The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA. To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coli expression vector. The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity. The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding. They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro. Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes. The possibility that domain B consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins.

ABSTRACT Most plant viruses rely on the production of subgenomic RNAs (sgRNAs) for the expression of their genes and survival in the plant. Although this is a widely adopted strategy among viruses, the mechanism(s) whereby sgRNA production occurs remains poorly defined. Turnip yellow mosaic tymovirus (TYMV) is a positive-stranded RNA virus that produces an sgRNA for the expression of its coat protein. Here we report that the subgenomic promoter sequence of TYMV is located on a 494-nucleotide fragment, containing previously identified highly conserved sequence elements, which are shown here to be essential for promoter function. After duplication, the subgenomic promoter can be inserted into the coat protein open reading frame, giving rise to the in vivo production of a second sgRNA. It is suggested that this promoter can function when contained on a different molecule than viral genomic RNA. This interesting trait may be of general use for plant and plant virus research.

Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro.

The first approximately 60 amino acids of the N-terminal part of the potyvirus helper component-proteinase (HC-Pro) include highly conserved residues comprising a Cys-rich region. In the present study, the domain in Potato virus Y sufficient for self-interaction was mapped using the yeast two-hybrid system to the 83 N-terminal amino acids of HC-Pro. Mutations in the conserved His and two Cys residues within the Cys-rich region have a strong debilitating effect on self-interaction when introduced in the full-length HC-Pro, but not when introduced in the N-terminal fragment.

Because of the small size of their genome, viral genes have been forerunners in helping us understand gene expression. It is also because of their small size that viruses have elaborated the amazing variety of strategies that enables them to produce all the proteins they require for their multiplication. As a consequence, many of the strategies of expression known to occur in cell systems were first demonstrated in viruses. The aim of this review is to highlight the contribution of viruses to our knowledge of cell processes.

Introduction. The genus Potyvirus, family Potyviridae, is the largest genus of plant viruses with 180 members or possible members (Brunt, 1992). Potyviruses are flexuous filamentous particles which contain a single-stranded RNA genome of positive polarity possessing a covalently linked 5′-terminal viral protein (VPg) and a 3′-terminal poly(A) tail (reviewed in Riechmann et al., 1992). They are transmitted from plant to plant by aphids in a non-persistent manner, and this process is dependent on the presence of two virus-encoded proteins (reviewed in Pirone, 1991). One of these, the helper component-proteinase (HC-Pro) has attracted renewed attention during the last few years due to its multifunctionality and to it being implicated in different steps of the potyvirus life cycle. The properties, as well as the established and postulated functions of this protein, are reviewed.

A poorly aphid-transmissible potato virus Y (PVY-PAT) variant emerged after several cycles of mechanical transmission of an initially aphid-transmissible (AT) isolate. Sequence analysis of the N-terminal region of the helper component-proteinase (HC-Pro) gene revealed a Lys to Glu change at a position previously found to abolish the HC-Pro aphid transmission activity in several potyviruses. Two cycles of aphid transmission allowed the virus population to evolve towards an AT form (PVY-ATnew) where a Glu to Lys change was observed. PVY-PAT produced lower amounts of coat protein and the accumulation of its HC-Pro in infected plants decreased from 7 to 28 days post-inoculation, as compared to PVY-ATnew. RT-PCR and restriction analysis showed that the two virus populations co-existed in the PVY-AT isolate and that the AT form was counter-selected during mechanical transmission. These observations suggest that the Lys to Glu substitution leads to decreased stability of HC-Pro resulting in poor transmissions by aphids, and further strengthen the idea that HC-Pro is involved in the accumulation of potyvirus in infected plants.

The potyvirus helper component-proteinase (HC-Pro) is a multifunctional protein previously reported to have affinity for polyribonucleotides. To investigate further the ability of HC-Pro to bind nucleic acids, the potato virus Y (PVY) LYE84 isolate HC-Pro gene was amplified, cloned in an Escherichia coli expression vector and sequenced. HC-Pro was expressed as a fusion with the maltose-binding protein and purified by affinity chromatography. Electrophoretic mobility-shift assays demonstrated that HC-Pro acts as a sequence non-specific RNA-binding protein and suggest that more than one molecule of protein was bound per molecule of RNA. The HC-Pro RNA-binding activity was stable in 400 mm-NaCl and temperature sensitive. The recombinant protein preferentially bound ssRNA over DNA or dsRNA and showed little, if any, affinity for poly(A). The possible implications of the RNA-binding activity of HC-Pro in potyvirus replication and movement are discussed.

Analyses of populations of viruses with RNA genomes find a large amount of variability that results from re-assortment of genome segments of those viruses that have multipartite genomes, recombination events, point mutations and small deletions or insertions. The reverse transcriptases (RT) and the RNA-dependent RNA polymerases (RdRp) cause the last three types of change. These polymerases are error-prone owing to their intrinsic low level of fidelity and to their lack of correction mechanisms. In addition, host factors can also be responsible for certain mutations.

Since removal of the exposed N-terminus of the coat protein of some potyviruses abolishes aphid transmission, the role of this coat protein region of maize dwarf mosaic potyvirus (MDMV) in aphid transmission was investigated. The viral cDNA encoding this region was cloned and expressed as a fusion protein in bacteria. The resulting purified N-terminus of the coat protein was used in controlled aphid transmission experiments in competition with MDMV. The results show that this region inhibits aphid transmission of MDMV, indicating a direct involvement of the N-terminal region of the coat protein in aphid transmission.

We have completed the pSC101 sequence. The coding capacities of the newly sequenced regions show the presence of two large open reading frames close to the oriT region. Their size and localization suggest that these polypeptide chains could be involved in the transfer process of pSC101.

Recently a new insertion element (IS102)b ha been described in plasmid pSC101. We have determined its complete sequence: it consists of 1057 bp; 338 bp at one end are identical to those already determined for the kanamycin resistance transposon Tn903. It is not flanked by any direct repeat. Its coding capabilities are discussed, and compared to those of IS903.

Little is known of the detailed mechanisms of the polymerization reactions carried out by RNA and DNA polymerases. Besides technical reasons, there are mathematical difficulties not encountered in traditional enzymology. The product of the reaction after one polymerization step is also the substrate of the next step. A number of polymerases, isolated from various sources, have an exonuclease activity. The chain which is being synthesized may be either elongated or trimmed, and its growth has the character of a random walk. In this case, although the overall reaction scheme is more complex, the experiments are more informative, as every dNTP may be transformed into two distinct products: incorporated, or free dNMP.

Experimental studies have shown that the fidelity of DNA replication can be affected by the concentrations of free deoxyribonucleotides present in the cell. Replication of mammalian chromosomes is achieved using pools of newly-synthesized deoxyribonucleotides which fluctuate during the cell cycle. Since regions of mammalian chromosomes are replicated sequentially, there is the potential for differences among mammalian loci in both the relative and absolute frequencies of the various transitional and transversional mutations which may occur. Where these mutations are effectively neutral, at silent sites in genes and in non-coding sequences, this may result in different rates of evolution and in different base compositions, as have been observed in data from mammalian genes. A simple model of the DNA replication process is developed to describe how the mutation rate could be affected by the G+C contents of the deoxyribonucleotide pools and of the replicating DNA. Mutation rates are predicted to vary from locus to locus; only in the particular case of identical G+C contents in the DNA locus and the deoxyribonucleotide pools, and no proofreading, will the mutation rate be uniform over all loci.

FEBS LettersVolume 57, Issue 2 p. 139-144 Full-length articleFree Access On the mechanism of nucleotide incorporation into DNA and RNA Jacques Ninio, Jacques Ninio Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorFrançoise Bernardi, Françoise Bernardi Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorGilbert Brun, Gilbert Brun Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorLiliane Assairi, Liliane Assairi Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorMarc Lauber, Marc Lauber Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorFrançois Chapeville, François Chapeville Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this author Jacques Ninio, Jacques Ninio Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorFrançoise Bernardi, Françoise Bernardi Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorGilbert Brun, Gilbert Brun Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorLiliane Assairi, Liliane Assairi Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorMarc Lauber, Marc Lauber Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this authorFrançois Chapeville, François Chapeville Laboratoire de Biochimie du Dévéloppement, Institut de Biologie Moléculaire du C.N.R.S., 2 Place Jussieu, 75005 Paris, FranceSearch for more papers by this author First published: September 15, 1975 https://doi.org/10.1016/0014-5793(75)80702-2Citations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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