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L Pinck

Publications and source records attributed to L Pinck.

At least 19 recordsLinked to original sources
PubMed2001

Involvement of RNA2-encoded proteins in the specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index.

The nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by the nematode Xiphinema index. To identify the RNA2-encoded proteins involved in X. index-mediated spread of GFLV, chimeric RNA2 constructs were engineered by replacing the 2A, 2B(MP), and/or 2C(CP) sequences of GFLV with their counterparts in Arabis mosaic virus (ArMV), a closely related nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index. Among the recombinant viruses obtained from transcripts of GFLV RNA1 and chimeric RNA2, only those which contained the 2C(CP) gene (504 aa) and 2B(MP) contiguous 9 C-terminal residues of GFLV were transmitted by X. index as efficiently as natural and synthetic wild-type GFLV, regardless of the origin of the 2A and 2B(MP) genes. As expected, ArMV was not transmitted probably because it is not retained by X. index. These results indicate that the determinants responsible for the specific spread of GFLV by X. index are located within the 513 C-terminal residues of the polyprotein encoded by RNA2.

Animals↗
PubMed2000

GFLV replication in electroporated grapevine protoplasts.

Grapevine fanleaf virus (GFLV), responsible for the economically important court-noué disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 µg) or viral RNA (10 ng) in both protoplast populations, as shown by anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation.

Journal Article↗
PubMed1999

Protein 2A of grapevine fanleaf nepovirus is implicated in RNA2 replication and colocalizes to the replication site.

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

Base Sequence↗
PubMed1999

The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread.

The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

Amino Acid Sequence↗
PubMed1995

The 119 kDa and 124 kDa polyproteins of arabis mosaic nepovirus (isolate S) are encoded by two distinct RNA2 species.

Arabis mosaic virus (ArMV) is a nepovirus that is serologically distantly related to grapevine fanleaf virus (GFLV). Both ArMV and GFLV induce grapevine degeneration disease. Several ArMV isolates, unlike isolates of GFLV, produce upon in vitro translation of RNA2 a polyprotein (P2) that forms a double band in polyacrylamide-SDS gels. Cloning of full-length copies of RNA2 of an ArMV isolate from grapevine (ArMV-S) revealed that this isolate contained two RNA2s of different length, called RNA2-U and RNA2-L. The two species were not readily separated by electrophoresis of the virion RNA under denaturing gel electrophoresis conditions but could be distinguished by analysis of primer extension and in vitro translation products. The size difference of the two RNA2s is due mostly if not exclusively to differences in their coding regions. The 124 kDa RNA2-U-encoded polyprotein P2' and the 119 kDa RNA2-L-encoded polyprotein P2", which co-migrate, respectively, with the upper and lower polyprotein bands produced by RNA2 of ArMV-S, were more than 95% identical except in their N-terminal domains. In vitro maturation experiments and sequence comparisons indicate that the N-terminal products of P2' and P2" have a molecular mass of 31 kDa and 26 kDa. The genomic organization proposed is similar to that of GFLV RNA2.

Amino Acid Sequence↗
PubMed1995

Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo.

The putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38). This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38. Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein. This protein was detected as early as 18 h post-inoculation in GFLV-infected Chenopodium quinoa protoplasts and accumulated to very high levels. Tissue-prints and time course experiments on infected C. quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo. P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations. No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro. Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes. Thought to be the GFLV movement protein, P38 would thus behave in an 'atypical' manner.

Amino Acid Sequence↗
PubMed1994

Differential proteolytic activities of precursor and mature forms of the 24K proteinase of grapevine fanleaf nepovirus.

The presence of a genome-linked protein (VPg) at the RNA 5'-end of the genome is a characteristic of different groups of animal and plant positive-sense single-stranded RNA viruses. These viruses express their structural and functional proteins from polyproteins that are sequentially processed by at least one viral proteinase. The grapevine fanleaf nepovirus 24K chymotrypsin-like cysteine proteinase, located between the VPg and the RNA polymerase in the RNA-1 encoded polyprotein P1, is active in its free form and in various precursors forms. The VPg proteinase precursor (VPg-Pro) constitutes a stable protein and its maturation in the reticulocyte lysate system occurs at a very low rate. Differences on cleavage activity were observed between the proteinase and its VPg-Pro precursor forms, depending upon the cleavage site considered. The proteinase alone has a greater cleavage efficiency than VPg-Pro at the Arg605/Gly606 and Cys257/Ala258 sites of polyprotein P2. On the other hand, the presumed Cys415/Ala416 site, present at the amino terminus of polyprotein P1, was preferentially cleaved by the VPg-Pro precursor. During their in vitro maturation, proteins containing the VPg proteinase-polymerase coding region or the proteinase-polymerase region were similar in their ability to cleave in cis between the proteinase and the RNA polymerase.

Base Sequence↗
PubMed1993

VPg Northern-immunoblots as a means for detection of viral RNAs in protoplasts or plants infected with grapevine fanleaf nepovirus.

Anti-genome-linked viral protein (anti-VPg) antibodies were produced from a synthetic peptide corresponding to the integral VPg sequence of grapevine fanleaf nepovirus-F13. These antibodies allowed detection of viral VPg-linked proteins which occur during the processing of viral polyproteins and of viral RNAs in total RNA extracts from infected protoplasts or plants after Northern blotting. These highly specific antibodies recognised RNAs from two grapevine fanleaf virus strains but not from arabis mosaic virus.

Antibodies, Viral↗
PubMed1993

Location of the replication determinants of the satellite RNA associated with grapevine fanleaf nepovirus (strain F13).

A large satellite RNA of 1114 nucleotides, named RNA3, is always found associated with the genomic RNAs of grapevine fanleaf virus, isolate F13 (GFLV-F13). RNA3 encodes a non-structural protein (P3) of M(r) 37K to which no function has previously been assigned. Full-length cDNA clones of RNA3 were mutated in the 5' and 3' non-coding regions and in the 37K open reading frame. The ability of transcripts obtained from these clones to be replicated was investigated by protoplast infection in the presence of a helper virus. We demonstrate that the 5' and 3' non-coding regions as well as the satellite-encoded P3 protein are essential for replication of the GFLV-F13 satellite RNA. Our results suggest that two hydrophobic regions located at the N- and C-extremity of P3 and a zinc-finger motif near the C-terminal extremity of P3 are probably involved in the replication of this satellite. Analysis of the in vitro translation products from transcripts of RNA3 clones of different lengths indicates that the double band formed by P3 could result from phosphorylation of a part of this protein.

Amino Acid Sequence↗
PubMed1993

Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs.

Transcripts were produced in vitro by run-off transcription from full-length cDNA of RNA1 and RNA2 of grapevine fanleaf nepovirus (GFLV; isolate F13) cloned downstream from a bacteriophage RNA polymerase promoter. These transcripts, which possess a 5' terminal cap structure and a non-viral G residue instead of the naturally occurring genome-linked viral protein (VPg), are infectious to Chenopodium quinoa protoplasts when inoculated by electroporation. Synthetic RNA1 alone replicated in protoplasts. Inoculation of C. quinoa plants with synthetic RNA1 plus RNA2 produced symptoms similar to, but weaker, than those observed in plants infected with natural GFLV 6 to 8 days post-inoculation. Co-inoculated RNA1 and RNA2 were able to replicate and spread systemically in plants but RNA1 alone produced no symptoms and was not detected in non-inoculated leaves, suggesting that virus spread requires RNA2. Analysis of the genomic RNAs in plants infected with transcripts showed that the non-viral G at their 5' ends was not retained in the progeny.

Base Sequence↗
PubMed1993

Genome organization of grapevine fanleaf nepovirus RNA2 deduced from the 122K polyprotein P2 in vitro cleavage products.

The full-length transcript of grapevine fanleaf virus (GFLV) RNA2 produces a primary product of 122K when translated in the rabbit reticulocyte system. This 122K polyprotein is completely processed in vitro by the RNA1-encoded 24K proteinase. The positions of the cleavage sites within the polyprotein have been mapped and the genome organization of GFLV-F13 RNA2 has been established. The order of mature proteins in the 122K polyprotein is the amino-terminal 28K protein, the 38K protein followed by the 56K coat protein at the carboxy terminus. These proteins represent the final cleavage products of the 122K polyprotein. A 66K protein which yields 28K and 38K proteins constitutes the major maturation intermediate. Microsequencing of the amino extremity of radioactively labelled 38K protein allowed identification of the Cys257/Ala258 site as the cleavage site recognized by the GFLV proteinase between the 28K and the 38K proteins in the 66K protein in addition to the Arg605/Gly606 site between the 38K protein and the coat protein.

Amino Acid Sequence↗
PubMed1992

Effects of site-directed mutagenesis on the presumed catalytic triad and substrate-binding pocket of grapevine fanleaf nepovirus 24-kDa proteinase.

Grapevine fanleaf nepovirus (GFLV) has a bipartite plus-sense RNA genome. Its structural and functional proteins originate from polyprotein maturation by at least one virus-encoded proteinase. Here we describe the cloning of the 24-kDa proteinase cistron located between the virus-linked protein (VPg) and the RNA-dependent RNA polymerase cistron in GFLV RNA1 (nucleotides 3966 to 4622). Proteinase expressed from this clone is able to cleave GFLV polyprotein P2 in order to produce the coat protein and a 66-kDa protein which is further processed to the 38-kDa presumed movement protein. The GFLV 24-kDa proteinase sequence contains sequence similarities with other nepovirus and comovirus proteinases, particularly at the level of the conserved domains corresponding to the hypothetical catalytic triad and to the substrate-binding pocket (amino acids 192 to 200). Site-directed mutagenesis of residues His43, Glu87, and Leu197 abolished proteinase activity. Inactivation of the enzyme is also observed if the catalytic residue Cys179 was substituted by isoleucine, but replacement by a serine at the same position produced a mutant with an activity identical to that of native proteinase. All our data show that GFLV cysteine proteinase presents structure similarities to the proteinases of cowpea mosaic virus and potyviruses but is most closely related to trypsin.

Amino Acid Sequence↗
PubMed1992

Replication of grapevine fanleaf virus satellite RNA transcripts in Chenopodium quinoa protoplasts.

A set of full-length cDNA clones of the satellite RNA of grapevine fanleaf nepovirus isolate F13 (GFLV-F13) was constructed with a variable number of additional, non-viral nucleotides at the 5' and 3' ends. The biological activity of the RNAs transcribed from these constructs was tested in Chenopodium quinoa protoplasts using a helper virus. When inoculated with arabis mosaic virus S (ArMV-S) RNA as helper, transcripts with 33 non-viral nucleotides at the 5' end (tr45p4) did not replicate, whereas transcripts with only one non-viral nucleotide at the 5' end (tr3S and tr3M) did replicate. Capping of the transcripts enhanced their replication. On the other hand, the presence of extra nucleotides at the 3' end had little influence on the biological activity of the in vitro transcripts. In contrast with ArMV-S, GFLV isolate 24 was not a helper for tr3M transcripts, indicating a specific interaction between the helper strain and the satellite RNA.

Base Sequence↗
PubMed1992

Immunodetection of grapevine fanleaf virus satellite RNA-encoded protein in infected Chenopodium quinoa.

An antiserum was raised against a fusion protein containing the C-terminal half of the protein (P3) encoded by the satellite RNA of grapevine fanleaf virus (GFLV; F13 isolate) and the N-terminal portion of the CI repressor of phage lambda. This antiserum specifically recognized P3 synthesized in the in vitro wheatgerm translation system and also in infected Chenopodium quinoa plants. In these plants, the amount of virus increased for 10 days, then remained constant for up to 21 days, whereas P3 was detected transiently, reaching its maximum on day 10.

Antibodies, Viral↗
PubMed1991

In vitro expression of a chimeric coat protein gene from Grapevine Fanleaf virus (strain F 13).

The coat protein (CP) cistron of Grapevine Fanleaf virus strain F13 (GFLV-F13) has been located in the C-terminal region of the 122k polyprotein encoded by the genomic RNA 2 [Serghini et al. (1990) J. Gen. Virol. 71: 1433-1441]. A chimeric CP gene of GFLV-F13 including a short sequence corresponding to 3 restriction sites, the leader sequence of the GFLV-F13 satellite RNA and an initiation codon was constructed. Transcripts from this construct were translated in wheat germ extract with equal efficiency to form a 56k protein which comigrates on PAGE with the GFLV-F13 CP and a protein of 52k. Both species react with GFLV-F13 CP-specific antibodies. Deletions in the 5' region of the CP gene show that the 56k protein is initiated at the first AUG after the satellite leader and the 52k protein at the second in-frame AUG. Transcripts with a 142 nt deletion including the two AUG codons from the 5' end of the CP gene are not efficiently expressed in vitro, no major translation product being detected.

Base Sequence↗
PubMed1991

Cloning and in vitro characterization of the grapevine fanleaf virus proteinase cistron.

The region of the genomic RNA-1 from grapevine fanleaf virus isolate F13 (GFLV-F13), containing the proteinase cistron and flanking sequences (nucleotides 3894 to 4789) of the GFLV polyprotein, was modified by PCR mutagenesis to create a start codon and cloned in a transcription vector. The transcripts from the resulting clone (pVP7) produced, upon translation in rabbit reticulocyte lysate, a 37.8-kDa protein which was subsequently cleaved to a stable 28-kDa product. Autocleavage was maximal at pH 7.0-8.5 and at 30 degrees. Inhibition of the activity was greater than 80% when translation was performed in the wheat germ system. In rabbit reticulocyte lysate, inhibition was also obtained with PMSF, EDTA, E-64, Ca+2, Zn+2, and Co+2. The pVP7 translation product acts in cis, in the case of its autocleavage, or in trans in the processing of the viral 122-kDa polyprotein from GFLV RNA-2 into a 66-kDa protein and the 56-kDa coat protein. The carboxy extremity of the complete pVP7 translation product, encoded by nucleotides 4633 to 4789 of RNA-1, was not required for the proteinase activity, at least in trans.

Amino Acid Sequence↗
PubMed1991

Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1.

The nucleotide sequence of the genomic RNA1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-F13) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picronavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-F13 RNA1. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the substrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease cofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.

Amino Acid Sequence↗