Françoise Bernardi

Potyviral HC-Pro: a multifunctional protein

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.

Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase

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.

DNA Helicase Activity Is Associated with the Replication Initiator Protein Rep of Tomato Yellow Leaf Curl Geminivirus

ABSTRACT The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA+ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA- or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA+ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3′ overhang and 3′-to-5′ polarity of unwinding, with some distinct features and with a minimal AAA+ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.

Gene expression from viral RNA genomes

This review is centered on the major strategies used by plant RNA viruses to produce the proteins required for virus multiplication. The strategies at the level of transcription presented here are synthesis of mRNA or subgenomic RNAs from viral RNA templates, and ‘cap-snatching’. At the level of translation, several strategies have been evolved by viruses at the steps of initiation, elongation and termination. At the initiation step, the classical scanning mode is the most frequent strategy employed by viruses; however in a vast number of cases, leaky scanning of the initiation complex allows expression of more than one protein from the same RNA sequence. During elongation, frameshift allows the formation of two proteins differing in their carboxy terminus. At the termination step, suppression of termination produces a protein with an elongated carboxy terminus. The last strategy that will be described is co-and/or post-translational cleavage of a polyprotein precursor by virally encoded proteinases. Most (+)-stranded RNA viruses utilize a combination of various strategies.

Identification and Functional Analysis of the Turnip Yellow Mosaic Tymovirus Subgenomic Promoter

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.

Switches in the mode of transmission select for or against a poorly aphid-transmissible strain of potato virus Y with reduced helper component and virus accumulation

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.

Effect of mutations within the Cys-rich region of potyvirus helper component-proteinase on self-interaction

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.

Viruses: Exquisite models for cell strategies

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.

Strategies of Expression of Turnip Yellow Mosaic Virus in Vivo: Developmental Approach for the Study of the Autocatalytic Cleavage of the 206k Polyprotein

Turnip yellow mosaic virus (TYMV) is the type-member of the tymovirus group which belongs to the Sindbis-like virus supergroup of (+)RNA viruses. TYMV infects members of the Cruciferae family. The genomic RNA directs the synthesis of two nonstructural overlapping proteins. It has recently been demonstrated that the larger one of 206K undergoes autocleavage yielding at least two products of 150K and 78K respectively, in vitro (1). The Smaller one of 69K is probably the movement protein (2).