Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana (original) (raw)
PLoS Pathogens, 2011
To search for host proteins involved in systemic spreading of Tomato spotted wilt virus (TSWV), the virus-encoded NSm movement protein has been utilized as a bait in yeast two-hybrid interaction trap assays. J-domain chaperones from different host species and a protein denominated At-4/1 from Arabidopsis thaliana showing homologies to myosins and kinesins were identified as NSm-interacting partners. In this communication we illustrate that following TSWV infection, J-domain proteins accumulated in systemically infected leaves of A. thaliana, whereas At-4/1 was constitutively detected in leaves of A. thaliana and Nicotiana rustica.
Virology, 1998
1997, J. Virol. 71, 6650±6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3Ј untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3Ј untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.
Virus Research, 1995
Studies on the molecular basis of flavivirus neutralisation, attenuation and tropism indicate that amino acid substitutions, in different parts of the envelope gene, may be responsible for the altered phenotypes. However, the association of particular substitutions with individual characteristics has proven difficult. Comparative analysis of all known tick-borne flavivirus envelope proteins through sequence alignment and a sliding window, reveals clusters of amino acid variation distributed throughout the envelope protein coding region. Further comparison with mosquito-borne flaviviruses reveals essentially the same profile of variability throughout the envelope protein sequence although there is a major difference within the postulated B domain of these viruses which may reflect their different evolutionary development. Most phenotypically variant properties, such as serotypic differences, variants characteristic of vaccine strains, altered tropisms and neutralisation escape mutants, map within the variable clusters. Thus, we propose that natural mutagenesis and selection may occur at specific sites that do not destroy the secondary and tertiary E protein structure and that the variable clusters represent the exposed surface amino acids of the envelope protein defining antigenicity, tropicity and pathogenesis.
Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus
Archives of Virology, 2003
We have described in the accompanying paper by Sang, et al.,, Arch Virol 2003: 1085-1093) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5 and 3 non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.
Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle
Scientific Reports, 2013
Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses. M embers of the genus Flavivirus within the family Flaviviridae comprise of several medically important pathogens including the Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV) which cause significant morbidity and mortality in humans, animals and birds 1. Flaviviruses are enveloped viruses with a positive-sense, single-strand RNA genome of approximately 11 kb that is translated as a single polyprotein precursor of ,3300 amino acids in length and proteolytically cleaved into 10 viral proteins: three structural (capsid, pre-membrane/membrane (prM-M), and envelope) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins 1. Flaviviruses enter their host cells through a process of receptor-mediated endocytosis followed by subsequent fusion within the endosomal compartment to release the viral genome into the cytoplasm for translation and replication 2. Replication of the viral RNA genome occurs on virus-induced host cell membranes. Such structures may serve as a scaffold for anchoring the viral replication complexes, which consist of viral RNA, viral proteins, and host cell factors. Virus assembly occurs within the endoplasmic reticulum-derived membrane compartments and non-infectious virions traverse through the Golgi stack to reach the trans-Golgi network (TGN) where furin-mediated cleavage between prM-M leads to conformational changes rendering the virion infectious. Infectious virus is subsequently released from cells via the secretory pathway 1,3. Adaptor complexes (AP-1 through 5) are heterotetrameric protein complexes comprising one each of the two large sub-units c, a, d, e and f and b1-5 and one medium sub-unit m1-5 and one small sub-unit s1-5 respectively. APs are involved in distinct intracellular vesicular transport pathways which play a vital role in maintaining cellular homeostasis 4-6. AP-1 is involved in the trafficking of cargo molecules in the biosynthetic pathway from the trans-Golgi network (TGN) to endosomes and back. AP-2 which is one of the most extensively-studied adaptor complex has been shown to be involved in the endocytic pathway at the plasma membrane. AP-3 is reported to function in the transport of selected proteins in the endo-lysosomal pathway. AP-4 is involved in sorting of proteins destined to basolateral surface in polarized cells. AP-5 is the newest member of the family discovered recently and has been proposed to function at the late endosomes 6. Despite the prominent role played by APs in intracellular trafficking pathways, their involvement in flavivirus life-cycle has not been characterized. A number of earlier studies investigating the internalization of flaviviruses have shown the involvement of clathrin-dependent and lipid raft-dependent pathways for virus entry but the role of APs in stages post-entry has not been investigated 2. A genome-wide RNA interference screen for identifying cellular proteins associated with WNV infection identified AP-1 m1 subunit and AP-3 s2 subunit as some of the host factors required for both WNV and DENV infection 7. Similarly, another study identified AP1M1 as one of the genes required for
Identification and analysis of truncated and elongated species of the flavivirus NS1 protein
Virus Research, 1999
The flavivirus non-structural glycoprotein NS1 is often detected in Western blots as a heterogeneous cluster of bands due to glycosylation variations, precursor-product relationships and/or alternative cleavage sites in the viral polyprotein. In this study, we determined the basis of structural heterogeneity of the NS1 protein of Murray Valley encephalitis virus (MVE) by glycosylation analysis, pulse-chase experiments and terminal amino acid sequencing. Inhibition of N-linked glycosylation by tunicamycin revealed that NS1 synthesised in MVE-infected C6/36 cells was derived from two polypeptide backbones of 39 kDa (NS1 o ) and 47 kDa (NS1%). Pulse-chase experiments established that no precursor-product relationship existed between NS1 o and NS1% and that both were stable end products. Terminal sequencing revealed that the N-and C-termini of NS1 o were located at amino acid positions 714 and 1145 in the polyprotein respectively, consistent with the predicted sites based upon sequence homology with other flaviviruses. Expression of the NS1 gene alone or in conjunction with NS2A by recombinant baculoviruses demonstrated that the production of NS1% was dependent on the presence of NS2A, indicating that the C-terminus of the larger protein was generated within NS2A. A smaller form (31 kDa) of NS1 (DNS1) was also identified in MVE-infected Vero cultures, and amino acid sequencing revealed a 120-residue truncation at the N-terminus of this protein. This corresponds closely with the in-frame 121-codon deletion at the 5% end of the NS1 gene of defective MVE viral RNA (described by , suggesting that DNS1 may be a translation product of defective viral RNA. : S 0 1 6 8 -1 7 0 2 ( 9 9 ) 0 0 0 0 3 -9
Molecular evolution of the insect-specific flaviviruses
Journal of General Virology, 2012
There has been an explosion in the discovery of ‘insect-specific’ flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all ‘insect-specific’ flavivirus sequences currently available and conduct phylogenetic analyses of both the ‘insect-specific’ flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus–mosquito co-divergence, suggesting that the ‘insect-specific’ flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on ‘insect-specific’ flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its...
The Journal of general virology, 2014
In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated 'Nhumirim virus'; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus's capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to v...
Virus Genes, 2013
We describe the isolation and characterization of an insect-specific flavivirus (ISF) from Ochlerotatus caspius (Pallas, 1771) mosquitoes collected in southern Portugal. The RNA genome of this virus, tentatively designated OCFV PT , for O. caspius flavivirus from Portugal, encodes a polyprotein showing all the features expected for a flavivirus. As frequently observed for ISF, the viral genomes seems to encode a putative Fairly Interesting Flavivirus ORF (FIFO)-like product, the synthesis of which would occur as a result of a-1 translation frameshift event. OCFV PT was isolated in the C6/36 Stegomyia albopicta (= Aedes albopictus) cell line where it replicates rapidly, but failed to replicate in Vero cells in common with other ISFs. Unlike some of the latter, however, the OCFV PT genome does not seem to be integrated in the mosquito cells we tested. Phylogenetic analyses based on partial ISF NS5 nucleotide sequences placed OCFV PT among recently published viral strains documented from mosquitoes collected in the Iberian Peninsula, while analyses of ORF/E/NS3/or NS5 amino acid sequences cluster OCFV PT with HANKV (Hanko virus), an ISF recently isolated from O. caspius mosquitoes collected in Finland. Taking into account the genetic relatedness with this virus, OCFV PT is not expected to be overtly cytopathic to C6/36 cells. The cytopathic effects associated with its presence in culture supernatants are postulated to be the result of the replication of a co-isolated putative new Negev-like virus.
Molecular Plant Pathology, 2017
Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2A HP , particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2A HP fused to an enhanced green fluorescent protein (EGFP) tag in N. occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N. occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression.
Secondary structure of the 3' untranslated region of flaviviruses: similarities and differences
Nucleic Acids Research, 1997
A genetic algorithm-based RNA secondary structure prediction was combined with comparative sequence analysis to construct models of folding for the distal 380 nucleotides of the 3h-untranslated region (3h-UTR) of yellow fever virus (YFV). A number of structural elements that are thermodynamically stable, conserved in shape, and confirmed by compensatory mutations were revealed. At the same time structural polymorphisms were observed among strains of YFV. These polymorphisms showed an association with virulence : all wild and pathogenic strains were likely to be folded in a signifi-0001-4553 # 1997 SGM BFED
Flavitrack: an annotated database of flavivirus sequences
Bioinformatics, 2007
Motivation-Properly annotated sequence data for flaviviruses, which cause diseases, such as tickborne encephalitis (TBE), dengue fever (DF), West Nile (WN) and yellow fever (YF), can aid in the design of antiviral drugs and vaccines to prevent their spread. Flavitrack was designed to help identify conserved sequence motifs, interpret mutational and structural data and track evolution of phenotypic properties.
Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus
Virology, 2013
Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.
Complete nucleotide sequence and genome organization of Grapevine fleck virus
Journal of General Virology, 2001
The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3h-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50 %), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5h and 3h ends, respectively. ORF 1 potentially encodes a 215n4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24n3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3h end of the viral genome and encode proline-rich proteins of 31n4 kDa (p31) and 15n9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.
Structural Basis of a Flavivirus Recognized by Its Neutralizing Antibody
Journal of Biological Chemistry, 2003
The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese encephalitis virus (JEV) envelope protein. The JEV domain III forms a -barrel type structure composed of six antiparallel -strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. Site-directed mutagenesis experiments are performed to confirm the NMR results. Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses. Flaviviruses are small (50 nm) positive-strand RNA viruses that contain a lipid-bilayer membrane. Forty species of the flavivirus family have been associated with human diseases, and most of them are transmitted to their vertebrate hosts by infected mosquitoes or ticks (1). Among these, yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue are the most important viral arboviruses in the world. Japanese encephalitis virus is transmitted by mosquitoes and is widely distributed in Asia, including Japan, China, Taiwan, Korea, Philippines, the far eastern former Soviet Union, all of Southeast Asia, and India (1). There are about 50,000 cases and 10,000 deaths reported annually throughout Asia, but these numbers are believed to be greatly underestimated. Like other flaviviruses, JEV 1 contains a single-stranded RNA genome of ϳ11 kb in size. The virion of JEV contains
Archives of Virology, 1997
Transgenic plants of Nicotiana benthamiana expressing single chain antibody fragments (scFv) speci®c for the coat protein of beet necrotic yellow vein virus (BNYVV) and non-expressing control plants were inoculated with BNYVV mechanically and by means of the vector Polymyxa betae. The scFv were presumably expressed in the endoplasmic reticulum (ER). The average time needed for infections to become detectable was longer in the scFvexpressing plants than in the non-expressing control plants. In addition, the scFv-expressing plants were partially protected against the pathogenic effects exerted by the virus on N. benthamiana plants in the late stages of infection. * Antibody-mediated resistance in transgenic plants is an attractive alternative to the various forms of pathogen-derived resistances, because it circumvents the danger of unintended side effects such as heteroencapsidations and recombinations of viral genomes . Voss et al. [19] have achieved partial resistance to tobacco mosaic virus (TMV) infections in transgenic tobacco by means of TMV coat protein-speci®c antibodies which were targeted to the endoplasmic reticulum (ER) by means of the immunoglobulin signal peptide, in order to enable their assembly and correct folding. Tavladoraki et al. [17] succeeded to obtain partial resistance to artichoke mottled crinkle virus (AMCV) infections in transgenic Nicotiana benthamiana by means of AMCV coat protein-speci®c single chain antibody fragments (scFv) which were expressed in the cytoplasm