Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana (original) (raw)
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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.
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...