Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

2011 ◽  
Vol 392 (10) ◽  
pp. 937-945 ◽  
Author(s):  
Kajohn Boonrod ◽  
Marc W. Füllgrabe ◽  
Gabi Krczal ◽  
Michael Wassenegger
Keyword(s):  
Escherichia Coli ◽  
Mosaic Virus ◽  
Zucchini Yellow Mosaic Virus ◽  
Point Mutations ◽  
Tobacco Etch Virus ◽  
Amino Acid Sequences ◽  
Yellow Mosaic Virus ◽  
Helper Component ◽  
C Terminus ◽  
Helper Component Proteinase

AbstractThe multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. SeveralEscherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.

scholarly journals The helper component-proteinase of the Zucchini yellow mosaic virus inhibits the Hua Enhancer 1 methyltransferase activity in vitro

2011 ◽  
Vol 92 (9) ◽  
pp. 2222-2226 ◽  
Author(s):  
Rana M. Jamous ◽  
Kajohn Boonrod ◽  
Marc W. Fuellgrabe ◽  
Mohammed S. Ali-Shtayeh ◽  
Gabi Krczal ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Small Rna ◽  
Rna Binding ◽  
Zucchini Yellow Mosaic Virus ◽  
Indirect Evidence ◽  
Yellow Mosaic Virus ◽  
Methyltransferase Activity ◽  
Helper Component ◽  
Helper Component Proteinase

The helper component-proteinase (HC-Pro) is a multifunctional protein found among potyviruses. With respect to its silencing suppressor function, small RNA binding appears to be the major activity of HC-Pro. HC-Pro could also exhibit other suppressor activities. HC-Pro may inhibit the Hua Enhancer 1 (HEN1) activity. There is indirect evidence showing that either transient or stable expression of HC-Pro in plants results in an increase of non-methylated small RNAs. Here, we demonstrated that recombinant Zucchini yellow mosaic virus (ZYMV) HC-Pro inhibited the methyltransferase activity of HEN1 in vitro. Moreover, we found that the HC-ProFINK mutant, which has lost small RNA-binding activity, inhibited HEN1 activity, while the truncated proteins and total soluble bacterial proteins did not. Using the ELISA-binding assay, we provided evidence that the HC-ProFRNK wild-type and HC-ProFINK both bound to HEN1, with HC-ProFRNK binding stronger than HC-ProFINK. Motif mapping analysis revealed that the amino acids located between positions 139 and 320 of ZYMV HC-Pro were associated with HEN1 interaction.

scholarly journals In vitro association between the helper component–proteinase of zucchini yellow mosaic virus and cuticle proteins of Myzus persicae

2007 ◽  
Vol 88 (5) ◽  
pp. 1602-1610 ◽  
Author(s):  
Aviv Dombrovsky ◽  
Natan Gollop ◽  
Songbi Chen ◽  
Nor Chejanovsky ◽  
Benjamin Raccah
Keyword(s):  
Myzus Persicae ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Two Dimensional Gel Electrophoresis ◽  
Helper Component ◽  
Molecular Masses ◽  
Cuticle Proteins ◽  
Helper Component Proteinase ◽  
Persistently Transmitted Viruses

Potyviruses, as typical non-persistently transmitted viruses, are carried within the stylets of aphids. Cuticle proteins (CuPs), which are a major component of the insect cuticle, were examined for in vitro binding to the potyviral helper component–proteinase (HC–Pro). Proteins in 8 M urea extracts from Myzus persicae were separated by SDS-PAGE, electroblotted onto membranes and identified as CuPs by using specific antibodies to M. persicae CuP. Blotted M. persicae protein extracts were overlaid with two HC–Pros, differing by the presence of K or E in the KLSC domain. The HC–Pro with KLSC, known to assist transmission, was found to bind M. persicae proteins, whereas the HC–Pro with ELSC, being deficient in assisting transmission, did not. To identify CuPs that react with HC–Pro, protein extracts were separated by two-dimensional gel electrophoresis. Nine proteins reacting with HC–Pro were sequenced by mass spectrometry. Sequences of peptides in four proteins, of molecular masses between 22 and 31 kDa, were identified as CuPs according to comparison with sequences in GenBank. The putative CuPs from M. persicae that bind HC–Pro are potentially of interest in locating receptors for virions bound to HC–Pro in aphids’ stylets.

scholarly journals Modifications of the Helper Component-Protease of Zucchini yellow mosaic virus for Generation of Attenuated Mutants for Cross Protection Against Severe Infection

2007 ◽  
Vol 97 (3) ◽  
pp. 287-296 ◽  
Author(s):  
Shih-Shun Lin ◽  
Hui-Wen Wu ◽  
Fuh-Jyh Jan ◽  
Roger F. Hou ◽  
Shyi-Dong Yeh
Keyword(s):  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Zucchini Yellow Mosaic Virus ◽  
Chenopodium Quinoa ◽  
Severe Infection ◽  
Plant Viruses ◽  
Cross Protection ◽  
Yellow Mosaic Virus ◽  
Mild Strain ◽  
Helper Component

A nonpathogenic mild strain is essential for control of plant viruses by cross protection. Three amino acid changes, Arg180→Ile180 (GA mutation), Phe205→Leu205 (GB mutation), and Glu396→Asn396 (GC mutation), of the conserved motifs of the helper component-protease (HC-Pro) of a severe strain TW-TN3 of Zucchini yellow mosaic virus (ZYMV), a member of the genus Potyvirus, were generated from an infectious cDNA clone that carried a green fluorescent protein reporter. The infectivity of individual mutants containing single, double, or triple mutations was assayed on local and systemic hosts. On Chenopodium quinoa plants, the GB mutant induced necrotic lesions; the GA, GC, and GBC mutants induced chlorotic spots; and the GAB and GAC mutants induced local infection only visualized by fluorescence microscopy. On squash plants, the GA, GB, GC, and GBC mutants caused milder mosaic; the GAC mutant induced slight leaf mottling followed by recovering; and the GAB mutant did not induce conspicuous symptoms. Also, the GAC mutant, but not the GAB mutant, conferred complete cross protection against the parental virus carrying a mite allergen as a reporter. When tested on transgene-silenced transgenic squash, the ability of posttranscriptional gene silencing suppression of the mutated HC-Pro of GAC was not significantly affected. We concluded that the mutations of the HC-Pro of ZYMV reduce the degrees of pathogenicity on squash and also abolish the ability for eliciting the hypersensitive reaction on C. quinoa, and that the mutant GAC is a useful mild strain for cross protection.

scholarly journals Mutations in zucchini yellow mosaic virus helper component protein associated with loss of aphid transmissibility

1993 ◽  
Vol 74 (12) ◽  
pp. 2737-2742 ◽  
Author(s):  
F. Granier ◽  
M. Durand-Tardif ◽  
F. Casse-Delbart ◽  
H. Lecoq ◽  
C. Robaglia
Keyword(s):  
Mosaic Virus ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Helper Component ◽  
Component Protein

scholarly journals Fevillea trilobata as a Natural Host of Zucchini yellow mosaic virus in Brazil

Plant Disease ◽  
2013 ◽  
Vol 97 (9) ◽  
pp. 1261-1261 ◽  
Author(s):  
L. S. Boiteux ◽  
D. M. A. Spadotti ◽  
J. A. M. Rezende ◽  
E. W. Kitajima
Keyword(s):  
Amino Acid ◽  
Coat Protein ◽  
Mosaic Virus ◽  
Direct Sequencing ◽  
Zucchini Yellow Mosaic Virus ◽  
Amino Acid Sequences ◽  
Natural Host ◽  
Yellow Mosaic Virus ◽  
Biodiesel Fuel ◽  
Thin Sections

The antidote vines or nhandirobas (Fevillea trilobata L. [Cucurbitaceae]) are dioecious plant species native to the South American Neotropics (1). Genetic materials of these species are now being domesticated and evaluated as potential crops for seed-oil extraction aiming to produce biodiesel fuel (2). Plants of F. trilobata (Accession No. CNPH-001) were cultivated from seeds under open field conditions during the years 2008 through 2011 in Brasília-DF, Brazil. Approximately 200 plants exhibiting mosaic symptoms and severe leaf malformation (with typical bubble-like patches) were found in all fields every year. Apical mosaic was slightly more severe in female than in male plants. Electron microscopy examination of negatively stained extracts of symptomatic leaf tissue showed the presence of filamentous particles about 700 to 800 nm long. Analysis of ultra-thin sections of the same tissues revealed the presence of lamellar inclusions typical of a potyvirus infection. No aphid colonies were observed on field-grown F. trilobata plants. The virus was mechanically transmitted to healthy Cucurbita pepo cv. Caserta and Luffa cylindrica, causing systemic mosaic. Sap from these infected plants reacted in PTA-ELISA with polyclonal antiserum against Zucchini yellow mosaic virus (ZYMV), but not with antisera against Papaya ringspot virus – type W (PRSV-W), Cucumber mosaic virus (CMV), and Zucchini lethal chlorosis virus (ZLCV). Total RNA extracted from experimentally infected C. pepo was analyzed by RT-PCR using specific pairs of primers for the coat protein gene of ZYMV (3). A cDNA fragment of approximately 1,186 bp was amplified and the nucleotide sequence obtained by direct sequencing. Comparisons of the nucleotide (837 nt) and deduced amino acid (279 aa) sequences of the coat protein genomic segment (GenBank Accession No. JX502677) revealed 93 to 98% and 97 to 98% identity, respectively, with the corresponding nucleotide and amino acid sequences of a group of ZYMV isolates from distinct hosts (AY188994, AY279000, and NC_003224). The infection by ZYMV might cause fruit yield losses to F. trilobata. In addition, the infected F. trilobata crops might work as a reservoir of ZYMV providing inoculum to other cucurbit hosts since it has been managed as a semi-perennial crop. To our knowledge, this is the first report of the genus Fevillea as a natural host of ZYMV. References: (1) M. Nee et al. Syst. Bot. 34:704, 2009. (2) E. G. Shay. Biomass Bioenergy 4:227, 1993. (3) K. G. Thomson et al. J. Virol. Meth. 55:83, 1995.

scholarly journals First Report of Papaya ringspot virus–Type W and Zucchini yellow mosaic virus Infecting Trichosanthes cucumerina in Brazil

Plant Disease ◽  
2010 ◽  
Vol 94 (6) ◽  
pp. 789-789 ◽  
Author(s):  
A. S. Jadão ◽  
J. E. Buriola ◽  
J. A. M. Rezende
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Virus Type ◽  
Zucchini Yellow Mosaic Virus ◽  
Amino Acid Sequences ◽  
Ringspot Virus ◽  
Yellow Mosaic Virus ◽  
Papaya Ringspot Virus ◽  
Mechanical Inoculation ◽  
Cp Gene

Trichosanthes cucumerina L., known as snake gourd, is a cucurbitaceous plant that is probably native to and originally domesticated in India. It is cultivated in humid subtropical and tropical countries of Australia, Latin America, and Africa (2). Plants of this species exhibiting symptoms of mosaic and leaf malformation were found during November 2008 near an experimental field of the Departamento de Fitopatologia e Nematologia, Universidade de São Paulo, Piracicaba, State of São Paulo, Brazil. Electron microscopy examination of negatively stained extract of infected tissue showed the presence of filamentous potyvirus-like particles. Sap from these infected plants reacted in plate-trapped antigen (PTA)-ELISA with the antiserum against Papaya ringspot virus–type W (PRSV-W) or Zucchini yellow mosaic virus (ZYMV), but not with the antiserum against Cucumber mosaic virus (CMV) or Zucchini lethal chlorosis virus (ZLCV). PRSV-W and ZYMV were simultaneously transmitted by mechanical inoculation to four plants of Cucurbita pepo cv. Caserta and one plant of T. cucumerina, causing mosaic. In addition, PRSV-W and ZYMV isolates from our virus collection separately infected one plant of T. cucumerina after mechanical inoculation. Infections were confirmed by PTA-ELISA. Total RNA extracted from infected and healthy T. cucumerina was analyzed by reverse transcription (RT)-PCR using a primer pair specific to the coat protein (CP) gene of PRSV-W (4) or ZYMV (3). Fragments of 864 bp and 1,045 bp were amplified with each pair of primers, respectively. Nucleotide sequences directly obtained from purified PCR products were used for further identification of these potyviruses. The nucleotide and deduced amino acid sequences of part of the CP gene (792 nt) of PRSV-W (GenBank Accession No. GU586789) shared 99 and 98% identity, respectively, with that of the Brazilian isolate PRSV-W-C (GenBank Accession No. 4152). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of ZYMV (GenBank Accession No. 6790) shared 91 to 98% and 94 to 100% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AB004640, D13914, AB004641, and AJ420019). Natural infection of T. cucumerina by PRSV-W was reported in Nepal (1). To our knowledge, this is the first report of T. cucumerina infected by PRSV-W and ZYMV in Brazil. References: (1) G. Dahal et al. Ann. Appl. Biol. 130:491, 1997. (2) R. W. Robinson and D. S. Decker-Walters. Cucurbits. CAB International, Wallingford, UK. 1997. (3) K. G. Thomson et al. J. Virol. Methods 55:83, 1995. (4) M. G. S. D. Vechia. Fitopatol. Bras. 28:678, 2003.

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