scholarly journals Nucleolar localization of potato leafroll virus capsid proteins

2005 ◽  
Vol 86 (10) ◽  
pp. 2891-2896 ◽  
Author(s):  
Sophie Haupt ◽  
Tanya Stroganova ◽  
Eugene Ryabov ◽  
Sang Hyon Kim ◽  
Gill Fraser ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Potato Leafroll Virus ◽  
Capsid Proteins ◽  
Immunogold Electron Microscopy ◽  
Nucleolar Localization ◽  
Major Protein ◽  
Translational Readthrough ◽  
Minor Protein ◽  
Leafroll Virus

Potato leafroll virus (PLRV) encodes two capsid proteins, major protein (CP) and minor protein (P5), an extended version of the CP produced by occasional translational ‘readthrough’ of the CP gene. Immunogold electron microscopy showed that PLRV CP is located in the cytoplasm and also localized in the nucleus, preferentially targeting the nucleolus. The nucleolar localization of PLRV CP was also confirmed when it was expressed as a fusion with green fluorescent protein (GFP) via an Agrobacterium vector. Mutational analysis identified a particular sequence within PLRV CP involved in nucleolar targeting [the nucleolar localization signal (NoLS)]. Minor protein P5 also contains the same NoLS, and was targeted to the nucleolus when it was expressed as a fusion with GFP from Agrobacterium. However, P5–GFP lost its nucleolar localization in the presence of replicating PLRV.

scholarly journals A novel cleavage site within the potato leafroll virus P1 polyprotein

2007 ◽  
Vol 88 (5) ◽  
pp. 1620-1623 ◽  
Author(s):  
Xuejun Li ◽  
Claire Halpin ◽  
Martin D. Ryan
Keyword(s):  
Serine Protease ◽  
Cleavage Site ◽  
Mutational Analysis ◽  
Peptide Sequencing ◽  
Potato Leafroll Virus ◽  
Proteolytic Processing ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Sequencing Studies ◽  
Leafroll Virus

To study the proteolytic processing of the potato leafroll virus replicase proteins, the multidomain P1 protein with a c-myc epitope tag attached at the N terminus was expressed in insect cells by using the baculovirus system. Western blotting showed that P1 was cleaved at a site upstream of the serine protease domain, in addition to the cleavage site downstream of the protease domain. Mutational analysis showed that the serine protease domain within P1 was responsible for this cleavage. To characterize this novel cleavage site further, a portion of the P1 protein comprising the protease domain and the two cleavage sites was expressed in Escherichia coli. A similar cleavage event was observed in bacteria and was abolished when the P1 protease was inactivated by mutation. Peptide-sequencing studies indicated that this cleavage occurred at a Glu/Arg junction, separating the N-terminal 204 residues from the serine protease domain of P1.

scholarly journals Evidence for RNA-mediated defence effects on the accumulation of Potato leafroll virus

2001 ◽  
Vol 82 (12) ◽  
pp. 3099-3106 ◽  
Author(s):  
Hugh Barker ◽  
Kara D. McGeachy ◽  
Eugene V. Ryabov ◽  
Uli Commandeur ◽  
Mike A. Mayo ◽  
...  
Keyword(s):  
Coat Protein ◽  
Transgenic Plants ◽  
Vascular Tissue ◽  
Fluorescent Protein ◽  
Vascular System ◽  
Potato Leafroll Virus ◽  
Transcriptional Gene Silencing ◽  
Mesophyll Cells ◽  
Tobacco Plants ◽  
Leafroll Virus

In plants infected with Potato leafroll virus (PLRV), or other luteoviruses, infection is very largely confined to cells in the vascular system. Even in tobacco plants transformed with PLRV full-length cDNA, in which all mesophyll cells should synthesize infectious PLRV RNA transcripts, only a minority of the mesophyll cells accumulate detectable amounts of virus. We have explored this phenomenon further by transforming a better PLRV host, Nicotiana benthamiana, with the same transgene, by superinfecting transformed plants with Potato virus Y and by producing tobacco plants in which cells contained both PLRV cDNA and DNA encoding the P1/HC-Pro genes of the potyvirus Tobacco etch virus. A greater proportion of cells in superinfected plants or in doubly transgenic plants accumulated PLRV than did in singly transgenic tobacco plants. However, most cells in these plants did not accumulate virus. To investigate restriction of the multiplication of viruses containing PLRV sequences, transgenic plants were infected with a chimeric virus that consisted of Tobacco mosaic virus (TMV) containing genes for either the coat protein (CP) of PLRV or jellyfish green fluorescent protein (GFP) in place of the TMV coat protein. The virus that encoded PLRV CP spread more slowly and accumulated less extensively than did the virus that expressed GFP. The results support the suggestion that an RNA-mediated form of resistance that resembles post-transcriptional gene silencing operates in non-vascular cells and may be part of the mechanism that restricts PLRV to vascular tissue in conventionally infected plants.

scholarly journals Tagging Potato leafroll virus with the jellyfish green fluorescent protein gene

2000 ◽  
Vol 81 (3) ◽  
pp. 617-626 ◽  
Author(s):  
Kulpash M. Nurkiyanova ◽  
Stewart M. Gray ◽  
Ulrich Commandeur ◽  
Michael E. Taliansky ◽  
George H. Duncan ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Potato Leafroll Virus ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent ◽  
Leafroll Virus

scholarly journals The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 585 ◽  
Author(s):  
Stacy DeBlasio ◽  
Yi Xu ◽  
Richard Johnson ◽  
Ana Rebelo ◽  
Michael MacCoss ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Affinity Purification ◽  
Potato Leafroll Virus ◽  
Structural Proteins ◽  
Mitochondrial Outer Membrane ◽  
Virus Movement ◽  
Cell Periphery ◽  
Movement Proteins ◽  
C Terminus ◽  
Leafroll Virus

The Luteoviridae is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of Potato leafroll virus complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the virus to the cell periphery.

scholarly journals Potato Leafroll Virus Binds to the Equatorial Domain of the Aphid Endosymbiotic GroEL Homolog

1998 ◽  
Vol 72 (1) ◽  
pp. 358-365 ◽  
Author(s):  
Saskia A. Hogenhout ◽  
Frank van der Wilk ◽  
Martin Verbeek ◽  
Rob W. Goldbach ◽  
Johannes F. J. M. van den Heuvel
Keyword(s):  
Mutational Analysis ◽  
Sequence Similarity ◽  
Promoter Sequence ◽  
Potato Leafroll Virus ◽  
Spatial Proximity ◽  
Virus Binding ◽  
E Coli ◽  
Capsid Integrity ◽  
Leafroll Virus

ABSTRACT A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) ofMyzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis. MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family. The genomic organization of the Buchnera groE operon is similar to that of thegroE operon of E. coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding. Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding.

scholarly journals Identifying the Determinants in the Equatorial Domain ofBuchnera GroEL Implicated in Binding Potato Leafroll Virus

2000 ◽  
Vol 74 (10) ◽  
pp. 4541-4548 ◽  
Author(s):  
Saskia A. Hogenhout ◽  
Frank van der Wilk ◽  
Martin Verbeek ◽  
Rob W. Goldbach ◽  
Johannes F. J. M. van den Heuvel
Keyword(s):  
Tertiary Structure ◽  
Mutational Analysis ◽  
Binding Capacity ◽  
Potato Leafroll Virus ◽  
Terminal Region ◽  
The Other ◽  
Deletion Mutants ◽  
Virus Binding ◽  
Aphid Vector ◽  
Leafroll Virus

ABSTRACT Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV;Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.

Green peach aphid and potato leafroll virus: transmission and control

2020 ◽  
Author(s):  
R.A. Bagrov ◽  
◽  
V.I. Leunov
Keyword(s):  
Myzus Persicae ◽  
Green Peach Aphid ◽  
Virus Transmission ◽  
Potato Leafroll Virus ◽  
Factors Affecting ◽  
Potato Viruses ◽  
Tuber Necrosis ◽  
Aphid Vectors ◽  
Leafroll Virus ◽  
And Control

The mechanisms of transmission of potato viruses from plants to aphid vectors and from aphids to uninfected plants are described, including the example of the green peach aphid (Myzus persicae, GPA). Factors affecting the spreading of tuber necrosis and its manifestation on plants infected with potato leafroll virus (PLRV) are discussed. Recommendations for PLRV and GPA control in the field are given.

scholarly journals Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Adam Taylor ◽  
Xiang Liu ◽  
Ali Zaid ◽  
Lucas Y. H. Goh ◽  
Jody Hobson-Peters ◽  
...  
Keyword(s):  
Host Cell ◽  
Capsid Protein ◽  
Chikungunya Virus ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Vaccine Design ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Nucleolar Localization ◽  
Localization Sequence

ABSTRACTMosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of theTogaviridaefamily responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infectionin vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.IMPORTANCECHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain affecting millions of people. After a series of major outbreaks over the last 10 years, CHIKV and its mosquito vectors have been able to expand their range extensively, now making CHIKV a human pathogen of global importance. With no licensed vaccine or antiviral therapy for the treatment of CHIKV disease, there is a growing need to understand the molecular determinants of viral pathogenesis. These studies identify a previously uncharacterized nucleolar localization sequence (NoLS) in CHIKV capsid protein, begin a functional analysis of site-directed mutants of the capsid protein NoLS, and examine the effect of the NoLS mutation on CHIKV pathogenesisin vivoand its potential to influence CHIKV vaccine design. A better understanding of the pathobiology of CHIKV disease will aid the development of effective therapeutic strategies.

scholarly journals Nucleotide sequence of the potato leafroll virus coat protein gene

1989 ◽  
Vol 17 (4) ◽  
pp. 1768-1768 ◽  
Author(s):  
B. Prill ◽  
E. Maiss ◽  
U. Timpe ◽  
R. Casper
Keyword(s):  
Nucleotide Sequence ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Potato Leafroll Virus ◽  
Virus Coat Protein ◽  
Virus Coat ◽  
Leafroll Virus

scholarly journals Host-Dependent Requirement for the Potato leafroll virus 17-kDa Protein in Virus Movement

2002 ◽  
Vol 15 (10) ◽  
pp. 1086-1094 ◽  
Author(s):  
Lawrence Lee ◽  
Peter Palukaitis ◽  
Stewart M. Gray
Keyword(s):  
Amino Acids ◽  
Solanum Tuberosum ◽  
Nicotiana Benthamiana ◽  
Systemic Infection ◽  
Potato Leafroll Virus ◽  
Virus Movement ◽  
Wild Type ◽  
Mature Leaves ◽  
Leafroll Virus ◽  
Virus Distribution

The requirement for the 17-kDa protein (P17) of Potato leafroll virus (PLRV) in virus movement was investigated in four plant species: potato (Solanum tuberosum), Physalis floridana, Nicotiana benthamiana, and N. clevelandii. Two PLRV P17 mutants were characterized, one that does not translate the P17 and another that expresses a P17 missing the first four amino acids. The P17 mutants were able to replicate and accumulate in agroinoculated leaves of potato and P. floridana, but they were unable to move into vascular tissues and initiate a systemic infection in these plants. In contrast, the P17 mutants were able to spread systemically from inoculated leaves in both Nicotiana spp., although the efficiency of infection was reduced relative to wild-type PLRV. Examination of virus distribution in N. benthamiana plants using tissue immunoblotting techniques revealed that the wild-type PLRV and P17 mutants followed a similar movement pathway out of the inoculated leaves. Virus first moved upward to the apical tissues and then downward. The P17 mutants, however, infected fewer phloem-associated cells, were slower than wild-type PLRV in moving out of the inoculated tissue and into apical tissues, and were unable to infect any mature leaves present on the plant at the time of inoculation.

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