The C-terminal region but not the Arg-X-Pro repeat of Epstein-Barr virus protein EB2 is required for its effect on RNA splicing and transport

The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seem s to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3' processi ng, I,ut no direct proof of EB2 being involved in RNA processing and transp ort has been provided, and no specific functional domain of EB2 has been ma pped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if th ey are generated from precursors containing weak (cryptic) 5' splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated f rom precursors containing constitutive splice sites, and (iii) EB2 has no e ffect on the 3' processing of precursor RNAs containing canonical and nonca nonical cleavage-polyadenylation signalls, We also show that in the presenc e of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm , EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to tha t described as an RNA-binding domain in the herpes simplex virus type 1 pro tein US11, As glutathione S-transferase fusion proteins, both EB? and the A rg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requi res the C-terminal half of the protein but not the Arg-X-Pro repeat.