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etiological agents of viral meningitis in children from a dengue-endemic area, southeast region of brazil.meningitis is a disease with a global distribution that constitutes a worldwide burden, with viruses as the primary etiologic agents. the range of viral meningitis severity depends mainly on age, immune status and etiological agent. the aim of this work was to investigate the suspected cases of viral meningitis using molecular techniques to confirm the viral infection. the diagnosed virus was correlated with clinical findings and cytochemical parameters in cerebrospinal liquid (csf) of patients. ...201728320174
emergence of a viral rna polymerase variant during gene copy number amplification promotes rapid evolution of vaccinia virus.viruses are under relentless selective pressure from host immune defenses. to study how poxviruses adapt to innate immune detection pathways, we performed serial vaccinia virus infections in primary human cells. independent courses of experimental evolution with a recombinant strain lacking e3l revealed several high-frequency point mutations in conserved poxvirus genes, suggesting important roles for essential poxvirus proteins in innate immune subversion. two distinct mutations were identified ...201727928012
evidence that hsc70 is associated with cucumber necrosis virus particles and plays a role in particle disassembly.uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. in the present study, we investigated the role of plant hsp70 homologs in the uncoating process of cucumber necrosis virus (cnv), a nonenveloped positive-sense single-stranded rna [(+)ssrna] virus having a t=3 icosahedral capsid. we have found through western blot analysis and mass spectrometry that the hsp70 homolog hsc70-2 copur ...201727807229
retinal pigment epithelial cell expression of active rap 1a by scaav2 inhibits choroidal neovascularization.to test the hypothesis that increased rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (cnv), self-complementary adeno-associated virus 2 (scaav2) with rpe65-promoter-driven gfp vectors were generated and introduced subretinally into rap1b-deficient mice. six-week-old mice that received subretinal control (scaav2-con) or constitutively active rap1a (scaav2-carap1a) showed strong gfp at the 5 × 10(8) viral particle/µl dose 5 weeks later without ...201627606349
copy number variation of genes involved in the hepatitis c virus-human interactome.copy number variation (cnv) is a newly discovered form of intra-species genetic polymorphism that is defined as deletions or duplications of genome segments ranging from 1 kbp to several mbp. cnv accounts for the majority of the genetic variation observed in humans (cnv regions cover more than 10% of the human genome); therefore, it may significantly influence both the phenotype and susceptibility to various diseases. unfortunately, the impact of cnv on a number of diseases, including hepatitis ...201627510840
a multifaceted computational report on the variants effect on kir2dl3 and ifnl3 candidate gene in hcv clearance.hcv infection causes acute and chronic liver diseases including, cirrhosis and hepatocellular carcinoma. following hcv infection, spontaneous clearance occurs in approximately 20 % of the population dependant upon hcv genotype. in this study, functional and non-functional variant analysis was executed for the classical and the latest hcv clearance candidate genes namely, kir2dl3 and ifnl3. initially, the functional effects of non-synonymous snps were assigned on exposing to homology based tools, ...201627461217
a new member of the family totiviridae associated with arboreal ants (camponotus nipponicus).a putative new member of the family totiviridae was identified in arboreal ants (camponotus nipponicus). the viral dsrna consisted of 5,713 nt with two overlapping open reading frames (orfs). orf1 encodes a putative capsid protein. orf2 encodes a viral rna-dependent rna polymerase (rdrp). orf2 could be translated as a fusion protein with the orf1 product through a -1 frameshift in the overlapping orf1. phylogenetic analysis based on the rdrp revealed that the virus from c. nipponicus is closely ...201627138551
biofilter as a functional annotation pipeline for common and rare copy number burden.recent studies on copy number variation (cnv) have suggested that an increasing burden of cnvs is associated with susceptibility or resistance to disease. a large number of genes or genomic loci contribute to complex diseases such as autism. thus, total genomic copy number burden, as an accumulation of copy number change, is a meaningful measure of genomic instability to identify the association between global genetic effects and phenotypes of interest. however, no systematic annotation pipeline ...201626776200
subretinal aav2.comp-ang1 suppresses choroidal neovascularization and vascular endothelial growth factor in a murine model of age-related macular degeneration.to assess whether tie2-mediated vascular stabilization ameliorates neovascular age-related macular degeneration (amd), we investigated the impact of adeno-associated virus-mediated gene therapy with cartilage oligomeric matrix protein angiopoietin-1 (aav2.comp-ang1) on choroidal neovascularization (cnv), vascular endothelial growth factor (vegf), and hypoxia-inducible factor (hif) in a mouse model of the disease. we treated mice with subretinal injections of aav2.comp-ang1 or control (aav2.acgfp ...201626775053
gene copy number variations in the leukocyte genome of hepatocellular carcinoma patients with integrated hepatitis b virus dna.integration of hepatitis b virus (hbv) dna into the human liver cell genome is believed to promote hbv-related carcinogenesis. this study aimed to quantify the integration of hbv dna into the leukocyte genome in hepatocellular carcinoma (hcc) patients in order to identify potential biomarkers for hbv-related diseases. whole-genome comparative genomic hybridization (cgh) chip array analyses were performed to screen gene copy number variations (cnv) in the leukocyte genome, and the results were co ...201626769853
pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from chandipura and piry viruses.while the envelope glycoprotein of vesicular stomatitis virus (vsv-g) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. to find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from chandipura (cnv-g) and piry (prv-g) vesiculoviruses. both permitted generation of high titer psuedotyped lentiviral vectors ...201626650691
cucumber necrosis virus recruits cellular heat shock protein 70 homologs at several stages of infection.rna viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. one such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (hsp70) family homologs which have been shown to play roles for different viruses in viral rna replication, viral assembly, disassembly, and cell-to-cell movement. using next generation sequence analysis, we reveal that several isoforms of hsp70 a ...201626719261
copy number variation in cep57l1 predisposes to congenital absence of bilateral acl and pcl ligaments.absence of the anterior (acl) or posterior cruciate ligament (pcl) are rare congenital malformations that result in knee joint instability, with a prevalence of 1.7 per 100,000 live births and can be associated with other lower-limb abnormalities such as acl agnesia and absence of the menisci of the knee. while a few cases of absence of acl/pcl are reported in the literature, a number of large familial case series of related conditions such as acl agnesia suggest a potential underlying monogenic ...201526561035
genome-wide identification of copy number variations between two chicken lines that differ in genetic resistance to marek's disease.copy number variation (cnv) is a major source of genome polymorphism that directly contributes to phenotypic variation such as resistance to infectious diseases. lines 63 and 72 are two highly inbred experimental chicken lines that differ greatly in susceptibility to marek's disease (md), and have been used extensively in efforts to identify the genetic and molecular basis for genetic resistance to md. using next generation sequencing, we present a genome-wide assessment of cnvs that are potenti ...201526492869
the role of magt1 in genetic syndromes.disturbances in magnesium homeostasis, often linked to altered expression and/or function of magnesium channels, have been implicated in a plethora of diseases. this review focuses on magnesium transporter 1 (magt1), as recently described changes in this gene have further extended our understanding of the role of magnesium in human health and disease. the identification of genetic changes and their functional consequences in patients with immunodeficiency revealed that magnesium and magt1 are ke ...201526422833
encapsidation of host rnas by cucumber necrosis virus coat protein during both agroinfiltration and infection.next-generation sequence analysis of virus-like particles (vlps) produced during agroinfiltration of cucumber necrosis virus (cnv) coat protein (cp) and of authentic cnv virions was conducted to assess if host rnas can be encapsidated by cnv cp. vlps containing host rnas were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that cnv virions accumulated during infection. vlps contained a variety of host rna species, including the major rrnas as well as cy ...201526269190
aav2 delivery of flt23k intraceptors inhibits murine choroidal neovascularization.long-term inhibition of extracellular vascular endothelial growth factor (vegf) in the treatment of age-related macular degeneration (amd) may induce retinal neuronal toxicity and risk other side effects. we developed a novel strategy which inhibits retinal pigment epithelium (rpe)-derived vegf, sparing other highly sensitive retinal tissues. flt23k, an intraceptor inhibitor of vegf, was able to inhibit vegf in vitro. adeno-associated virus type 2 (aav2)-mediated expression of flt23k was maintai ...201525306972
genogroup iv and vi canine noroviruses interact with histo-blood group antigens.human noroviruses (hunv) are a significant cause of viral gastroenteritis in humans worldwide. hunv attaches to cell surface carbohydrate structures known as histo-blood group antigens (hbgas) prior to internalization, and hbga polymorphism among human populations is closely linked to susceptibility to hunv. noroviruses are divided into 6 genogroups, with human strains grouped into genogroups i (gi), ii, and iv. canine norovirus (cnv) is a recently discovered pathogen in dogs, with strains class ...201425008923
development of gene therapy for treatment of age-related macular degeneration.intraocular neovascular diseases are the leading cause of blindness in the western world in individuals over the age of 50. age-related macular degeneration (amd) is one of these diseases. exudative amd, the late-stage form, is characterized by abnormal neovessel development, sprouting from the choroid into the avascular subretinal space, where it can suddenly cause irreversible damage to the vulnerable photoreceptor (pr) cells essential for our high-resolution, central vision. the molecular bas ...201424953666
adeno-associated virus type 8 vector-mediated expression of sirna targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model.to assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (cnv), we generated an adeno-associated virus type 8 vector (aav2/8) encoding an sirna targeting vascular endothelial growth factor (vegf), and determined the aav2/8 vector's ability to inhibit angiogenesis.201424744609
messenger rna- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.the use of synthetic messenger rnas to generate human induced pluripotent stem cells (ipscs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of dna-based or virus-based reprogramming strategies, including transgene integration in particular. we compared the genomic integrity of mrna-derived ipscs with that of retrovirus-derived ipscs generated in strictly comparable conditions, by single-nucleotide polymorphism (snp) and copy ...201424736403
the p33 auxiliary replicase protein of cucumber necrosis virus targets peroxisomes and infection induces de novo peroxisome formation from the endoplasmic reticulum.tombusviruses replicate on pre-existing organelles such as peroxisomes or mitochondria, the membranes of which become extensively reorganized into multivesicular bodies (mvbs) during the infection process. cucumber necrosis virus (cnv) has previously been shown to replicate in association with peroxisomes in yeast. we show that cnv induces mvbs from peroxisomes in infected plants and that gfp-tagged p33 auxiliary replicase protein colocalizes with yfp(skl), a peroxisomal marker. most remarkably, ...201424606690
the hop-like stress-induced protein 1 cochaperone is a novel cell-intrinsic restriction factor for mitochondrial tombusvirus replication.recent genome-wide screens reveal that the host cells express an arsenal of proteins that inhibit replication of plus-stranded rna viruses by functioning as cell-intrinsic restriction factors of viral infections. one group of cell-intrinsic restriction factors against tombusviruses contains tetratricopeptide repeat (tpr) domains that directly interact with the viral replication proteins. in this paper, we find that the tpr domain-containing hop-like stress-inducible protein 1 (sti1p) cochaperone ...201424920799
the effect of functional mapkapk2 copy number variation cnv-30450 on elevating nasopharyngeal carcinoma risk is modulated by ebv infection.mitogen-activated protein kinase-activated protein kinase 2 (mapkapk2) is recognized as oncogenic and simulative role on tumorigenesis by virtue of abnormal expression in cancer including nasopharyngeal carcinoma (npc). we hypothesized that the copy number variation (cnv)-30450, which duplicates the mapkapk2 promoter, may affect mapkapk2 expression and be associated with npc risk. in two independent case-control panels of southern and eastern chinese with a total of 1590 npc patients and 1979 ca ...201424056810
screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in göttingen minipigs.to establish the safety of xenotransplantation when cells, tissues, or organs of pigs are used, an effective screening for potential zoonotic microorganisms has to be performed. in doing so, special attendance has to be paid to porcine endogenous retroviruses (pervs) that are widely distributed as proviruses in the genome of pigs. perv-a and perv-b are present in all pigs, they infect human cells in vitro and therefore represent a direct risk. perv-c infects only pig cells; however, recombinant ...201423551867
kir2dl4 copy number variation is associated with cd4+ t-cell depletion and function of cytokine-producing nk cell subsets in siv-infected mamu-a*01-negative rhesus macaques.here, we demonstrate that kir2dl4 copy number variation (cnv) is associated with cd4(+) t-cell decline and functionality of cytokine-producing nk cells during primary simian immunodeficiency virus (siv) infection in mamu-a*01(-) indian-origin rhesus macaques, with higher kir2dl4 copy numbers being associated with a better preservation of cd4(+) t cells and an increased gamma interferon (ifn-γ) production from stimulated cytokine-producing nk cell subsets during acute sivmac251 infection. these f ...201323449795
epstein-barr virus latent membrane protein 1 increases genomic instability through egr-1-mediated up-regulation of activation-induced cytidine deaminase in b-cell lymphoma.epstein-barr virus (ebv)-encoded latent membrane protein-1 (lmp1) is a transmembrane protein essential for ebv-induced immortalization and transformation of b cells. activation-induced cytidine deaminase (aid) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. here, we report an intracellular mechanism by which lmp1 contributes to b cell lymphomagenesis via aid expression. in our experiments, lmp1 increased aid mrna expression and promoter activity. the ai ...201323363221
characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly.the assembly of the viral replicase complex (vrc) on subcellular membranes is a key step in the replication process of plus-stranded rna viruses. in this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of cucumber necrosis virus, a tombusvirus. mutations within the p33:p33/p92 interaction domain also affected viral rna recombination in yeast model host. an in vitro approach base ...201323332599
a survey of resistance to tomato bushy stunt virus in the genus nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.in this study, we screened 22 nicotiana spp. for resistance to the tombusviruses tomato bushy stunt virus (tbsv), cucumber necrosis virus, and cymbidium ringspot virus. eighteen species were resistant, and resistance was manifested in at least two different categories. in all, 13 species responded with a hypersensitive response (hr)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. three diffe ...201323075040
atomic structure of cucumber necrosis virus and the role of the capsid in vector transmission.cucumber necrosis virus (cnv) is a member of the genus tombusvirus and has a monopartite positive-sense rna genome packaged in a t=3 icosahedral particle. cnv is transmitted in nature via zoospores of the fungus olpidium bornovanus. cnv undergoes a conformational change upon binding to the zoospore that is required for transmission, and specific polysaccharides on the zoospore surface have been implicated in binding. to better understand this transmission process, we have determined the atomic s ...201324006433
proliferation and copy number variation of bel-like long terminal repeat retrotransposons within the diabrotica virgifera virgifera genome.the proliferation of retrotransposons within a genome can contribute to increased size and affect the function of eukaryotic genes. bel/pao-like long-terminal repeat (ltr) retrotransposons were annotated from the highly adaptable insect species diabrotica virgifera virgifera, the western corn rootworm, using survey sequences from bacterial artificial chromosome (bac) inserts and contigs derived from a low coverage next-generation genome sequence assembly. eleven unique d. v. virgifera bel elemen ...201324513336
serological evidence for multiple strains of canine norovirus in the uk dog population.noroviruses are associated with intestinal disease in humans, cows, pigs, mice, and, more recently, dogs. in 2007, the first canine norovirus (cnv) was identified and characterized in italy. subsequent studies have identified cnv in stools of dogs from portugal, greece, and the united states. to investigate the prevalence of cnv in the uk dog population, 228 canine stool samples were screened for cnv by qpcr, and 396 serum samples were screened for anti-cnv antibodies. qpcr of rna extracted from ...201324339947
macrophage activation associated with chronic murine cytomegalovirus infection results in more severe experimental choroidal neovascularization.the neovascular (wet) form of age-related macular degeneration (amd) leads to vision loss due to choroidal neovascularization (cnv). since macrophages are important in cnv development, and cytomegalovirus (cmv)-specific igg serum titers in patients with wet amd are elevated, we hypothesized that chronic cmv infection contributes to wet amd, possibly by pro-angiogenic macrophage activation. this hypothesis was tested using an established mouse model of experimental cnv. at 6 days, 6 weeks, or 12 ...201222570607
ccl3l gene copy number and survival in an hiv-1 infected zimbabwean population.the c-c motif chemokine ligand 3-like (ccl3l) protein is a potent chemoattractant which by binding to c-c chemokine receptor type 5 (ccr5) inhibits human immunodeficiency virus (hiv) entry. copy number variation (cnv) of the ccl3l has been shown to be associated with hiv susceptibility and progression to aids, but these results have been inconsistent. we examined a zimbabwean study population for an association of ccl3l cnv with hiv status, progression (cd4 t-cells and viral load), and survival. ...201222484760
morphometric analysis of postoperative corneal neovascularization after high-risk keratoplasty: herpetic versus non-herpetic disease.postoperative complications after high-risk corneal grafting are decisively associated with corneal neovascularization (cnv). this study aimed to identify the incidence, extent, speed, localization, and influence of surgery-related factors on cnv after high-risk penetrating keratoplasty (pk) and to evaluate the effect of removing the angiogenic stimulus, i.e., residual components of herpes simplex virus (hsv) on postkeratoplasty cnv in patients with herpetic stromal keratitis (hsk).201222419035
micrornas in human lymphoblastoid cell lines.human lymphoblastoid cell lines (lcls) are generated by ebv-mediated b-cell transformation to provide unlimited genomic resources for human genetics and immunological studies. the lcl is a good in vitro cell model for assessing population differences in the basal expression of genes and mirnas as well as in cellular responses to various stimulators. recently, the utility of lcls was extended to pharmacogenomic studies to discover genetic factors underlying individual variations in response to ch ...201223140160
inhibition of choroidal and corneal pathologic neovascularization by plgf1-de gene transfer.ocular neovascularization (nv), the primary cause of blindness, typically is treated via inhibition of vegf-a activity. however, besides vegf-a, other proteins of the same family, including vegf-b and placental growth factor (plgf, all together vegfs), have a crucial role in the angiogenesis process. plgf and vegf, which form heterodimers if co-expressed, both are required for pathologic angiogenesis. we generated a plgf1 variant, named plgf1-de, which is unable to bind and activate vegfr-1, but ...201223139276
reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin rna.strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (amd). the present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (vegf), which is identified as the key player in exudative amd.201223080553
a worldwide analysis of beta-defensin copy number variation suggests recent selection of a high-expressing defb103 gene copy in east asia.beta-defensins are a family of multifunctional genes with roles in defense against pathogens, reproduction, and pigmentation. in humans, six beta-defensin genes are clustered in a repeated region which is copy-number variable (cnv) as a block, with a diploid copy number between 1 and 12. the role in host defense makes the evolutionary history of this cnv particularly interesting, because morbidity due to infectious disease is likely to have been an important selective force in human evolution, a ...201121387465
a non membrane-targeted human soluble cd59 attenuates choroidal neovascularization in a model of age related macular degeneration.age related macular degeneration (amd) is the most common cause of blindness amongst the elderly. approximately 10% of amd patients suffer from an advanced form of amd characterized by choroidal neovascularization (cnv). recent evidence implicates a significant role for complement in the pathogenesis of amd. activation of complement terminates in the incorporation of the membrane attack complex (mac) in biological membranes and subsequent cell lysis. elevated levels of mac have been documented o ...201121552568
Targeted decorin gene therapy delivered with adeno-associated virus effectively retards corneal neovascularization in vivo.Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×10 ...201122039486
comparative analysis of the capacity of tombusvirus p22 and p19 proteins to function as avirulence determinants in nicotiana species.we have used an agroinfiltration assay for a comparative study of the roles of tombusvirus p22 and p19 proteins in elicitation of hypersensitive response (hr)-like necrosis and the role of p19 in silencing suppression in nicotiana species. the advantage of agroinfiltration rather than expression in plant virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. we found that tombusvirus p2 ...201120977306
genetic variations at loci involved in the immune response are risk factors for hepatocellular carcinoma.primary liver cancer is the third most common cause of cancer-related death worldwide, with a rising incidence in western countries. little is known about the genetic etiology of this disease. to identify genetic factors associated with hepatocellular carcinoma (hcc) and liver cirrhosis (lc), we conducted a comprehensive, genome-wide variation analysis in a population of unrelated asian individuals. copy number variation (cnv) and single nucleotide polymorphisms (snps) were assayed in peripheral ...201021105107
cucumber necrosis virus p20 is a viral suppressor of rna silencing.the p20 protein encoded by the tombusvirus, cucumber necrosis virus has previously been shown to be involved in host pathogenicity and shares sequence similarity with the tomato bushy stunt virus p19 suppressor of silencing. using a virus-induced gene silencing (vigs) assay, we show that p20 is a viral suppressor of rna silencing (vsr) in infected plants. in addition, a cnv p20-knockout mutant showed a decline in viral rna accumulation in infected plants, consistent with the role of p20 in suppr ...201021144871
contributions of mamu-a*01 status and trim5 allele expression, but not ccl3l copy number variation, to the control of sivmac251 replication in indian-origin rhesus monkeys.ccl3 is a ligand for the hiv-1 co-receptor ccr5. there have recently been conflicting reports in the literature concerning whether ccl3-like gene (ccl3l) copy number variation (cnv) is associated with resistance to hiv-1 acquisition and with both viral load and disease progression following infection with hiv-1. an association has also been reported between ccl3l cnv and clinical sequelae of the simian immunodeficiency virus (siv) infection in vivo in rhesus monkeys. the present study was initia ...201020585621
distinct regions at the n-terminus of the cucumber necrosis virus coat protein target chloroplasts and mitochondria.cucumber necrosis virus (cnv) is a spherical virus consisting of 180 identical coat protein (cp) subunits. the n-terminus of the cp subunit contains a 58aa rna binding (r) domain and a 34aa arm that connects the r domain to the shell. these regions are known to play critical roles in virus assembly and disassembly. it has recently been shown that a region encompassing the arm can function as a chloroplast transit peptide (tp) in infected plants and that targeting may represent a means for virus ...201020600385
copy number variation in chemokine superfamily: the complex scene of ccl3l-ccl4l genes in health and disease.genome copy number changes (copy number variations: cnvs) include inherited, de novo and somatically acquired deviations from a diploid state within a particular chromosomal segment. cnvs are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. cnvs are distributed widely in the genomes of apparently healthy individuals and thus constitute significant amounts of population-based genomic variation. human cnv loci are en ...201020659124
inhibition of choroidal neovascularization via brief subretinal exposure to a newly developed lentiviral vector pseudotyped with sendai viral envelope proteins.lentiviral vectors are promising tools for the treatment of chronic retinal diseases, including age-related macular degeneration (amd), as they enable stable transgene expression. on the other hand, sendai virus (sev) vectors provide the unique advantage of rapid gene transfer. here we show that novel simian immunodeficiency viral vectors pseudotyped with sev envelope proteins (sev-f/hn-siv) achieved rapid, efficient, and long-lasting gene transfer in the mouse retina. subretinal exposure to sev ...201019778186
a highly basic kgkkgk sequence in the rna-binding domain of the cucumber necrosis virus coat protein is associated with encapsidation of full-length cnv rna during infection.the cucumber necrosis virus particle is a t=3 icosahedron consisting of 180 identical coat protein (cp) subunits. the n-terminal 58 aa residue segment of the cp r domain is believed to bind viral rna within virions and during assembly. we report results of in vivo experiments that examine the role of the r domain in assembly. deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. a highly basic kgkkgk sequence was found to be both necessary and sufficient for encapsi ...201020483445
rna-rna recombination in plant virus replication and evolution.rna-rna recombination is one of the strongest forces shaping the genomes of plant rna viruses. the detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. in the divided genome brome mosaic virus system, both inter- and intrasegmental crossovers are described. other systems utilize satellite or defective interfering rnas of turnip crinkle virus, tomato bushy stunt virus, cucumber necrosis virus, and potato virus x. these a ...201021529157
induction of particle polymorphism by cucumber necrosis virus coat protein mutants in vivo.the cucumber necrosis virus (cnv) particle is a t=3 icosahedron consisting of 180 identical coat protein (cp) subunits. plants infected with wild-type cnv accumulate a high number of t=3 particles, but other particle forms have not been observed. particle polymorphism in several t=3 icosahedral viruses has been observed in vitro following the removal of an extended n-terminal region of the cp subunit. in the case of cnv, we have recently described the structure of t=1 particles that accumulate i ...200818032493
downregulation of p22phox in retinal pigment epithelial cells inhibits choroidal neovascularization in mice.choroidal neovascularization (cnv) occurs in a variety of chorioretinal diseases including age-related macular degeneration (amd), and is the major cause of severe visual loss in patients with amd. oxidative stress has been thought to play an important role in the development of cnv. nicotinamide adenine dinucleotide phosphate (nadph) oxidase is one of the major intracellular sources of reactive oxygen species (ros) in the vascular system. in this study, we examined the expression of p22phox, an ...200818665154
structures of t=1 and t=3 particles of cucumber necrosis virus: evidence of internal scaffolding.cucumber necrosis virus (cnv) is a member of the genus tombusvirus, of which tomato bushy stunt virus (tbsv) is the type member. the capsid protein for this group of viruses is composed of three major domains: the r domain, which interacts with the rna genome: the s domain, which forms the tight capsid shell: and the protruding p domain, which extends approximately 40 angstrom from the surface. here, we present the cryo-transmission electron microscopy structures of both the t=1 and t=3 capsids ...200717049553
expression of the arabidopsis xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus rna and promotes rapid emergence of viral variants in plants.rapid rna virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic rna viruses. a previous genome-wide screen for host factors affecting recombination in tomato bushy stunt tombusvirus (tbsv), a small monopartite plant virus, identified xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [serviene, e., shapka, n., cheng, c.p., panavas, t., phuangrat, b., baker, j., nagy, p.d., (2005). ...200717688902
kinetics and functional studies on interaction between the replicase proteins of tomato bushy stunt virus: requirement of p33:p92 interaction for replicase assembly.the assembly of the functional replicase complex via protein:protein and rna:protein interactions among the viral-coded proteins, host factors and the viral rna on cellular membranes is a key step in the replication process of plus-stranded rna viruses. in this work, we have characterized essential interactions between p33:p33 and p33:p92 replication proteins of tomato bushy stunt virus (tbsv), a tombusvirus with a non-segmented, plus-stranded rna genome. surface plasmon resonance (spr) measurem ...200616242746
proteomics analysis of the tombusvirus replicase: hsp70 molecular chaperone is associated with the replicase and enhances viral rna replication.plus-strand rna virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. to identify host proteins present in the cucumber necrosis tombusvirus (cnv) replicase, we affinity purified functional viral replicase complexes from yeast. mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of cnv p33 and p92 replicase proteins as well as four major host proteins in the cnv replicase. the ho ...200616474124
eiav vector-mediated delivery of endostatin or angiostatin inhibits angiogenesis and vascular hyperpermeability in experimental cnv.we evaluated the efficacy of equine infectious anaemia virus (eiav)-based lentiviral vectors encoding endostatin (eiav.endostatin) or angiostatin (eiav.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laser-induced model of choroidal neovascularisation (cnv). equine infectious anaemia virus.endostatin, eiav.angiostatin or control (eiav.null) vectors were administered into the subretinal space of c57bl/6j mice. two weeks after laser injury cnv areas and the degree of ...200616572190
evaluation of the roles of specific regions of the cucumber necrosis virus coat protein arm in particle accumulation and fungus transmission.the cucumber necrosis virus (cnv) particle is a t=3 icosahedron composed of 180 identical coat protein (cp) subunits. each cp subunit includes a 34-amino-acid (aa) arm which connects the rna binding and shell domains. the arm is comprised of an 18-aa "beta" region and a 16-aa "epsilon" region, with the former contributing to a beta-annular structure involved in particle stability and the latter contributing to quasiequivalence and virion rna binding. previous work has shown that specific regions ...200616731935
[antisense vascular endothelial growth factor suppressed corneal neovascularization in rats].to explore whether recombinant adeno-associated virus mediated antisense vascular endothelial growth factor (raav-avegf) gene transfer inhibits the development of corneal neovascularization (cnv) in a rat model.200616762237
use of double-stranded rna templates by the tombusvirus replicase in vitro: implications for the mechanism of plus-strand initiation.plus-stranded rna viruses replicate efficiently in infected hosts producing numerous copies of the viral rna. one of the long-standing mysteries in rna virus replication is the occurrence and possible role of the double-stranded (ds)rna formed between minus- and plus-strands. using the partially purified cucumber necrosis virus (cnv) replicase from plants and the recombinant rna-dependent rna polymerase (rdrp) of turnip crinkle virus (tcv), in this paper, we demonstrate that both cnv replicase a ...200616765402
a 38-amino-acid sequence encompassing the arm domain of the cucumber necrosis virus coat protein functions as a chloroplast transit peptide in infected plants.experiments to determine the subcellular location of the coat protein (cp) of the tombusvirus cucumber necrosis virus (cnv) have been conducted. by confocal microscopy, it was found that an agroinfiltrated cnv cp-green fluorescent protein (gfp) fusion targets chloroplasts in nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete cp arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. western blot analyses of purified and ...200616873252
development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration.angiogenesis is a key aspect of the wet form of age-related neovascular (amd), the leading cause of blindness in the elderly population. substantial evidence indicated that vascular endothelial growth factor (vegf)-a is a major mediator of angiogenesis and vascular leakage in wet amd. vegf-a is the prototype member of a gene family that includes also plgf, vegf-b, vegf-c, vegf-d and the orf virus-encoded vegf-e. several isoforms of vegf-a can be generated due to alternative mrna splicing. variou ...200617031284
introgression of a tombusvirus resistance locus from nicotiana edwardsonii var. columbia to n. clevelandii.abstract a new variety of nicotiana, n. edwardsonii var. columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. columbia was developed from a hybridization between n. glutinosa and n. clevelandii, the same parents used for the formation of the original n. edwardsonii. however, in contrast to the original n. edwardsonii, crosses between columbia and either of its parents are fertile. thus, the inheritance of virus resistance genes present in n. glutinosa cou ...200618944304
[detection of direct markers of cytomegalovirus. research of the spectrum and avidity of antiviral antibodies in infants and preschool children].thirty-three children aged 1 month to 3 years were examined within the case study. spelisa, immunoblot (ib), shell vial method (svm) and pcr, were used for the detection of anti-cmv igm and igg, in the diagnosis of cytomegalovirus (cmv). clinical signs of cmv infection (cmvi) were registered in 20 children (group 1); no cmvi specific signs were detected in the remaining 13 children (group 2). class m antibodies were identified in 50% of group-1 sera. around 80% of children in the group had anti- ...200515747865
the role of the p33:p33/p92 interaction domain in rna replication and intracellular localization of p33 and p92 proteins of cucumber necrosis tombusvirus.replication of plus-stranded rna viruses is performed by the viral replicase complex, which, together with the viral rna, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. using replication-competent fluorescent protein-tagged p33 of cucumber necrosis virus (cnv), we determined that two domains affected p33 targeting to peroxis ...200515936051
role of an internal and two 3'-terminal rna elements in assembly of tombusvirus replicase.plus-strand rna virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. the replicase of cucumber necrosis virus (cnv), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. in addition, the assembly of cnv replicase is stimulated in the presence of plus-stranded viral rna (z. panaviene et al., j. virol. 78:8254-8263, 2004). to define cis-acting viral rna sequences that stimulate replicase asse ...200516051853
heterologous rna replication enhancer stimulates in vitro rna synthesis and template-switching by the carmovirus, but not by the tombusvirus, rna-dependent rna polymerase: implication for modular evolution of rna viruses.the viral rna plays multiple roles during replication of rna viruses, serving as a template for complementary rna synthesis and facilitating the assembly of the viral replicase complex. these roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (ren). to test if these rna elements can be used by related viral rna-dependent rna polymerases (rdrp), we compared the potential stimulatory effects of homologous and heterologous ren elements on complement ...200516083933
phosphorylation of the p33 replication protein of cucumber necrosis tombusvirus adjacent to the rna binding site affects viral rna replication.replication of the nonsegmented, plus-stranded rna genome of cucumber necrosis tombusvirus (cnv) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 rna-dependent rna polymerase. in this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase c. the related p28 replication protein of turnip crinkle virus ...200516154610
inhibition of in vitro rna binding and replicase activity by phosphorylation of the p33 replication protein of cucumber necrosis tombusvirus.tombusviruses, which are small plus-strand rna viruses of plants, require the viral-coded p33 replication co-factor for template selection and recruitment into replication in infected cells. as presented in the accompanying paper [shapka, n., stork, j., nagy, p.d., 2005. phosphorylation of the p33 replication protein of cucumber necrosis tombusvirus adjacent to the rna binding site affects viral rna replication. j. virol. 343, 65-78.], p33 can be phosphorylated in vitro at serine and threonine r ...200516154612
sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type 1 (hiv-1) infection and dendritic-cell-directed hiv-1 transfer.both endocytic uptake and viral fusion can lead to human immunodeficiency virus type 1 (hiv-1) transfer to cd4+ lymphocytes, either through directional regurgitation (infectious transfer in trans [i-it]) or through de novo viral production in dendritic cells (dcs) resulting in a second-phase transfer to cd4+ lymphocytes (infectious second-phase transfer [i-spt]). we have evaluated in immature monocyte-derived dcs both pathways of transfer with regard to their susceptibilities to being blocked by ...200516227272
evidence that vector transmission of a plant virus requires conformational change in virus particles.transmission of cucumber necrosis virus (cnv) by zoospores of its fungal vector, olpidium bornovanus, involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus-bound zoospores. previous work has shown that specific components of both cnv and zoospores are required for successful cnv/zoospore recognition. here, we show that limited trypsin digestion of cnv following in vitro cnv/zoospore binding assays, results in the production ...200415081809
purification of the cucumber necrosis virus replicase from yeast cells: role of coexpressed viral rna in stimulation of replicase activity.purified recombinant viral replicases are useful for studying the mechanism of viral rna replication in vitro. in this work, we obtained a highly active template-dependent replicase complex for cucumber necrosis tombusvirus (cnv), which is a plus-stranded rna virus, from saccharomyces cerevisiae. the recombinant cnv replicase showed properties similar to those of the plant-derived cnv replicase (p. d. nagy and j. pogany, virology 276:279-288, 2000), including the ability (i). to initiate crna sy ...200415254197
molecular aspects of plant virus transmission by olpidium and plasmodiophorid vectors.the genome structures of a large number of viruses transmitted by olpidium and plasmodiophorid vectors have been determined. the viruses are highly diverse, belonging to 12 genera in at least 4 families. plasmodiophorids are now classified as protists rather than true fungi. this finding, along with the recognition of the great variety of viruses transmitted by olpidium and plasmodiophorid vectors, will likely lead to an elaboration of the details of in vitro and in vivo transmission mechanisms. ...200415283666
[progress in pathogenesis and therapeutic research in retinitis pigmentosa and age-related macular degeneration].retinitis pigmentosa (rp) and age-related macular degeneration (amd) are designated special targeted eye diseases by the welfare and labor ministry of japan. we have been studying the pathogenesis, diagnosis, treatment, and evaluation of these diseases. the development of molecular genetic analyses of rp revealed that the type and frequency of mutations varied with the ethnic population. in our present study, we focused on the genetic analysis and clinical examinations for autosomal dominant ret ...200415656086
aav-mediated expression of vascular endothelial growth factor induces choroidal neovascularization in rat.to develop a small-animal model of choroidal neovascularization (cnv) by injecting adeno-associated virus (aav)-vegf into the subretinal space (srs) of rats.200312556414
evidence that binding of cucumber necrosis virus to vector zoospores involves recognition of oligosaccharides.despite the importance of vectors in natural dissemination of plant viruses, relatively little is known about the molecular features of viruses and vectors that permit their interaction in nature. cucumber necrosis virus (cnv) is a small spherical virus whose transmission in nature is facilitated by zoospores of the fungus olpidium bornovanus. previous studies have shown that specific regions of the cnv capsid are involved in transmission and that transmission defects in several cnv transmission ...200312634352
the overlapping rna-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication.two of the five viral-coded proteins of tombusviruses, which are small, nonsegmented, plus-stranded rna viruses of plants, are required for replication in infected cells. these replicase proteins, namely, p33 and p92, of cucumber necrosis virus are expressed directly from the genomic rna via a readthrough mechanism. their overlapping domains contain an arginine/proline-rich rna-binding motif (termed rpr, which has the sequence rprrrp). site-directed mutagenesis of p33 expressed in escherichia co ...200312706102
yeast as a model host to study replication and recombination of defective interfering rna of tomato bushy stunt virus.defective interfering (di) rna associated with tomato bushy stunt virus (tbsv), which is a plus-strand rna virus, requires p33 and p92 proteins of tbsv or the related cucumber necrosis virus (cnv), for replication in plants. to test if di rna can replicate in a model host, we coexpressed tbsv di rna and p33/p92 of cnv in yeast. we show evidence for replication of di rna in yeast, including (i) dependence on p33 and p92 for di replication; (ii) presence of active cnv rna-dependent rna polymerase ...200314517084
mutations in the rna-binding domains of tombusvirus replicase proteins affect rna recombination in vivo.rna recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. in this article, we show evidence that the replicase proteins of cucumber necrosis virus, a tombusvirus, are directly involved in rna recombination in vivo. mutations within the rna-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (di) rna, a model template for reco ...200314698673
expression of open reading frame 5 protein of porcine reproductive and respiratory syndrome virus using semliki forest virus expression system.the orf5 gene encodes a major envelope glycoprotein (gp5), which is one of the three major proteins of porcine reproductive and respiratory syndrome virus (prrsv). the gp5 protein has been known to be a 24.5-26 kda n-glycosylated envelope protein. the gp5 is involved in inducing neutralizing antibodies. for this reason, the gp5 is primary candidate for the prrsv subunit vaccine. to produce the native form of gp5 in mammalian cells, we have cloned the orf5 gene from prrsv cnv-1 into the semliki f ...200214614267
analysis of minimal promoter sequences for plus-strand synthesis by the cucumber necrosis virus rna-dependent rna polymerase.tombusviruses are small, plus-sense, single-stranded rna viruses of plants. a partially purified rna-dependent rna polymerase (rdrp) preparation of cucumber necrosis virus (cnv), which is capable of de novo initiation of complementary rna synthesis from either plus-strand or minus-strand templates, was used to dissect minimal promoter sequences for tombusviruses and their defective interfering (di) rnas. in vitro rdrp assay revealed that the core plus-strand initiation promoter included only the ...200212069525
internal initiation by the cucumber necrosis virus rna-dependent rna polymerase is facilitated by promoter-like sequences.tombusviruses, small positive sense rna viruses of plants, are replicated by the viral-coded rna-dependent rna polymerase (rdrp) in infected cells. an unusual feature of the tombusvirus rdrp that is partially purified from cucumber necrosis virus (cnv)-infected plants is the ability to initiate complementary rna synthesis from several internal positions on minus-strand templates derived from di rnas ( nagy and pogany, 2000 ). in this study, we used template deletion, mutagenesis, and oligo-based ...200212069526
identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment.cucumber necrosis virus (cnv) is naturally transmitted in the soil by zoospores of the fungal vector olpidium bornovanus. successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. mechanically passaged cnv was screened for mutants deficient in fungus transmission. we found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (wt) cnv. results of in vitro virus-zo ...200111356965
inhibition of choroidal neovascularization by intravenous injection of adenoviral vectors expressing secretable endostatin.endostatin is a cleavage product of collagen xviii that inhibits tumor angiogenesis and growth. interferon alpha2a blocks tumor angiogenesis and causes regression of hemangiomas, but has no effect on choroidal neovascularization (cnv). therefore, inhibitors of tumor angiogenesis do not necessarily inhibit ocular neovascularization. in this study, we used an intravenous injection of adenoviral vectors containing a sig-mendo transgene consisting of murine immunoglobulin kappa-chain leader sequence ...200111438478
suppression of choroidal neovascularization by adeno-associated virus vector expressing angiostatin.to test the efficacy of a recombinant adeno-associated virus (raav) vector that expresses mouse angiostatin in suppressing experimental choroidal neovascularization (cnv) in a rat model.200111527956
characterization of distinct tombusviruses that cause diseases of lettuce and tomato in the western united states.a soilborne disease of lettuce, associated with necrosis and dieback, has been found with increasing frequency in california and arizona over the last 10 years. an isometric virus, serologically related to tomato bushy stunt virus (tbsv), was consistently isolated from lettuce plants with these disease symptoms. back-inoculation to healthy lettuce plants and subsequent reisolation of the virus from symptomatic lettuce leaves suggested that this virus was the causal agent of this disease. a tombu ...200118944038
spontaneous deletion enhances movement of a cucumber necrosis virus based chimera expressing the red clover necrotic mosaic virus movement protein genedagger.summary the 35-kda movement protein (mp) gene of red clover necrotic mosaic virus (rcnmv) and 3' flanking sequence were inserted in a cucumber necrosis virus (cnv) deletion mutant lacking a large portion of the coding region for the mp. nicotiana benthamiana plants inoculated with chimeric synthetic transcripts of the resulting hybrid cdna clone (m5/rm2) developed both local and systemic symptoms and accumulated high levels of chimeric viral rna. reverse transcriptase polymerase chain reaction ( ...200120572988
the role of vascular endothelial growth factor (vegf) in abnormal vascular changes in the adult rat eye.the aim of this project was to determine if the subretinal delivery of a recombinant adenovirus encoding vascular endothelial growth factor (vegf) was sufficient to induce changes resembling choroidal neovascularisation (cnv) in a rat model. a recombinant adenovirus was produced encoding vegf164 cdna (ad.rsv.vegf). transduction of cultured rpe cells confirmed vegf expression and ensured the absence of ad.rsv.vegf-related toxicity. following subretinal injection into rat eyes, fluorescein angiogr ...200010801078
partial purification and characterization of cucumber necrosis virus and tomato bushy stunt virus rna-dependent rna polymerases: similarities and differences in template usage between tombusvirus and carmovirus rna-dependent rna polymerases.tombusviruses are small, plus-sense, single-stranded rna viruses of plants. rna-dependent rna polymerases (rdrp) of two tombusviruses, tomato bushy stunt virus (tbsv) and cucumber necrosis virus (cnv), have been partially purified from infected nicotiana benthamiana plants. the obtained rdrp complexes are capable of de novo initiation of complementary rna synthesis using either plus- or minus-strand templates derived from tombusvirus defective interfering (di) rnas. in addition to template-sized ...200011040120
restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene.nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (tbsv) failed to elicit effective virus resistance when inoculated with wildtype virus. subsequently, r1 and r2 progeny from 13 transgenic lines were inoculated with a tbsv mutant containing a defective coat protein gene. mild symptoms typical of those elicited in nontransformed plants infected with the tbsv mutant initially appeared. however, within 2 to 4 weeks, up to 20% of the transgenic plants sp ...19999926415
both rna rearrangement and point mutation contribute to repair of defective chimeric viral genomes to form functional hybrid viruses in plants.the putative movement protein gene (p27) plus 5' and 3' flanking sequences of cucumber leaf spot aureusvirus (clsv) was inserted into an infectious cucumber necrosis tombusvirus (cnv) cdna clone containing a deletion in the cell-to-cell movement protein gene. approximately 5% of plants inoculated with synthetic transcripts of two such defective chimeric cnv/clsv cdna clones developed systemic symptoms 7-19 days postinoculation. reverse transcription-polymerase chain reaction and sequence analysi ...199910366559
a cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain.little is currently known regarding the specific interactions that govern transmission of plant viruses by their vectors. a cucumber necrosis virus (cnv) variant (ll5) deficient in fungal transmissibility has been isolated from mechanically passaged cnv and characterized. although ll5 accumulates to wild-type (wt) levels, is capable of rapid systemic infection, and produces stable, highly infectious particles, it is only inefficiently transmitted by olpidium bornovanus zoospores. the ll5 coat pr ...19979234955
genome structure of cucumber leaf spot virus: sequence analysis suggests it belongs to a distinct species within the tombusviridae.the complete nucleotide sequence of cucumber leaf spot virus (clsv) has been determined and the sizes and locations of predicted viral proteins deduced. the genome consists of 4432 nucleotides and contains five long orfs. the 5' proximal orf encodes a 25 kda product that terminates in an amber codon which may be readthrough to produce an 84 kda protein (orf 2). orf 3 codes for the 41 kda coat protein (cp). orfs 4 and 5 are completely overlapping at the 3' terminus and code for 27 and 17 kda prod ...19979453144
both codon context and leader length contribute to efficient expression of two overlapping open reading frames of a cucumber necrosis virus bifunctional subgenomic mrna.the importance of codon context and leader length in the translational regulation of p20 and p21 from the bifunctional 0.9-kb subgenomic mrna cucumber necrosis virus was investigated. nucleotide substitutions introduced into the -3 and +4 positions of the p21 aug codon (where the a of the aug is +1) verified that purines in these positions are favored and demonstrated the similar contribution of the -3 and +4 positions to the efficiency of initiation codon selection in plants. the effect of nucl ...19968661432
deletion analysis of the promoter for the cucumber necrosis virus 0.9-kb subgenomic rna.sequences comprising the core promoter for the cucumber necrosis virus (cnv) 0.9-kb subgenomic rna have been determined using deletion analysis and site-directed mutagenesis. the deletion studies indicate that the promoter lies within a region located 20 nucleotides upstream and 6 nucleotides downstream and including the subgenomic start site. sequences further upstream or downstream of the core promoter do not appear to strongly affect promoter activity or viral rna accumulation. results of sit ...19958525604
host effects and sequences essential for accumulation of defective interfering rnas of cucumber necrosis and tomato bushy stunt tombusviruses.passage of cucumber necrosis virus (cnv) containing defective interfering (di) rnas through cucumber plants decreased the accumulation of di rnas to undetectable levels. subsequent passages in two nicotiana species (nicotiana benthamiana or n. clevelandii) resulted in the appearance of di rna species that were larger than the di rnas observed during exclusive serial passages of cnv through the nicotiana species. sequence analysis of cloned cdnas corresponding to the two di rna populations indica ...19957793079
mutational analysis of the cucumber necrosis virus coat protein gene.a series of frameshift, deletion, and inversion mutations were made in the coat protein (cp) gene of the icosahedral cucumber necrosis tombusvirus (cnv) to investigate the role of the cp protruding (p) domain in the production of virus particles and, also, to investigate the basis for the accumulation of cp deletion derivatives previously reported in plants inoculated with pd(-), a p-domainless cnv cp mutant. p-domainless coat protein subunit could be detected in extracts of cp mutant-infected p ...19957831793
characterization and biological activity of di rna dimers formed during cucumber necrosis virus coinfections.coinfections of synthetic transcripts from a cdna clone of cucumber necrosis virus (cnv) and cdna clones of defective interfering (di) rnas were previously demonstrated to contain di-like rnas approximately twice the size of the di rna used for co-inoculation. analysis of these rnas revealed that they are head-to-tail repeats of cnv di rna sequence (dimers). sequence analysis of 21 cloned dimer junctions indicated that approximately half of the junction sequences correspond to precise fusions of ...19957871739
involvement of the cucumber necrosis virus coat protein in the specificity of fungus transmission by olpidium bornovanus.cucumber necrosis (cnv) and the cherry strain of tomato bushy stunt (tbsv-ch) are tombusviruses which differ in transmissibility by the fungus olpidium bornovanus (sahtiyanci) karling (= o. radicale schwartz and cook). zoospores acquire and transmit cnv, but not tbsv-ch, in the in vitro manner. to assess the role of the coat protein in the specificity of fungus transmission, reciprocal exchanges were made between the coat protein genes of these two viruses in full-length infectious cdna clones. ...19947941356
coat protein of cucumber necrosis virus is not required for efficient generation or accumulation of defective interfering rnas.it is generally assumed that defective interfering (di) forms of viruses are encapsidated in structural proteins encoded by the helper virus. virion rna extracts from cucumber necrosis virus (cnv) infections showing high levels of cellular di rnas contain barely detectable levels of di rnas, suggesting that di rnas are encapsidated very inefficiently. in addition, accumulation of cnv di rnas occurs at equal efficiency in coinoculated plants using either synthetic wild-type cnv genomic rna as hel ...19948077954
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