MULTIPLE BINDING-SITES FOR CELLULAR PROTEINS IN THE 3' END OF SINDBISALPHAVIRUS MINUS-SENSE RNA

The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The fi rst binding domain was found in the first 60 nucleotides, which repres ents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and th e fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, K(r), of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Delet ion mapping of the first domain showed that the first 10 nucleotides w ere critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides w as deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40 , or 41 to 55 had little effect on the binding, and deletion of nucleo tides 15 to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross Riv er virus and Semliki Forest virus, and from rubella virus were also ab le to interact with the 50- and 52-kDa proteins. The K(r) value for th e Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times great er. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replica tion. We suggest that the binding of the 50- and 52-kDa proteins to th e 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.