Biophysical and biochemical properties of baculovirus-expressed CaMV P1 protein.

Cauliflower mosaic virus (CaMV) gene I encodes a protein (P1) that has been implicated in the control of virus movement in infected plants. To assist in the characterization of the mechanism of action of P1, gene I has been expressed efficiently in Spodoptera frugiperda (Sf) cells using recombinant baculovirus. Control of the expression of CaMV gene I by the polyhedrin late promoter in the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) resulted in very high levels of P1 accumulation late in the infection cycle. This was predominantly as insoluble inclusion bodies within the cytoplasm of infected Sf cells, and not extracellularly. Evidence from anomalous gel migration and sequence homology with an analogous viral protein (tobacco mosaic virus 30K) indicated that P1 may be post-translationally processed. However, neither phosphorylation nor glycosylation of P1 occurred in this system, suggesting a functional distinction between P1 and TMV 30K. P1 from insect cells and native P1 from infected plants were immunologically related, allowing the expressed product to be used in the preparation of anti-P1 serum for detecting P1 in plant extracts. The full-size (46 kD) P1 product from insect cells, from plants, and from in vitro translations of in vitro gene I transcripts all showed similar behavior on two-dimensional protein gels, with a major pI of 7.0. Using a combination of 4 M urea, 1 M NaCl, and high temperature, P1 was solubilized. Approximately 5% of the starting material remained in solution after dialysis and remained stable to freeze/thawing. This preparation should enable us to identify the biochemical function of P1 and to resolve its role in controlling virus spread.

Nucleotide sequence of potato virus Y (N Strain) genomic 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.