Potato Leafroll Virus Coat Protein Research Articles

Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of plants, including staple food crops. Previous cryo-electron microscopy studies of virus-like particles show that luteovirid viral capsids are built from a structural coat protein that organizes with T = 3 icosahedral symmetry. Here, we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.80-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation and suggest that the CP N-terminus and its interactions with RNA play an important role in generating capsid curvature.

RT-PCR Based Cloning and Sequencing of Potato Leaf Roll Virus-Coat Protein (PLRV-CP) Gene for Characterization as a Bangladeshi PLRV Isolate and Its Phylogenetic Analysis

An experiment was conducted in Molecular Biology and Plant Virology Laboratory under the Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh. Total RNA was extracted from Potato leaf roll virus (PLRV) positive leaves and complementary DNA (cDNA) were synthesized from total RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection conditions were optimized by using coat protein (CP) gene specific primers. In PCR amplification cDNA and in nucleotide sequencing PCR product was used as a template. A 346 bp amplicon of PLRV- CP gene was amplified and amplified gene region was sequenced. The expected nucleotide sequence of amplified PLRV-CP gene showed 95 to 98% homology when compared with the isolates sequences reported in Gene Bank database. This explored novel PLRV-CP gene was characterized as a PLRV Bangladeshi isolate (Accession number, Bankit 2274496, MN605963). PLRV-CP gene protein modeling was carried out using Expert Protein Analysis System (ExPaSy), DNASTAR’s protein tools server used for 3D protein modeling. Phylogenetic analysis was also carried out, the tree was made by using MEGA 4.0 software and maximum parsimony method was selected to construct phylogenetic tree. The RT-PCR based molecular technique optimized in this study, would be a useful for early detection, epidemiological studies of PLRV as well as in seed tubers certification program and the novel hyper variable sequenced region of PLRV-CP gene will be useful in pathogen derived resistance breeding program against the PLRV local strain.

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically.

A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Diagnóstico serológico y molecular del Potato leafroll virus (PLRV) en tubérculos-semilla de papa en Antioquia, Colombia

<p>Las virosis son uno de los problemas fitosanitarios más limitantes para la producción de papa, siendo los virus Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus S (PVS) y Potato ye-llow vein virus (PYVV) los que generan mayores pérdidas económicas en la región andina. Gene-ralmente estos virus son transmitidos por insectos hemípteros y por tubérculos-semilla. En esta investigación se evaluó la presencia y distribución del PLRV en tubérculos de <em>Solanum tuberosum</em> variedad Diacol-Capiro y S. phureja variedad Criolla-Colombia obtenidos en Antioquia, utilizando pruebas serológicas y moleculares. Mediante DAS-ELISA se evaluaron 128 muestras consisten-tes en tres tipos de tejidos (piel, yemas latentes y brotes), mientras que con RT-PCR en tiempo real (RT-qPCR), se utilizó un subgrupo de 12 muestras de cada variedad. Utilizando DAS-ELISA, el PLRV se encontró en el 3,1% y 10,1% de las muestras de Diacol-Capiro y Criolla-Colombia, respectivamente; siendo evidente una mayor detección del virus en la piel de los tubérculos de ésta última variedad. Las pruebas de RT-qPCR detectaron el PLRV en 83,3% y 91,6% de las 12 muestras de Diacol-Capiro y de Criolla-Colombia, respectivamente; con valores de Ciclo umbral (CT) entre 17,72 y 30,59. La naturaleza de los amplicones obtenidos por RT-qPCR fue confirmada por secuenciación como parte de una región codificante de la cápside del PLRV (Identidad=99%-100%). Estos resultados enfatizan en la necesidad de utilizar tubérculo-semilla certificados por su sanidad viral en los cultivos de papa del país y de fortalecer dichos programas a partir del empleo de técnicas de detección altamente sensibles como RT-qPCR.</p>

Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission

The coat protein (CP) of potato leafroll virus (PLRV) is the primary component of the capsid, and is a multifunctional protein known to be involved in vector transmission and virus movement within plant hosts, in addition to particle assembly. Thirteen mutations were generated in various regions of the CP and tested for their ability to affect virus-host and virus-vector interactions. Nine of the mutations prevented the assembly of stable virions. These mutants were unable to infect systemically four different host species. Furthermore, although virus replication and translation of the CP were similar for the mutants and wild-type virus in individual plant cells, the translation of the CP readthrough product was affected in several of the mutants. Four of the mutants were able to assemble stable particles and infect host plants systemically, similarly to the wild-type virus; however, two of the mutants were transmitted less efficiently by aphid vectors. Based on a computer-generated model of the PLRV CP, the mutations that prevented virion assembly were associated with subunit interfaces, while the amino acid alterations in the assembly-competent mutants were associated with surface loops. This and previous work indicates that the CP structural model has value in predicting the structural architecture of the virion.

A Surface Loop of the Potato Leafroll Virus Coat Protein Is Involved in Virion Assembly, Systemic Movement, and Aphid Transmission

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.

Cloning and sequencing of coat protein gene of an Indian potato leaf roll virus (PLRV) isolate and its similarity with other members of Luteoviridae.

An Indian strain of potato leaf roll virus (PLRV) was purified to generate complementary DNA corresponding to the coat protein (CP) gene. Virus cDNA was synthesized from purified viral RNA using oligo (dT)-anchor primer and virus specific primers. The viral sequence encoding the coat protein was specifically amplified by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was A-T cloned into the pGEM-T Easy vector and the authenticity of the cloned gene verified by dot blot hybridization and sequence analysis. Run-way-transcripts of the cloned CP gene could detect PLRV in tissue imprints and tissue dilution. The nucleotide sequences and the deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99% identical at both the nucleotide and amino acid sequence level of other isolates. Multiple sequence alignment of deduced amino acid sequences revealed considerable homology to other luteoviruses. A nuclear localization signal located close to the N-terminus of the CP gene was predicted. This is the first report of PLRV coat protein sequence from an Indian strain.

Analysis of a Three-Dimensional Structure of Potato leafroll virus Coat Protein Obtained by Homology Modeling

Viruses of the family Luteoviridae are ssRNA plant viruses that have particles that exhibit icosahedral symmetry. To identify the residues that might be exposed on the surface of the Potato leafroll virus (PLRV; genus Polerovirus, family Luteoviridae) capsid, and therefore involved in biological interactions, we performed a structural analysis of the PLRV coat protein (CP) on the basis of comparisons with protein sequences and known crystal structures of CPs of other viruses. The CP of PLRV displays 33% sequence similarity with that of Rice yellow mottle virus (genus Sobemovirus) when the sequences were aligned by using the hidden Markov model method. A structure model for PLRV CP was designed by protein homology modeling, using the crystal structure of RYMV as a template. The resulting model is consistent with immunological and site-directed mutagenesis data previously reported. On the basis of this model it is possible to predict some surface properties of the PLRV CP and also speculate about the structural evolution of small icosahedral viruses.

Reduced Field Spread of Potato Leafroll Virus in Potatoes Transformed with the Potato Leafroll Virus Coat Protein Gene.

Russet Burbank potato was transformed with plant expression vectors containing the potato leafroll luteovirus (PLRV) coat protein (CP) gene. Transgenic potato lines contained a gene expression cassette with two copies of a PLRV CP gene in which the nucleotide sequence was modified to improve expression of the gene. In addition, the two copies of the PLRV CP gene were each driven by a different promoter. Field test screening for PLRV resistance identified 15 lines which showed moderate resistance to PLRV infection and virus titer build-up and a longer incubation period for systemic infection. By conducting field resistance assays during a period when the vector of PLRV was not present, it was possible to test whether the observed resistance was sufficient to restrict aphid transmission of PLRV in a field test. Two years of field testing demonstrated that PLRV-spread from an infected plant to adjacent healthy plants of the same line was severely restricted in nearly all the transgenic lines in the field. These lines have useful resistance to PLRV and could aid in managing PLRV disease in Russet Burbank potato.

Suppression of PLRV Titer in transgenic Russet Burbank and Ranger Russet

Potato varieties Russet Burbank and Ranger Russet were transformed with a cDNA version of the 23 kD coat protein cistron (CP) of the potato leafroll virus (PLRV) using anAgrobacterium-mediated procedure. Clones were assayed for presence of thenpt- II and CP genes by Southern analysis, for expression of CP mRNA by Northern analysis, and for presence of PLRV coat protein in uninoculated and aphid inoculated plants by ELISA and Western blot analysis in uninoculated plants. Two putative Russet Burbank transformants were escapes, lacking eithernptll or CP, while one putative Ranger Russet transformant possessed thenpt- II gene but not the CP gene. In Russet Burbank and Ranger Russet, some transformants had statistically lower virus titer. The lowered titer was consistent throughout assays at three times during primary infection and one assay of secondary infection. The ranking of virus titer across all tests was statistically consistent. Transformants with the lowest secondary titers had virus contents 15 and 31 % of the titers of untransformed controls for Russet Burbank and Ranger Russet, respectively. The virus titer of the two Russet Burbank escapes and the Ranger Russet with only thenpt- II gene did not differ significantly from their respective untransformed controls.

Use of the polymerase chain reaction to clone the potato leafroll virus coat protein gene directly from the total RNA of infected plants

The potato leafroll virus (PLRV) coat protein (CP) gene was directly cloned from the total RNA extracted from virus-infected plants. First strand cDNA synthesis was not necessarily specific; it was equally efficient using either random or CP-specific primers. The viral sequence encoding the coat protein was specifically amplified from the total population of cDNA molecules by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was cloned blunt-end into the pT7T318U plasmid vector, and the authenticity of the cloned gene verified by sequence analysis. This cloning strategy obviates the need for virus purification. Sequence comparison of the CP gene of the Italian isolate and those of five other PLRV isolates revealed a close similarity to the three European and the Canadian isolates, and a more distant relationship with the Australian one.

Enhancement of resistance to potato leafroll virus multiplication in potato by combining the effects of host genes and transgenes.

Four potato clones with host gene-mediated resistance to potato leafroll virus (PLRV) multiplication were transformed with the PLRV coat protein (CP) gene. Plants of lines expressing high levels of transcript were highly resistant to PLRV multiplication; virus concentration was only 20-40 ng/g of leaf, which is approximately 1% of the concentration reached in susceptible cultivars. The effects of the transgenic and host-derived resistance genes appear to be additive.

Relationship between transcript production and virus resistance in transgenic tobacco expressing the potato leafroll virus coat protein gene

The coat protein (CP) gene of potato leafroll luteovims (PLRV) was inserted into tobacco (Nicotiana tabacum) using disarmed Agrobacterium tumefaciens (LBA4404) containing a binary expression vector. PLRV CP gene transcript was detected in transgenic plants but its abundance differed between transformed lines. CP was not detected in virus-free transgenic plants. The segregation of kanamycin resistance in S1 seedling progenies (obtained by selfing transformed plants) indicated that multiple (up to five) integration events involving vector T-DNA had occurred in most transformants. However, the amount of detectable CP transcript was not related to the neomycin phosphotransferase II gene copy number. Multiplication of PLRV in mature transgenic plants was diminished by up to 6-fold; the greatest diminution was in those transformed lines in which most CP gene transcript was detected. However, S1 progeny seedlings of transgenic plants were no more resistant to infection, following inoculation with viruliferous aphids, than seedlings of non-transformed control plants.

A characterization of epitopes on potato leafroll virus coat protein

A panel of ten stable hybridoma cell lines secreting monoclonal antibodies (MAbs) specific for potato leafroll virus (PLRV) antigen, was produced in two fusion experiments with murine splenic and myeloma cells. Using different ELISA procedures and Western blotting it was shown that one MAb detected a continuous epitope and nine MAbs reacted with conformation-dependent ones. The conformation-dependent epitopes could be separated into two groups after alkaline treatment of the virions. The MAbs were further differentiated in competitive binding assays. Within the group of MAbs reacting with epitopes not sensitive to alkaline degradation, only two MAbs were directed to the same epitope. The MAbs detecting epitopes formed by the quaternary protein structure or by a protein subunit configuration sensitive to alkaline degradation, displayed positive cooperative binding among each other. In total, a minimum number of nine different, but overlapping, epitopes on the PLRV coat protein could be revealed. The immune response to PLRV antigen in rabbit appeared to be directed mainly towards epitopes recognized by three MAbs. Most MAbs displayed heterologous reactivity to other luteoviruses, i.e., tomato yellow top virus (TYTV), beet western yellow virus (BWYV), beet mild yellowing virus (BMYV), bean leafroll virus (BLRV), and different strains of barley yellow dwarf virus. Three MAbs solely reacted with PLRV and TYTV. Six MAbs gave different reaction patterns in these tests; one of these MAbs differentiated BMYV from BWYV, and another detected a common epitope on PLRV and BLRV, a serological relationship not reported previously to our knowledge.

Identification and Characterization of the Potato Leafroll Virus Putative Coat Protein Gene

Complementary DNA clones representing approximately 6100 nucleotides of potato leafroll virus (PLRV) were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) encoding a 23K protein was identified and further characterized. Amino acid sequence comparison of this protein showed significant homology (47.1%) with the barley yellow dwarf virus (BYDV-PAV) coat protein. This and other observations suggested that this gene encodes the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including an ORF of 17K within the ORF encoding the 23K putative coat protein.

Nucleotide sequence of the potato leafroll virus coat protein gene.

Nucleotide sequence of the potato leafroll virus coat protein gene.