...201323842448
active vaccination with vaccinia virus a33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.vaccinia virus protein a33 (a33vacv) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. in this study we show that single-dose vaccination with recombinant sindbis virus expressing a33vacv, is sufficient to protect mice against lethal challenge with vaccinia virus wr (vacv-wr) and ectromelia virus (ectv) but not against cowpox virus (cpxv), a closely related orthopoxvirus. moreover, a subunit vaccine based on the ...201323842430
mhc class ii-alpha chain knockout mice support increased viral replication that is independent of their lack of mhc class ii cell surface expression and associated immune function deficiencies.mhcii molecules are heterodimeric cell surface proteins composed of an α and β chain. these molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (apcs) and play a central role in the development and function of cd4 t cells. various mhc-ii knockout mice have been generated including mhc-iiaα(-/-) (i-aα(-/-)), mhc-iiaβ(-/-) (i-β(-/-)) and the double knockout (i-aαxβ(-/-)). here we report a very striking observation, namely that alphaviruses including the avi ...201323840854
visualization of dc-sign-mediated entry pathway of engineered lentiviral vectors in target cells.dendritic cells (dcs) are potent antigen-presenting cells and therefore have enormous potential as vaccine targets. we have previously developed an engineered lentiviral vector (lv) that is pseudotyped with a mutated sindbis virus glycoprotein (svgmu), which is capable of targeting dcs through dendritic cell-specific icam3-grabbing nonintegrin (dc-sign), a receptor that is predominantly expressed by dcs. in this study, we aimed to elucidate the internalization and trafficking mechanisms of this ...201323840690
specific nucleotide changes in the subgenomic promoter region influence infectivity of the sindbis virus.transcription of the subgenomic mrna of sindbis virus (sinv) is initiated at a subgenomic promoter (sp). alignment of sinv sequences identified a 68-nucleotide conserved domain spanning -19 to +49 relative to the subgenomic mrna start site. nucleotide t or c is present at -18 or +49 in all known sinvs while a sindbis-like virus xj-160 has an a or t at a corresponding position. our results indicate that deletion or substitution of the t at +49 decreased the activity of sp, while substituting t fo ...201323838061
degradation of high affinity hud targets releases kv1.1 mrna from mir-129 repression by mtorc1.little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. we provide evidence for a novel mechanism for mtorc1 kinase-dependent translational regulation of the voltage-gated potassium channel kv1.1 messenger rna (mrna). we identified a microrna, mir-129, that repressed kv1.1 mrna translation when mtorc1 was active. when mtorc1 was inactive, we found that the rna-binding protein, hud, bound to kv1.1 mrna and promoted its translation. un ...023836929
monitoring neuronal excitability at the synapse.mammalian target of rapamycin (mtor) is a key player at the synapse regulating local translation and long-lasting synaptic plasticity. now, a new study by sosanya et al. (2013. j. cell biol. http://dx.doi.org/10.1083/jcb.201212089) investigates the molecular mechanism of how mtor suppresses local protein synthesis of a key potassium channel at activated synapses.023836926
beclin 1 and nuclear factor-κbp65 are upregulated in hepatocellular carcinoma.there are no sensitive and specific biomarkers that aid in the clinical diagnosis and prognosis of hepatocellular carcinoma (hcc). the aim of the present study was to determine the mrna and protein expression levels of beclin 1 (becn1) and nuclear factor-κb (nf-κb)p65 in patients with hcc, to evaluate their value as potential diagnostic and prognostic biomarkers. immunohistochemistry and in situ hybridization were used to detect the expression of hepatic becn1 and nf-kbp65 in patients with hcc, ...201323833647
neuro-invasion of chandipura virus mediates pathogenesis in experimentally infected mice.neuro-tropism is a major feature in many viral infections. chandipura virus produces neurological symptoms in naturally infected young children and experimentally infected suckling mice. this study was undertaken to find out the neuro-invasive behaviour of chandipura virus in suckling mice. the suckling mice were infected with the virus via footpad injection. different tissues were collected at 24-h intervals up to 96-h post infection and processed for virus quantification and histological study ...201323826408
reconstruction of h3n2 influenza virus based virosome in-vitro.virosomes are virus like particles (vlp) assembled in-vitro. influenza virosomes maintain the cell binding and membrane fusion activity of the wild type virus but are devoid of viral genetic material or internal proteins. influenza virosomes mimic the natural antigen presentation route of the influenza virus.023825736
zebrafish isg15 exerts a strong antiviral activity against rna and dna viruses and regulates the interferon response.isg15, a 15-kda interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. in fish, as in mammals, viral infection and interferon treatment induce isg15 expression. the two ubiquitin-like domains of isg15 and the presence of a consensus lrlrgg sequence in the c-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. our data demonstrate that overexpression of zebrafish isg15 (zf ...201323824820
flaviviruses are sensitive to inhibition of thymidine synthesis pathways.dengue virus has emerged as a global health threat to over one-third of humankind. as a positive-strand rna virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. in a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and f ...201323824813
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