1.11.51
TRANSLATION STRATEGY OF THE POTATO VIRUS X TRIPLE GENE BLOCK PROTEINS REQUIRED FOR VIRAL CELL-TO-CELL MOVEMENT

J VERCHOT1, SM ANGELL2 AND DC BAULCOMBE2

1Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA; 2Sainsbury Laboratory, Colney Lane, Norwich, UK

Background and objectives
The triple gene block of potato virus X (PVX) encodes three proteins (25, 12 and 8 kDa) required for viral cell-to-cell and vascular transport. This block of proteins is conserved among viruses within the hordei-, furo-carla, and potexvirus families. Very little is known about how these proteins interact to facilitate viral movement. The 25-kDa protein is the best studied protein and accumulates to a high level in infected cells. On the other hand, the 12- and 8-kDa proteins are difficult to detect possibly because they are less abundant. Previous studies [1, 2] of PVX suggested that two viral 3' co-terminal subgenomic RNAs (sgRNAs) of 2.1 and 1.4 kb were necessary for translation of all three tgb proteins. This would explain the difference in protein expression levels observed in vivo. To determine if the tgb is translated from one or more mRNAs we devised two methods to analyse expression of the PVX tgb in vivo.

Results and conclusions
We produced tobacco plants carrying triple gene block transgene constructs corresponding to possible mono, di, and tricistronic mRNAs. Cell-to-cell movement of PVX containing mutations in each of these protein ORFs was complemented on plants whose transgenes encode the individual ORF. On plants containing polycistronic transgenes, mutations within the 25- and 12-kDa proteins were complemented when either of these proteins were the 5'-most ORF within the transgene, suggesting that separate RNAs are required for expression of these proteins. Mutations within the 8-kDa protein were complemented by transgenes expressing the 12- but not the 25-kDa protein. The ability of these transgenes to complement mutations in the triple gene block of PVX indicates that the first triple gene block ORFs (encoding a 25-kDa protein) are expressed from a functionally monocistronic mRNA, with the latter two ORFs (encoding the 12- and 8-kDa proteins) expressed from a functionally bicistronic mRNA. To analyze the translation strategy of the 8-kDa protein within the PVX genome, we introduced mutations into the 12-kDa protein ORF and tested their effects on expression of the 8-kDa protein. The type of mutation that would prevent 8-kDa protein expression depends on the mechanism by which the 8-kDa protein ORF is translated from a bicistronic mRNA. Three mutant viruses, 12FS, 121, and 12STOP, contain a frameshift mutation, substitution of the translation initiation codon, and translation stop codons inserted near the 5' end of the 12-kDa protein ORF. If the 8-kDa protein ORF was translated by ribosome frameshifting or by stop codon readthrough, then these mutations would all prevent translation of the 8-kDa protein ORF. These mutations prevent ribosome readthrough or frameshifting from the 12-kDa protein ORF into the 8-kDa protein ORF because the ribosomes would either not initiate translation of the 12-kDa protein or would disengage before reaching the 8-kDa protein ORF. In contrast, the 12D mutant, which lacks most of the 12-kDa protein ORF, would prevent internal entry of ribosomes translating the 8-kDa protein ORF by removal of the internal ribosome entry site (IRES). Finally the 12KOZ mutant contains several translation initiation codons at the start of the 12-kDa protein ORF and would prevent translation of the 8-kDa protein ORF by leaky ribosome scanning because the mutation introduces several initiation codons at the start of the 12kDa protein ORF.

Mutations affecting expression of only the 12-kDa protein were complemented on plants expressing the individual ORF. This includes the 12FS, 12STOP, 12D viruses. Only the 12KOZ mutation inhibited inhibiting expression of the 8-kDa protein and was complemented on plants expressing the bicistronic mRNA. These data indicate that the 8-kDa protein is translated by a leaky scanning mechanism from a functionally bicistronic mRNA.

References
1. Doija VV, Grama DP, Morozov SY, Atabekov JG, 1987. FEBS Letters 214, 308-312.
2. Morozov SY, Miroshnichenko NA, Solovyev AG et al., 1991. J. Gen. Virology 72, 2039-2042.