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.

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 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 GENETIC ENGINEERING OF THE ZUCCHINI YELLOW MOSAIC VIRUS COAT PROTEIN GENE FOR EXPRESSION IN PLANTS.

HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1130d-1130
Author(s):  
Guowei Fang ◽  
Rebecca Grumet
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Zucchini Yellow Mosaic Virus ◽  
In Vitro Transcription ◽  
Yellow Mosaic Virus ◽  
35S Promoter ◽  
Plant Expression Vector

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.

scholarly journals The Conserved FRNK Box in HC-Pro, a Plant Viral Suppressor of Gene Silencing, Is Required for Small RNA Binding and Mediates Symptom Development

2007 ◽  
Vol 81 (23) ◽  
pp. 13135-13148 ◽  
Author(s):  
Yoel Moshe Shiboleth ◽  
Elina Haronsky ◽  
Diana Leibman ◽  
Tzahi Arazi ◽  
Michael Wassenegger ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Small Rna ◽  
Rna Binding ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Developmental Defect ◽  
Symptom Development ◽  
Suppressor Activity ◽  
Protein Levels ◽  
Interfering Rna

ABSTRACT The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMVFRNK caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA “passenger” strands (miRNA*s), in systemically infected leaves than the attenuated ZYMVFINK did. HC-ProFRNK specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-ProFINK. Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HCFINK mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.

The asparagine residue in the FRNK box of potyviral helper-component protease is critical for its small RNA binding and subcellular localization

2014 ◽  
Vol 95 (5) ◽  
pp. 1167-1177 ◽  
Author(s):  
Nandita Sahana ◽  
Harpreet Kaur ◽  
R. K. Jain ◽  
Peter Palukaitis ◽  
Tomas Canto ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Small Rna ◽  
Rna Silencing ◽  
Rna Binding ◽  
Cytoplasmic Inclusion ◽  
Binding Potential ◽  
Variable Regions ◽  
Helper Component ◽  
Natural Variants

The multifunctional potyviral helper-component protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at the FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variants for the N182 residue are known to occur. Here, a mutant at HcProN182L was developed to investigate its role in natural populations. Using in vitro studies, we found an increase in the small RNA (sRNA) binding potential of HcProN182L without affecting its protein–protein interaction properties, suggesting that the presence of N182 is critical to maintain threshold levels of sRNAs, but does not interfere in the self-interaction of HcPro. Furthermore, we found that expression of HcProN182L in Nicotiana benthamiana affected plant growth. Transient expression of HcProN182L induced reporter gene expression in 16c GFP transgenic plants more than HcPro did, suggesting that replacement of asparagine in the FRNK box favours RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcProN182L was observed only in cytoplasmic inclusion bodies in N. benthamiana leaves, when fused to a GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerization of HcPro. These findings suggest that amino acid N182 of the conserved FRNK box may regulate RNA silencing mechanisms, and is required for maintenance of the subcellular localization of the protein for its multi-functionality. Hence, the N182 residue of the FRNK box seems to be indispensable for potyvirus infection during evolution.

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