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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.

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.Initiation of DNA replication is a highly specific and controlled event which depends on the coordinated assembly of large protein complexes at the origin of replication. This process, which is universal, is activated by recognition and binding of the initiator protein to its cognate replication origin on the chromosome. Other steps follow, such as melting at the origin of replication and further unwinding carried out by replicative helicases recruited at the site of replication (31). In the case of extrachromosomal elements, such as viruses and plasmids, the initiator protein is often multifunctional and performs several of these steps, as well as interacting with host factors necessary for replication (42).The Rep protein is the initiator protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a begomovirus of the Geminiviridae family of plant single-stranded DNA (ssDNA) viruses. The Rep proteins of geminiviruses are closely related and show substantial sequence conservation. Rep, also named C1 and AL1, is a multifunctional protein and the only viral protein absolutely required for virus replication. Four functional domains have been delineated for begomovirus Rep: the N-terminal domain (amino acids [aa] 1 to 120), which is involved in initiation of the rolling-circle replication (RCR) utilized by geminiviruses (4,34,35); the oligomerization domain (aa 121 to 180), leading to interactions with itself (34) and with host factors (17); the ATPase domain (aa 181 to 330), which is characterized by the presence of a P loop (discussed in more detail below); and a carboxyl-terminal domain (aa 331 to 359) of unknown function but shown to be required for viral replication in the case of tomato golden mosaic virus (35) (Fig. 1).The ATPase domain of geminivirus Rep proteins was identified as a common element among proteins encoded by...

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.

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.

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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 nonspecific 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 mu-NaC1 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.