Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E.

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.

Genetic variability of potato spindle tuber viroid RNA replicon.

The genetic continuity of the potato spindle tuber viroid (PSTVd) genome was analysed after infection of tomato plants with cloned cDNAs of parental strains. During the six weeks of the experiment, several new sequence variants appeared. The sequence variants detected in the progeny population induced sequence-specific disease symptoms. The PSTVd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. Assessing further the replicon continuity, a PSTVd cDNA mutant with a deletion in the central conserved region was constructed and proven to be non-infectious. Surprisingly, in a sub-population of potato transformants expressing the same deleted PSTVd RNA an infectious viroid was detected. This suggests specific transcript conversion followed by recovery of the full-length pathogen genome.

Restoration of secondary hairpin II is associated with restoration of infectivity of a non-viable recombinant viroid.

Mutagenesis and/or construction of recombinants by exchange of genomic regions between parental molecules constitute powerful tools for the study of viroids. However, a large proportion of such modifications results in molecules, which have lost their infectivity. Such is the case for a recombinant viroid named CECS, obtained by replacing the right half of a citrus exocortis viroid (CEVd) by the same region from chrysanthemum stunt viroid (CSVd). In an effort to recover viable infectious progeny from this recombinant, tomato plants were inoculated with an Agrobacterium strain carrying a dimer of the CECS viroid in positive orientation under the control of the CaMV 35S promoter. About 20% of the plants treated in this way were found to be infected with a replicating viroid, which was further propagated. Sequence analysis of six cloned full-length cDNAs derived from progeny molecules revealed the presence of mutations as compared with the parental CECS sequence. However, only two types of mutations were consistently recovered in all progeny molecules, the addition of a G in a string of four at positions 70-73, a mutation frequently observed in CEVd isolates and mutations leading to the restoration of the correct base pairing in secondary hairpin II. These results show that agro-infection is a suitable technique for the recovery of viable molecules from non-infectious viroid mutants and confirm that the ability to form secondary hairpin II is a prerequisite for viroid infectivity.

Effect of genomic and subgenomic leader sequences of potato leafroll virus on gene expression.

The effect of the genomic and subgenomic leader sequence of potato leafroll polerovirus on the efficiency of translation of the downstream located genes has been studied. The results obtained in vitro and in vivo indicate that neither leader sequence functions as translational enhancer, a generally important feature of leader sequences. Deletion analyses demonstrated that both leader sequences not only decrease translation of the downstream located genes but also alter the ratio of the synthesized proteins. A correlation between the in vitro and in vivo results can be established in the case of the subgenomic leader sequence.

Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism.

New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, genomic cDNA. Two different reverse transcriptases were used (AMV-RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.

The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants.

RNA viruses propagate as a population of genetically related entities composing a quasi-species. Specific representatives are the result of both a high mutation rate during replication and competition between the continuously arising sequence variants. Similar to other RNA pathogens, potato spindle tuber viroid (PSTVd) propagates as a population of similar but nonidentical sequences. The sequence of progeny molecules derived from cloned molecular variants of PSTVd were studied after one and six consecutive plant passages. Although the severe parental sequence S23 was found to be genetically stable, all five other parental sequences analyzed, irrespective of their pathogenicity, led to the appearance of complex populations. Divergence of the progeny was observed at the sequence level, but also, more surprisingly, at the level of the pathogenicity of individual progeny molecules. In two cases, the parental sequence was retained in the progeny population. In the other cases, it was completely out-competed and eliminated, sometimes in as little as one plant passage. Although it has been observed previously that artificially mutated PSTVd molecules may revert rapidly to the wild-type sequence, this study presents direct evidence for the rapid evolution of naturally occurring PSTVd sequence variants.

Tobacco veinal necrosis determinants are unlikely to be located within the 5' and 3' terminal sequences of the potato virus Y genome.

Three potato virus Y isolates, representatives of distinct PVY groups, identified in potato fields in northern Poland were submitted to biological and molecular analysis. Phenotypically, two isolates, PVYN-Ny and PVYN-Wi, belong to the necrotic strain and the third one (PVYO-LW) to the common strain. PVYN-Wi, however, did not react with monoclonal antibodies directed against the necrotic strain isolates which recognise PVYN-Ny. To characterise the isolates, coat protein genes were sequenced and compared with sequences from databases. The necrotic PVYN-Wi isolate showed 99% amino acid homology with the common one-PVYO-LW and significantly differed from the second necrotic isolate (PVYN-Ny). Sequence based homology matrix and phylogenetic analysis lead to classification of PVYN-Ny into group I, encompassing solely necrotic strain isolates, whereas PVYN-Wi falls into a phenotypically heterogeneous group II. The sequence analysis allowed for identification of putative group I-specific epitopes. 3'NTR (non-translated region) sequences were identical for PVYN-Wi and PVYO-LW. The 5'NTR, P1 gene, coat protein gene and 3'NTR sequences of the common (PVYO-LW) and the necrotic (PVYN-Wi) isolates are 99-100% homologous. This suggests that tobacco veinal necrosis determinants are located outside the 3' and 5' terminal sequences of the PVY genome.

Three-dimensional model of the potyviral genome-linked protein.

The full sequence of the genome-linked viral protein (VPg) cistron located in the central part of potato virus Y (common strain) genome has been identified. The VPg gene codes for a protein of 188 amino acids, with significant homology to other known potyviral VPg polypeptides. A three-dimensional model structure of VPg is proposed on the basis of similarity of hydrophobic-hydrophilic residue distribution to the sequence of malate dehydrogenase of known crystal structure. The 5' end of the viral RNA can be fitted to interact with the protein through the exposed hydroxyl group of Tyr-64, in agreement with experimental data. The complex favors stereochemically the formation of a phosphodiester bond [5'-(O4-tyrosylphospho)adenylate] typical for representatives of picornavirus-like viruses. The chemical mechanisms of viral RNA binding to VPg are discussed on the basis of the model structure of protein-RNA complex.

Use of intramolecular chimeras to map molecular determinants of symptom severity of potato spindle tuber viroid (PSTVd).

Nucleotide sequence comparison shows that sequence variations are mostly clustered in the P (pathogenicity) and V (variable) domains of the potato spindle tuber viroid (PSTVd) molecule. Although these comparisons suggest the P domain as the primary determinant of PSTVd symptom severity, the potential contribution of the V domain has never been analysed in detail. To investigate the relationship between the structure of these domains and pathogenicity, six intraspecific chimeric PSTVd variants were constructed by exchanging P and V domains between a mild and two different severe PSTVd isolates. Infectivity studies showed that the P domain is directly responsible for the severity of symptoms induced in tomato. The four recombinants containing a P domain from a severe isolate caused severe symptoms including severe epinasty, stunting and veinal necrosis, while the two chimeras containing the mild isolate P domain induced only mild symptoms. Quantitation of viroid accumulation in plants infected with the various recombinants suggests that, with the constructions used, symptom severity did not correlate with viroid accumulation, indicating that the P domain did not influence symptom production through this simple mechanism.

Nucleotide sequence of RNA of a Polish isolate of potato leafroll luteovirus.

The nucleotide sequence of the genomic RNA of a Polish isolate of a potato leafroll virus was determined. Some variations between the determined sequences were observed. A comparison of the frequency of sequence variants in particular regions of the genome is presented.

Digoxigenin-labelled molecular probe for the simultaneous detection of three potato pathogens: potato spindle tuber viroid (PSTVd), potato virus Y (PVY), and potato leafroll virus (PLRV).

A molecular probe, p3POT, was constructed of PSTVd, PVY, PLRV cDNA fragments introduced into pUC18 vector. Sequencing of the inserts revealed that cloned fragments covered conservative parts of pathogenic genomes. Dot-blot hybridization of digoxigenin-labelled construct to crude extracts from plants infected with different potato viruses proved high sensitivity and specificity of the p3POT probe. This makes p3POT probe an useful tool for the routine testing, and selection of virus-free potatoes.

Analysis of the population structure of three phenotypically different PSTVd isolates.

Phenotypically dissimilar greenhouse isolates from a Polish collection of potato spindle tuber viroid (PSTVd) were analysed. Partially purified PSTVd genomic RNAs from severe, intermediate and mild isolates was reverse transcribed and the resulting cDNAs enzymatically amplified. Abutting-primer PCR (Ab-P PCR) technology was used to obtain, in a single step, infectious full-length PSTVd cDNA monomers and these were sequenced. The mild isolate was found to be composed of a unique molecular variant (M), closely related to previously described PSTVd mild isolates. In the intermediate isolate, three variants, i2, i3 and i4, were detected. The severe isolate was found to be a mixture containing at least four molecular variants: s23, s27, i4 and i2. Infection of test plants with plasmids carrying monomeric cDNAs corresponding to each of the cloned variants confirmed that they are infectious. In addition, variant M produced mild symptoms, variants i2, i3, i4 intermediate symptoms and variants s23 and s27 severe symptoms. Therefore, the disease symptoms produced by a mixture are determined by the severe variants, masking the presence of milder ones. All the variants detected (except i2 which is identical to previously described PSTVd-DI) represent novel PSTVd sequences with point mutations located in the V and/or P domains. In particular, variants s23 and i4 represent shorter (358 nucleotides) versions of the PSTVd genome.

Purification and characterization of the Saccharomyces cerevisiae mitochondrial leucyl-tRNA synthetase.

We have purified the product of the NAM2 gene, the mitochondrial leucyl-tRNA synthetase, from yeast mitochondria. The purified protein cross-reacts with antibodies raised against the product of a LacZ/NAM2 gene fusion and antibodies raised against the purified Escherichia coli leucyl-tRNA synthetase. The mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 100 kDa, consistent with the size predicted by the gene sequence (102 kDa). The N-terminal sequence of the protein has been determined and shows that the first nine amino acids predicted by the gene sequence have been removed, probably during transport into the mitochondria.

Detection of potato spindle tuber viroid (PSTV) in dormant potato tubers by concatameric cDNA probe.

The 32P-labelled concatameric insert cut out from a plasmid pSPAv6.2(+), containing 6.2 copies of a full-length PSTV, was used to detect PSTV in dormant potato tubers by dot-blot hybridisation assay. The concatameric insert probe was 4 times more sensitive than the monomeric one. This allowed the detection of 0.5 pg of viroid RNA. The sensitivity makes th eoligomeric cDNA probe a useful alternative to cDNA probes.

Characterisation of synthetic DNA probe detecting potato spindle tuber viroid.

Chemically synthesized DNA fragments complementary to selected regions of the potato spindle tuber viroid (PSTV) genome were cloned into Escherichia coli plasmid pUC9. One of the recombinant plasmids (pIBB4) with a 87 bp insert representing the central region of the PSTV genome (nucleotides 88 to 174) was used after labelling by nick translation for detecting PSTV by dot-blot hybridization. The molecular probe was almost as sensitive as the one carrying the full genomic PSTV copy (pAV401), detecting down to 20 pg of PSTV RNA in 8-15 micrograms of infected tissue. The specificity of the test was high; no signals were created by extracts from healthy plants or plants infected with a variety of common potato viruses. The probe is potentially useful in studies on mixed infections of potatoes with different viruses, and in selection of certified seed material.

Stable preparation of yeast mitochondria and mitoplasts synthesizing specific polypeptides.

Yeast mitochondria isolated in the presence of 0.6 M sorbitol and 0.5% bovine serum albumin can be stored in liquid nitrogen without loss of translational activity. Frozen mitochondria retain the respiratory control and the mutant pattern of polypeptide synthesis identical to those detected for fresh preparations. Stored mitochondria may be efficiently transformed into a stable preparation of mitoplasts actively synthesizing mitochondrial polypeptides.

Nuclear omnipotent suppressors of premature termination codons in mitochondrial genes affect the 37S mitoribosomal subunit.

nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit- mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit. Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides. These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes.

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.

mim3 and nam3 omnipotent suppressor genes similarly affect the polypeptide composition of yeast mitoribosomes.

Yeast informational suppressors of mit- mutations coded for by nuclear (nam3-1, nam3-2) or by mitochondrial DNA (mim3-1) affect the mitoribosome. Nuclear mutations result in the appearance of an additional polypeptide called SI in the small mitoribosomal subunit. An identical polypeptide, not detected in the wild type 37S subunit, is present in crude preparations of mitoribosomes isolated from a mim3-1 suppressor carrying strain. Traces of the SI polypeptide may be found in highly purified small subunits from the mim3-1 strain. Therefore, mutations affecting either mitochondrial rRNA (mim3-1) or mitochondrial r-proteins (nam3-1, nam3-2) could be followed by similar changes in overall mitoribosome structure. This may explain the functional similarity of nuclear and mitochondrially coded suppressors.