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A unique feature of several T=3 icosahedral viruses is the presence of a structure called the beta-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T=3 capsid assembly. In order to examine the importance of the beta-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48-59 involved in the formation of the beta-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Delta48-59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of sizes close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6 A resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the beta-annulus. Therefore, the beta-annulus is not essential for T=3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the beta-annulus could be closely similar. 相似文献
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The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII-PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154-201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII-PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection. 相似文献
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V2 protein encoded by Tomato yellow leaf curl China virus is an RNA silencing suppressor 总被引:1,自引:0,他引:1
The V2 protein of Tomato yellow leaf curl China virus (TYLCCNV) was identified as an RNA silencing suppressor by Agrobacterium-mediated co-infiltration. The V2 protein could inhibit local RNA silencing, systemic RNA silencing of the green fluorescent protein (GFP) gene and the spread of a systemic GFP RNA silencing signal. However, the V2 could not interfere with the cell-to-cell spread of RNA silencing. Subcellular localization assay indicated that the V2 protein was distributed in the cytoplasm of Nicotiana benthamiana cells, and accumulated in irregular cytoplasmic bodies. The V2 bound 21 nt and 24 nt small interfering RNA (siRNA) duplexes and 24 nt single-stranded (ss)-siRNA but not 21 nt ss-siRNA in electrophoresis mobility shift assays. Expression of the V2 protein via the Potato virus X (PVX) vectors heterogenous system induced severe symptoms in N. benthamiana. In a yeast two-hybrid system, TYLCCNV V2 could interact with itself, but not with SlSGS3, which is known to been involved in RNA silencing pathway and to interact with a closely related Tomato yellow leaf curl virus (TYLCV) V2. These results indicate that TYLCCNV V2 is an RNA silencing suppressor, possibly through sequestering siRNA molecules. 相似文献
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To elucidate the mechanism of Apple latent spherical virus (ALSV) movement, various properties of its cell-to-cell movement protein (MP) were analyzed. ELISA and blot overlay assays demonstrated that the MP bound specifically to ALSV virions and in particular to one of the three coat proteins (VP25) but not to the other two coat proteins (VP20 and VP24). Mutational analyses have revealed that the MP contains two domains with independent VP25-binding activity (amino acid residues 1-188 and 189-281). Furthermore, nucleotide-binding experiments showed that the MP and VP25 bound to single-stranded RNA (ssRNA) and ssDNA without any sequence specificity, but these two proteins did not bind to double-stranded RNA (dsRNA) and dsDNA. The MP contains three potentially independent single-stranded nucleic acid-binding domains between amino acid residues 95-188, 189-281 and 277-376. The MP demonstrated cooperative and VP25 demonstrated non-cooperative binding to ssRNA in gel-retardation analyses. The cooperative RNA binding of the MP became non-cooperative when MP and VP25 were tested together in competition binding experiments, even though a sufficient amount of the MP for fully cooperative RNA binding the MP was supplied. The roles of the MP and VP25 interactions and nucleic acid binding activities in ALSV movement are discussed. 相似文献
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Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles 总被引:2,自引:1,他引:1
Dufresne PJ Thivierge K Cotton S Beauchemin C Ide C Ubalijoro E Laliberté JF Fortin MG 《Virology》2008,374(1):217-227
Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions. 相似文献
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Reovirus genomes from plant-feeding insects represent a newly discovered lineage within the family Reoviridae 总被引:1,自引:0,他引:1
A complex set of double-stranded RNAs (dsRNAs) was isolated from threecornered alfalfa hopper (Spissistilus festinus), a plant-feeding hemipteran pest. A subset of these dsRNAs constitute the genome of a new reovirus, provisionally designated Spissistilus festinus reovirus (SpFRV). SpFRV was present in threecornered alfalfa hopper populations in the San Joaquin Valley of California, with incidence ranging from 10% to 60% in 24 of 25 sample sets analyzed. The 10 dsRNA segments of SpFRV were completely sequenced and shown to share conserved terminal sequences (5′-AGAGA and CGAUGUUGU-3′) of the positive-sense strand that are distinct from known species of the family Reoviridae. Comparisons of the RNA directed RNA polymerase (RdRp) indicated SpFRV is most closely related (39.1% amino acid identity) to another new reovirus infecting the angulate leafhopper (Acinopterus angulatus) and provisionally designated Acinopterus angulatus reovirus (AcARV). The RdRp of both viruses was distantly related to Raspberry latent virus RdRp at 27.0% (SpFRV) and 30.0% (AcARV) or Rice ragged stunt virus RdRp at 26.2% (SpFRV) and 29.0% (AcARV) amino acid identity. RdRp phylogeny confirmed that SpFRV and AcARV are sister taxa sharing a most recent common ancestor. SpFRV segment 6 encodes a protein containing two NTP binding motifs that are conserved in homologs of reoviruses in the subfamily Spinareovirinae. The protein encoded by SpFRV segment 4 was identified as a guanylyltransferase homolog. SpFRV segments 1, 3, and 10 encode homologs of reovirus structural proteins. No homologs were identified for proteins encoded by SpFRV segments 5, 7, 8, and 9. Collectively, the low level of sequence identity with other reoviruses, similar segment terminal sequences, RdRp phylogeny, and host taxa indicate that SpFRV and AcARV may be considered members of a proposed new genus of the family Reoviridae (subfamily Spinareovirinae), with SpFRV assigned as the type species. 相似文献
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Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease. 相似文献