Lettuce infectious yellows virus

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Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants.

Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants.

Keywords: Lettuce infectious yellows virus ; P5; P9; small ORFs; virus infectivity; ER stress 1. Introduction RNA viruses have evolved to compress maximal coding and regulatory information into minimal sequence space. Small open reading frames (ORFs) that encode peptides of less than 100 amino acids, a strategy often employed by viruses to minimize the size of their genome, still perform essential roles in virus infection [ 1 ]. For example, the 6-kDa viral protein 6K2 of Turnip mosaic virus (TuMV, Potyvirus) is a membrane-associated protein that induces the formation of endoplasmic reticulum (ER)-derived vesicles for viral genome amplification, and mediates their export from the ER for virus systemic infection [ 2 , 3 ]. Another 6-kDa potyviral protein, 6K1, is also required for viral replication and targets the viral replication complex at the early stage of infection [ 4 ]. In another example, two consecutive small proteins of 7-kDa (P7A and P7B) encoded by Melon necrotic spot virus (MNSV) are involved in virus cell-to-cell movement, and P7B has shown to be a type II integral membrane protein that is essential for ER export, transport to the Golgi apparatus and finally to the plasmadesmata (PD) [ 5 – 7 ]. However, for many viruses, the very small ORFs/proteins are often overlooked due to their short sequences and uncertain significance. It is imperative to identify the coding regions and understand their functions to decipher how these small ORFs/proteins promote the infection cycle.
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A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

To test the hypothesis that a complete CPm is required for positive detection on virions, we analyzed p1-5b and the engi- neered mutants p1-5bM1 (p1-5b engineered to express a com- plete CPm) and pR6-5b (an engineered mutant in which the p1-5b CPm gene had been incorporated within the cloned WT [pR6] background [Fig. 1A and C]). As a comparison, LIYV virions purified from pR6-infected protoplasts and/or lettuce plants and greenhouse-maintained WT-infected lettuce plants were included in the study. Western blot analyses of pR6 virions identified proteins with sizes corresponding to those of the intact CP and CPm (Fig. 1B, lanes 1 and 3), consistent with our previously reported results (12). The complete CP and CPm were also identified for virions of p1-5bM1 in the same Western analyses (Fig. 1B, lane 4). Thus, the mutation engi- neered into the CPm gene of p1-5b removed the premature stop codon and resulted in the restoration of an intact CPm while retaining the other nucleotide polymorphisms of the p1-5b clone relative to the wild-type pR6 clone. However, Western blot analyses of purified pR6-5b virions positively FIG. 1. Predicted sizes of the CPm encoded by the WT and mutants of LIYV and immunoblot analysis of virions. (A) Genome organization of LIYV RNA 2 and amino acid positions of expected translation products encoded by the CPm gene. Expected translation products were encoded by (1) the full-length CPm gene of whitefly-transmissible cloned WT pR6 and the CPm restoration mutants p1-5bM1 and agro-pR6-5bM1 and (2) the CPm gene of the non-whitefly-transmissible mutants p1-5b, pR6-5b, and agro-pR6-5b, including 14 predicted novel amino acids (green) at the C terminus due to the frameshift-preceding termination. The complete CPm corresponds to an intact protein with 453 amino acids, whereas that from p1-5b, pR6-5b, and agro-pR6-5b corresponds to a truncated protein with 211 amino acids. The relative positions of the LIYV RNA 2 ORFs encoding P5, HSP70h, P59, P9, CP, CPm, and P26 are indicated. Arrows indicate NheI sites used for the construction of agroinfiltration plasmids. (B) Western blot analysis of virions. Shown are pR6 virions (lanes 1 and 3) purified from lettuce plants inoculated by whitefly transmission of virions from pR6-infected protoplasts and pR6-5b and p1-5bM1 virions (lanes 2 and 4) purified from inoculated protoplasts. Virion proteins were detected with antisera raised against the CPm (top) or whole virions (bottom). Positions of the CP (28 kDa) and CPm (52 kDa) are indicated. CPm antiserum frequently detected both the expected 52-kDa protein and a ca. 35-kDa protein, which may be a CPm degradation product (lanes 1, 3, and 4). Migrations of molecular mass markers are indicated with colored text and arrows. Separate arrows are shown for the 50-kDa and 25-kDa marker locations for each blot at the top. (C) Summary of constructs used for experiments described herein.
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Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans  Enhancer of RNA 2 Accumulation

Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation

Replication enhancers have been reported for several mul- tipartite plant viruses and include the ␥RNA-encoded ␥B of Barley stripe mosaic virus (BSMV; 19), the Cowpea mosaic virus (CPMV) M-RNA-encoded 58-kDa protein (4), the Beet ne- crotic yellow vein virus (BNYVV)-encoded P14 (8), and the Peanut clump virus (PCV) RNA 1-encoded P15 (9). The BSMV ␥B, the BNYVV P14, and the PCV P15 proteins all belong to a group of cysteine-rich proteins, and P14 shares weak but statistically significant similarity with other nucleic acid binding proteins (8). These cysteine-rich proteins influ- ence or enhance replication for all genomic components of their respective virus. In contrast, the CPMV 58-kDa protein is a template-selective replication enhancer. The CPMV 58-kDa protein is needed for efficient replication of the CPMV M- RNA but not the CPMV B-RNA; thus, it is a cis replication enhancer. Interestingly, a RNA sequence located within Red clover necrotic mosaic virus (RCNMV) genomic RNA 2 has recently been identified as a transcriptional enhancer, func- tioning in trans for the synthesis of an RCNMV RNA 1 sub- genomic RNA (22).
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The Hydrophobic Domain of Infectious Bronchitis Virus E Protein Alters the Host Secretory Pathway and Is Important for Release of Infectious Virus

The Hydrophobic Domain of Infectious Bronchitis Virus E Protein Alters the Host Secretory Pathway and Is Important for Release of Infectious Virus

We have shown that the HD of IBV E plays an important role in the release of infectious IBV particles from Vero cells. A recombinant virus with IBV E containing a heterologous HD (EG3) was competent for virus assembly but showed a defect in the release of infectious particles. The finding that the HD was not required for assembly is consistent with our earlier observation that the cytoplasmic tail of IBV E is sufficient for interaction with IBV M (4). Further characterization of the mutant virus showed that it accumulated intracellularly in vac- uole-like structures along with aberrant material. We hypoth- esized that the mutant virions were accumulating intracellu- larly and becoming damaged and were subsequently released as noninfectious particles. Thus, we initially thought that the HD of IBV E might alter the secretory pathway to promote anterograde trafficking. However, in overexpression experi- ments IBV E, but not EG3, caused a dramatic reduction in protein trafficking to the plasma membrane by impeding cargo trafficking through the Golgi complex. We also observed that overexpression of IBV E disrupted Golgi morphology but did not affect the ER or endosomal compartments. Finally, we observed that cells infected with IBV-EG3 had increased sur- face levels of IBV S, leading to larger syncytia.
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Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

The results with D172N provide another opportunity to test the Terradot structure. We have shown above that successful transmission of this mutant is accompanied either by reversion to the wild-type sequence or by the appearance in the progeny of a reverse second-site mutation, N137D. One possible inter- pretation of this finding is that the charge density on the capsid surface is important for virus-aphid interactions and that the viral variant with the second-site mutation is selected for be- cause it restores the wild-type surface charge density. This situation has been observed for a mutation introduced into the coat protein of Cucumber mosaic virus (11). In the context of our findings with P3, however, a similar hypothesis does not readily explain why the mutations D168N and E171Q, which FIG. 4. BWYV P3 S-domain residues predicted to be in ␤-sheets (horizontal lines) and ␣-helices (loops) in the Terradot model (symbols above the sequence) and in the alternative model proposed in this paper (symbols below the sequence). The positions of the BWYV counterparts of PLRV epitopes 5 and 10 are indicated by thick lines.
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Interaction between Long-Distance Transport Factor and Hsp70-Related Movement Protein of Beet Yellows Virus

Interaction between Long-Distance Transport Factor and Hsp70-Related Movement Protein of Beet Yellows Virus

Moreover, coexpression of Hsp70h, N42, or C23 with p20-GFP results in relocalization of the latter product, suggesting that p20 can interact with Hsp70h or its domains in live plant cells. p20 is a long-distance transport factor. What is the primary function of p20 in the BYV life cycle? Previous work demon- strated that p20 is dispensable for BYV replication (39) and is not essential for virus cell-to-cell movement, although it might have an accessory role in the latter process (2). The interaction of p20 with Hsp70h, which is one of the three BYV MPs (40), prompted us to revisit the role of p20 in cell-to-cell movement. A previously characterized p20 mutant, ⌬p20, was poten- tially capable of expressing C-terminal fragments of p20 (Fig. 5). It could not be excluded that this fragment was partially functional in the cell-to-cell movement of the corresponding mutant variant (2). Deletion of the RNA region encoding this fragment was impractical because it would inactivate a sub- genomic promoter that governs expression of a replication- associated BYV protein, p21 (39). Consequently, we designed two additional point mutations by introducing premature stop codons in place of the 49th and 81st p20 codons. These mu- tants could direct the translation of only short, N-terminal p20 peptides (Fig. 5). Each mutation was introduced into GFP- tagged BYV, and the resulting mutant BYV-GFP variants were inoculated into the local lesion host plants (40). Exami- nation of these mutants demonstrated that each of them was capable of cell-to-cell movement. The Stop-49 and Stop-81 mutants produced multicellular infection foci with mean diam- eters of 4.0 ⫾ 2.3 and 2.1 ⫾ 1.1 cells, respectively. The infec- tion foci formed by the parental BYV-GFP variant had mean diameters of 5.1 ⫾ 2.8 cells. These data confirmed our previous conclusion that p20 is not essential for the BYV cell-to-cell movement, although its inactivation results in a less efficient local spread.
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Identification and QTL mapping of resistance to Turnip yellows virus (TuYV) in oilseed rape, Brassica napus

Identification and QTL mapping of resistance to Turnip yellows virus (TuYV) in oilseed rape, Brassica napus

Abstract Turnip yellows virus (TuYV) is transmitted by the peach-potato aphid (Myzus persicae) and causes severe yield losses in commercial oilseed rape crops (Brassica napus). There is currently only one genetic resource for resistance to TuYV available in brassica, which was identified in the re-synthesised B. napus line ‘R54’. In our study, 27 mostly homozygous B. napus accessions, either doubled-haploid (DH) or inbred lines, representing a diverse subset of the B. napus genepool, were screened for TuYV resistance/susceptibility. Partial resistance to TuYV was identified in the Korean spring oilseed rape, B. napus variety Yudal, whilst the dwarf French winter oilseed rape line Darmor-bzh was susceptible. QTL mapping using the established Darmor-bzh × Yudal DH mapping population (DYDH) revealed one major QTL explaining 36% and 18% of the phenotypic variation in two independent experiments. A DYDH line was crossed to Yudal, and reciprocal backcross (BC 1 )
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Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements for infectious virus and incomplete particles.

Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements for infectious virus and incomplete particles.

384-392 0022-538X/82/100384-09$02.00/0 Copyright © 1982, American Society for Microbiology Biosynthesis of Virus-Specific Proteins in Cells Infected with Infectious Bursal Disease Virus [r]

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Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

One reason is the extraordinary lability of the infectious property Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest Collection of medium 10,000 50 Concentration with PE[r]

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Norwalk Virus RNA Is Infectious in Mammalian Cells

Norwalk Virus RNA Is Infectious in Mammalian Cells

replicate and produce NV. Huh-7.5.1 cells were derived from the parental Huh-7 cells that harbored a subgenomic hepatitis C virus (HCV) replicon, after curing them by prolonged treat- ment with alpha interferon (IFN- ␣ ). These cells are deficient in virus-activated signaling of IFN- ␤ synthesis through the in- tracellular retinoic acid-inducible gene I (RIG-I) pathway and are highly permissive for HCV replication (4, 41). To deter- mine whether there were quantitative differences in replication efficiencies between Huh-7 and Huh-7.5.1 cells, equal amounts of NV RNA were transfected in both cell lines in parallel, and kinetics of viral replication were compared after quantifying the released virus in the supernatant at different times post- transfection. The numbers of positive cells by IF at 24 hpt were similar for both cell lines, indicating that RNA transfection efficiencies were similar for both cell lines. NV particle release into the supernatant of transfected Huh-7 cells was slightly delayed compared to production by Huh-7.5.1 cells (Fig. 9), but the differences were not statistically significant. Similar levels of intracellular synthesized subgenomic RNA were de- tected by Northern blotting between both cell lines (data not shown). These results demonstrate that NV RNA replication efficiencies are similar between Huh-7 and Huh-7.5.1 cells and suggest that an inactivating mutation in RIG-I does not have any effect on NV RNA replication and that the RIG-I signaling FIG. 8. Analysis of virus binding and viral replication in Huh-7 cells overexpressing FUT2. (A) Effect of FUT2 transient expression on NV binding to Huh-7 cells. Huh-7 cells were transfected with a plasmid carrying the human FUT2 gene or the empty vector (pcDNAI), and 48 hpt, binding assays were performed. Binding of recombinant NV VLPs (5 ␮ g/ml) to Huh-7 cells was detected by IF using the MAb NV8812. (B) Effect of MAbs against NV VP1 on the binding of wild-type NV to FUT2-expressing Huh-7 cells. Purified NV from stool was incubated in three serial 10-fold dilutions of ascites fluids from MAbs NV8812 and NV3901, starting at a 10 ⫺ 3 dilution, for 2 h at room temperature before performing the
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The 64-Kilodalton Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails

The 64-Kilodalton Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails

The filamentous virion of the closterovirus Beet yellows virus (BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography–tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement.
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Recovery of Infectious Bluetongue Virus from RNA

Recovery of Infectious Bluetongue Virus from RNA

A calculation from the number of plaques recovered dem- onstrates that rescue of infectious virus occurs in approxi- mately 1 in 10,000 transfected cells when microgram quantities of viral ssRNA are used, whereas high levels of viral-protein expression are detectible in ⬃ 50% of the cells. One explana- tion for this difference is that there may be a critical amount of viral ssRNA that must be introduced into the cell for a pro- ductive infection to be initiated. Below this threshold, there would be no rescue, but viral proteins would be synthesized. In addition, if rescue is to occur, a transfected cell must receive copies of each of the 10 transcripts. Furthermore, enough copies of each transcript must be present so that both viral proteins are synthesized, and enough of each transcript must remain available for packaging and replication. Once progeny cores are assembled, the transcripts produced by these progeny cores will lead to an amplification of gene expression and so to further core particle and virion production. A second possibil- ity involves the poorly understood processes of genome pack- aging and genome replication, which in the Reoviridae occur within the viral inclusion bodies. The introduction of viral ssRNA into cells by transfection omits the presence of a core
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Intracellular ribonucleoprotein complexes of visna virus are infectious.

Intracellular ribonucleoprotein complexes of visna virus are infectious.

To verify that RNP complexes contained the same complete genetic information as parental virions and were able to induce a normal replication cycle of visna virus, the hybridization kine[r]

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Effects of Point Mutations in the Readthrough Domain of the Beet Western Yellows Virus Minor Capsid Protein on Virus Accumulation In Planta and on Transmission by Aphids

Effects of Point Mutations in the Readthrough Domain of the Beet Western Yellows Virus Minor Capsid Protein on Virus Accumulation In Planta and on Transmission by Aphids

(iv) BW5.127. As shown above, plants agroinfected with BW5.127 initially accumulated low levels of virus, but near- wild-type levels appeared at later times. This observation sug- gests that the primary K200A/Y201D mutation had affected a motif in the RTD important for virus accumulation but that modified forms of the virus which overcome this defect appear later in infection. To test this hypothesis, viral RT-PCR clones were produced from RNA extracted from leaves taken either early (4 weeks) or later (7 weeks) following agroinfection. The sequence analysis revealed that both of the primary mutations (K200A and Y201D) were present in 20 of 21 RT-PCR clones from three different plants at 4 weeks p.i. (Fig. 6A). In the single exception, D201 had been replaced by N in a clone derived from plant 9. We also detected the mutation S206L in two other clones from plant 9 at 4 weeks p.i. (Fig. 6A), but the significance, if any, of this particular second-site substitution has not been further investigated.
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Infectious Mononucleosis and Epstein Barr Virus in Childhood

Infectious Mononucleosis and Epstein Barr Virus in Childhood

The Epstein-Barr virus ( EBV) antibody titer was examined in 22 children whose atypical mononuclear cells appeared in the peripheral blood smear.. Paul-Bunnell.[r]

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Epstein-Barr Virus Infectious Mononucleosis in Children

Epstein-Barr Virus Infectious Mononucleosis in Children

The other nine children were determined to have an acute EBV infection due to the presence of serum IgG antibody to EBV- capsid antigen concurrent with the early absence and later emerge[r]

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Epstein-Barr Virus Infectious Mononucleosis in Children

Epstein-Barr Virus Infectious Mononucleosis in Children

above infants was unclear, but a dual EBV-cyto- megalovirus infection, possibly congenital, has been reported previously.29 The otitis media detected in five children during acute EBV-in[r]

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Electron Microscopy of Equine Infectious Anemia Virus

Electron Microscopy of Equine Infectious Anemia Virus

Electron Microscopy of Equine Infectious Anemia Virus MASANORI TAJIMA, HIDEO NAKAJIMA, AND YASUICHIRO ITO Vippon Instinitee for Biological Scielnce, Akeboniociho, Tacihikawa, Tokyo, Jalp[r]

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Biophysical studies of infectious pancreatic necrosis virus.

Biophysical studies of infectious pancreatic necrosis virus.

Consequently, it is not possible to determine the molecular weight of the virion by measuring the percent RNA content of purified virus preparations, a method that was used in earlier st[r]

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Structural proteins of equine infectious anemia virus.

Structural proteins of equine infectious anemia virus.

In summary, the results of this work indicate SDS-disrupted EIAV, our present working hythat EIA virions produced by persistently in- pothesis is that they are not coded by the viral fec[r]

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