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Sterically stabilized diblock copolymer nanoparticles with a well-defined spherical morphology and tunable diameter were prepared by RAFT aqueous emulsion polymerization of benzyl methacrylate at 70 °C. The steric stabilizer precursor used for these syntheses contained pendent cis -diol groups, which means that such nanoparticles can react with a suitable aldehyde-functional surface via acetal bond formation. This principle is examined herein by growing an aldehyde-functionalized polymer brush from a planar silicon wafer and studying the extent of nanoparticle adsorption onto this model substrate from aqueous solution at 25 °C using a quartz crystal microbalance (QCM). The adsorbed amount, Γ, depends on both the nanoparticle diameter and the solution pH, with minimal adsorption observed at pH 7 or 10 and substantial adsorption achieved at pH 4. Variable-temperature QCM studies provide strong evidence for chemical adsorption, while scanning electron microscopy images recorded for the nanoparticle-coated brush surface after drying indicate mean surface coverages of up to 62%. This fundamental study extends our understanding of the chemical adsorption of nanoparticles on soft substrates.

Ten PVY isolates representative of four PVY groups (YN, YNTN, YN-W, YO), differing by their ability to induce reactions of vein necrosis on tobacco and tuber necrosis on potato, were studied in order to research the regions of the viral genome involved in these necrosis phenomena. The whole genome of these isolates was amplified in two fragments (4,063 and 5,670 nucleotides) and was subjected to a restriction fragment length polymorphism (RFLP) study. In the first 4,063 nucleotides of the PVY genome, a phenetic analysis of RFLP data resulted in a clustering of our PVY isolates into three groups: PVYN isolates (group A); PVYNTN and PVYN-W isolates (group B) and PVYO isolates (group C). In the last 5,670 nucleotides, two groups were found: PVYN and PVYNTN isolates (group D) and PVYO and PVYN-W isolates (group E). From this clustering and the necrosing properties known for these isolates, the tobacco necrosis determinants seem more likely located in the 5' than in the 3' half part of the viral RNA, whereas it would be the opposite situation for the determinants of the necrosis on potato tubers. Moreover a recombination event seemed to have occurred in the genome of the PVYN-W isolates.

Potato virus Y (PVY) the type member of the genus Potyvirus, occurs world-wide as isolates which differ in host range and the type of symptoms caused. The sequences of a 5' segment of viral RNA overlapping the 5' non-translated region (5'NTR) alone (ten isolates) or the 5'NTR and the adjacent P1 coding region (eight isolates) were established. These data were used to quantify the polymorphism in the 5'-terminal part of the PVY genome. Nucleotide sequence identity between isolates ranged from 66-100% in the 5'NTR and from 70-100% in the P1 coding region. The lowest amino acid sequence similarity between PVY P1 was 77%, illustrating the high variability of this protein in the PVY species. Phylogenetic trees based on either 5'NTR or P1 sequence analyses resulted in the same clustering of the studied isolates into three groups. Group I comprises potato isolates all inducing 'tobacco veinal necrosis' symptoms. Group II contains isolates inducing either 'tobacco veinal necrosis' or mosaic symptoms in tobacco. Group III contains mainly pepper or tomato isolates inducing mosaic symptoms in tobacco and shows a geographical clustering of the Tunisian isolates. This clustering into three groups is discussed in comparison with phylogenetic trees previously obtained from capsid gene or 3'NTR sequence analysis in the PVY species. Multiple sequence alignment indicated conserved motifs potentially involved in viral functions.

Potato virus Y (PVY) is the type member of the potyvirus group. Potyviruses, like picorna-, como-, and nepoviruses, belong to the picornavirus-like supergroup. All these viral RNAs have a VPg at their 5' end, and for four picornaviruses and one comovirus internal initiation of translation has been reported. To know if such a translational mechanism holds true for potyviral RNAs, the 5' nontranslated region (NTR) of PVY RNA was placed in an internal position, either by adding 91 bases upstream of the PVY 5'NTR or by inserting the PVY 5'NTR into an intercistronic region. The addition of extra bases stimulates translation in a rabbit reticulocyte lysate, and the presence of the PVY 5'NTR in the intercistronic region allows the synthesis of the second cistron. These findings strongly suggest that PVY RNA initiates translation by an internal ribosome-binding mechanism. Furthermore, the use of antisense oligodeoxynucleotides indicates that the entire 5'NTR seems to be involved in such a mechanism.

Potato virus Y (PVY), a potyvirus, has an RNA genome containing 9704 nucleotides of which 185 belong to the 5' nontranslated region (NTR). Contrary to most eukaryotic mRNAs that have a cap structure, the potyvirus RNA has a genome-linked protein (VPg). In order to understand the mechanisms of PVY RNA translation initiation, hybrid-arrest translation was used to localize sequences involved in binding of proteins and/or ribosomes. The 5' NTR was fused to the beta-glucuronidase (GUS) reporter gene. Six antisense oligodeoxynucleotides were used for hybridization, and the efficiency of the in vitro translation of the hybridized mRNA was modified to different levels depending upon the position of the oligodeoxynucleotide used. The highest inhibition was obtained with an oligodeoxynucleotide hybridized to the 5' end. In addition, translation of GUS mRNA containing the PVY 5' NTR was greatly enhanced when this mRNA was capped. These results differ from those obtained with the tobacco etch virus (TEV) and three picornaviruses, but are similar to those obtained with capped mRNA.

DNA complementary to the 3' terminal 1651 nucleotides of the genome of the common strain of lettuce mosaic virus (LMV-O) has been cloned and sequenced. Microsequencing of the N-terminus enabled localization of the coat protein gene in this sequence. It showed also that the LMV coat protein coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein by cleavage at an unusual Q/V dipeptide between the polymerase and the coat protein. This is the first report of such a site for cleavage of a potyvirus polyprotein, where only Q/A, Q/S, and Q/G cleavage sites have been reported. The LMV coat protein gene encodes a 278 amino acid polypeptide with a calculated Mr of 31,171 and is flanked by a region which has a high degree of homology with the putative polymerase and a 3' untranslated region of 211 nucleotides in length. Percentage of homology with the coat protein of other potyviruses confirms that LMV is a distinct member of this group. Moreover, amino acid homologies noticed with the coat protein of potexvirus, bymovirus, and carlavirus elongated plant viruses suggest a functional significance for the conserved domains.

An RNA-dependent RNA polymerase has been completely purified from Cauliflower inflorescences. Analysis of the purified enzyme on SDS-polyacrylamide gels showed one polypeptide chain with a molecular weight of 140,000 dalton. The enzyme is monomeric in its native state. The in vitro activity was completely dependent on added RNA, the most efficient templates being poly (U), poly (U, C), poly (I) and viral RNA.

The complete nucleotide sequence of the genomic RNA of the potyvirus potato virus Y strain N (PVYn) was obtained from cloned cDNAs. This sequence is 9704 nucleotides long and can encode a polyprotein of 3063 amino acids. The positions of the cleavage sites at the N terminus of the capsid and cytoplasmic inclusion proteins have been determined. Other putative protein cleavage sites have been deduced by searching for consensus sequences and by analogy with the polyprotein of the tobacco vein mottling virus and of the tobacco etch virus. Comparison of the PVY polyprotein sequence with that of other potyvirus polyproteins shows similarities in genome organization and a high level of identity along most of the polyprotein, except for the putative proteins flanking the helper component. A search for specific protein motifs has revealed the existence of a potential metal-binding site at the putative N terminus of the helper component in potyviruses. The possible functions of this structure are discussed.

Turnip yellow mosaic virus (TYMV) possesses a monopartite single-stranded (+) sense RNA genome in which the coat protein (cp) gene is 3' proximal and is expressed in vivo via a subgenomic RNA. Evidence is presented here that this subgenomic RNA is synthesized in vivo by internal initiation of replication on (-) RNA strands of genomic length. The double-stranded RNAs (dsRNAs) from TYMV-infected plants have been isolated, purified, and characterized. Under native conditions, no dsRNAs (replicative intermediates and/or replicative forms) of subgenomic length corresponding to subgenomic cp RNA can be detected by ethidium bromide staining of RNA-sizing gels or by Northern blot hybridization using RNA probes. The presence of nascent subgenomic cp (+) RNA strands on the dsRNA of genomic length has been demonstrated using two different approaches: (1) Northern blot hybridization using (-) RNA probes under denaturing conditions and (2) characterization of the 5' ends of nascent (+) RNA strands upon labeling by vaccinia virus nucleoside-2'-methyltransferase.