Potato leafroll virus protein P1 contains a serine proteinase domain

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1857-1864 ◽  
Author(s):  
X. Li ◽  
M. D. Ryan ◽  
J. W. Lamb
Keyword(s):  
Insect Cells ◽  
Plant Cells ◽  
Serine Proteinase ◽  
Potato Leafroll Virus ◽  
Autographa Californica ◽  
Virus Protein ◽  
Conserved Residues ◽  
Intermolecular Reaction ◽  
Polyhedrin Promoter ◽  
Leafroll Virus

The multi-domain potato leafroll virus replicase protein P1 was expressed in insect cells from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Using antisera raised against P1, it was shown that P1 was cleaved near the VPg in insect cells in a manner similar to that in plant cells, to produce a ∼27 kDa C-terminal fragment. Furthermore, it was shown that the proposed serine proteinase-like domain within P1 is responsible for this processing and that this can occur in a trans (intermolecular) reaction. Four conserved residues within the serine proteinase domain that are essential for catalysis have been identified, consistent with the proposal that this domain comprises a serine proteinase.

scholarly journals Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

1985 ◽  
Vol 5 (10) ◽  
pp. 2860-2865 ◽  
Author(s):  
C Miyamoto ◽  
G E Smith ◽  
J Farrell-Towt ◽  
R Chizzonite ◽  
M D Summers ◽  
...  
Keyword(s):  
Insect Cells ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Autographa Californica ◽  
Baculovirus Expression ◽  
Polyhedrin Promoter ◽  
Baculovirus Expression Vector ◽  
Myc Gene ◽  
Infected Insect ◽  
Nuclear Polyhedrosis

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

scholarly journals Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids.

2005 ◽  
Vol 52 (3) ◽  
pp. 699-702 ◽  
Author(s):  
Elzbieta Sułuja ◽  
Ludmiła Strokowskaja ◽  
Włodzimierz Zagórski-Ostoja ◽  
Andrzej Pałucha
Keyword(s):  
Coat Protein ◽  
Nucleic Acids ◽  
Coat Protein Gene ◽  
Insect Cells ◽  
Protein Gene ◽  
Recombinant Baculovirus ◽  
Potato Leafroll Virus ◽  
Rnase A ◽  
Virus Like Particles ◽  
Leafroll Virus

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.

scholarly journals The innate immunity protein C1QBP functions as a negative regulator of circulative transmission of Potato leafroll virus by aphids

2020 ◽  
Author(s):  
Stacy L. DeBlasio ◽  
Jennifer Wilson ◽  
Cecilia Tamborindeguy ◽  
Richard S. Johnson ◽  
Patricia V. Pinheiro ◽  
...  
Keyword(s):  
Innate Immunity ◽  
High Resolution Mass Spectrometry ◽  
Affinity Purification ◽  
Plant Viruses ◽  
Potato Leafroll Virus ◽  
Negative Regulator ◽  
Virus Protein ◽  
Insect Vector ◽  
Immunity Protein ◽  
Leafroll Virus

ABSTRACTThe vast majority of plant viruses are transmitted by insect vectors with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Yet, very few vector proteins interacting with virus have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most effectively by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using an affinity purification strategy coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicae displaying high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Two of these proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid with an additional vector protein displaying binding specificity. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV within cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Chemical inhibition of C1QBP in the aphid leads to increased PLRV acquisition and subsequently increased titer in inoculated plants, supporting the role of C1QBP as a negative regulator of PLRV accumulation in M. persicae. We hypothesize that the innate immune function of C1QBP is conserved in aphids and represents the first instance of aphids mounting an immune response to a non-propagative plant virus. This study presents the first use of AP-MS for the in vivo isolation of functionally relevant insect vector-virus protein complexes.

scholarly journals Production of human c-myc protein in insect cells infected with a baculovirus expression vector

1985 ◽  
Vol 5 (10) ◽  
pp. 2860-2865
Author(s):  
C Miyamoto ◽  
G E Smith ◽  
J Farrell-Towt ◽  
R Chizzonite ◽  
M D Summers ◽  
...  
Keyword(s):  
Insect Cells ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Autographa Californica ◽  
Baculovirus Expression ◽  
Polyhedrin Promoter ◽  
Baculovirus Expression Vector ◽  
Myc Gene ◽  
Infected Insect ◽  
Nuclear Polyhedrosis

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

scholarly journals The ORF0 product of Potato leafroll virus is indispensable for virus accumulation

2001 ◽  
Vol 82 (6) ◽  
pp. 1529-1532 ◽  
Author(s):  
Ewa Sadowy ◽  
Anna Maasen ◽  
Marek Juszczuk ◽  
Chantal David ◽  
Wlodzimierz Zagórski-Ostoja ◽  
...  
Keyword(s):  
Plant Cells ◽  
Potato Leafroll Virus ◽  
Expression Cassette ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Potato Leaf ◽  
Leaf Discs ◽  
Virus Accumulation ◽  
Cdna Expression ◽  
Leafroll Virus

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.

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.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Bacmid Expression of Granulovirus Enhancin En3 Accumulates in Cell Soluble Fraction to Potentiate Nucleopolyhedrovirus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1233
Author(s):  
Adriana Ricarte-Bermejo ◽  
Oihane Simón ◽  
Ana Beatriz Fernández ◽  
Trevor Williams ◽  
Primitivo Caballero
Keyword(s):  
Trichoplusia Ni ◽  
Recombinant Virus ◽  
Spodoptera Exigua ◽  
Insect Cells ◽  
Soluble Fraction ◽  
Autographa Californica ◽  
Insect Midgut ◽  
Baculovirus Infection ◽  
Occlusion Bodies ◽  
Infected Cells

Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy.

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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