An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.
Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.
Sriskanda
V S
VS
Department of Biological Sciences, University of South Carolina, Columbia 29208, USA.
Pruss
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G
Ge
X
X
Vance
V B
VB
D017353:Gene Deletion; D016679:Genome, Viral; D010947:Plants, Toxic; D017863:Potexvirus / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D017423:Sequence Analysis, RNA; D014026:Nicotiana / Q000821:virology; D014779:Virus Replication / Q000235:genetics
D012367:RNA, Viral