Włodzimierz Zagórski

Virulence Factor of Potato Virus Y, Genome-attached Terminal Protein VPg, Is a Highly Disordered Protein

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5′ end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.

Analysis of the population structure of three phenotypically different PSTVd isolates

SummaryPhenotypically 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.

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

SummaryNucleotide 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.

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.

An antisense coat protein gene confers immunity to potato leafroll virus in a genetically engineered potato

The Bzura commercial potato cultivar was transformed by sense or antisense constructs which included the coat protein gene of potato leafroll virus RNA. In the sense construct, the coat protein gene was preceded by a leader sequence shorter than that in the subgenomic RNA formed in infected cells. The antisense construct consisted of a sequence complementary to the first 2020 nucleotides of the subgenomic RNA. Selected transformants expressing viral RNA were resistant to virus challenge by viruliferous aphids. In one line, expression of the antisense RNA prevented virus infection even after grafting with scions from infected plants and therefore this transformant might be regarded as virus immune.

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.

Protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes

Yeast mitochondria isolated from two different wild type strains (gal+ and gal-), whether grown on galactose or glucose, synthesize all mitochondrial polypeptides with similar efficiencies and in proportions approximating those detected in vivo. Mitochondria isolated from mit- mutants synthesize in vitro a mutant pattern of mitochondrial proteins, indistinguishable from the in vivo products. The mutant pattern is restored to the wild type one in mitochondria isolated from pseudorevertant strains carrying an additional nuclear (nam3-1 and R705) or mitochondrial (mim3-1) informational suppressor gene. Suppression is expressed in isolated mitochondria without the obligatory presence of cytosol at the level of both respiratory control and specific polypeptide synthesis. Translation in isolated mitochondria is sensitive to paromomycin. The antibiotic differentiates between translation in mitochondria from wild type strains and that in nam-type gene carrying strains. This strongly suggests that nam-type mutations affect the mitoribosome, enhancing ambiguity of translation, thus allowing for the pseudoreversion of mit- phenotypes.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases. RIO kinases contain a canonical eukaryotic protein kinase fold, but also display several unusual structural features that potentially create opportunity for the design of selective inhibitors. In an attempt to identify structural leads to target the RIO kinases, a series of pyridine caffeic acid benzyl amides (CABA) were tested for their ability to inhibit the autophosphorylation activity of Archeaoglobus fulgidus Rio1 (AfRio1). Screening of a small library of CABA molecules resulted in the identification of four compounds that measurably inhibited AfRio1 activity. Additional biochemical characterization of binding and inhibition activity of these compounds demonstrated an ATP competitive inhibition mode, and allowed identification of the functional groups that result in the highest binding affinity. In addition, docking of the compound to the structure of Rio1 and determination of the X-ray crystal structure of a model compound (WP1086) containing the desired functional groups allowed detailed analysis of the interactions between these compounds and the enzyme. Furthermore, the X-ray crystal structure demonstrated that these compounds stabilize an inactive form of the enzyme. Taken together, these results provide an important step in identification of a scaffold for the design of selective molecular probes to study molecular mechanisms of Rio1 kinases in vitro and in vivo. In addition, it provides a rationale for the future design of potent inhibitors with drug-like properties targeting an inactive form of the enzyme. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Use of randomly mutagenized genomic cDNA banks of potato spindle tuber viroid to screen for viable versions of the viroid genome

In an effort to study sequence space allowing the recovery of viable potato spindle tuber viroid (PSTVd) variants we have developed an in vivo selection (Selex) method to produce and bulk-inoculate by agroinfiltration large PSTVd cDNA banks in which a short stretch of the genome is mutagenized to saturation. This technique was applied to two highly conserved 6 nt-long regions of the PSTVd genome, the left terminal loop (TL bank) and part of the polypurine stretch in the upper strand of pre-melting loop 1 (PM1 bank). In each case, PSTVd accumulation was observed in a large fraction of bank-inoculated tomato plants. Characterization of the progeny molecules showed the recovery of the parental PSTVd sequence in 89 % (TL bank) and 18 % (PM1 bank) of the analysed plants. In addition, viable and genetically stable PSTVd variants with mutations outside of the known natural variability of PSTVd were recovered in both cases, although at different rates. In the case of the TL region, mutations were recovered at five of the six mutagenized positions (357, 358, 359, 1 and 3 of the genome) while for the PM1 region mutations were recovered at all six targeted positions (50-55), providing significant new insight on the plasticity of the PSTVd genome.

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.