A distinct host cellular protein binding element was mapped within a 38-nucleotide (nt) sequence 166-129 nucleotides upstream of the 3' end of the MHV-JHM genome using a RNase T-1 protection/gel mobility shift electrophoresis assay. The resultant RNA-protein complex contains six host cellular proteins, one protein of 120-kDa molecular mass, two poorly resolved species approximately 55 kDa in size, a second pair of poorly resolved 40-kDa proteins, and a minor component of 25 kDa. A series of RNA probes containing deletions or clustered transversion mutations were tested for their ability to form complexes with mock- and MHV-JHM-infected cytoplasmic extracts. Three mutant RNA probes (mA, mB, and mC) with deletions at 154-140, 139-129, and 128-118, respectively, expressed 4, 37, and 94% of the host protein binding activity exhibited by the wild-type RNA. Defective interfering (DI) RNAs (DlmA, DlmB, and DlmC) containing corresponding deletions at 154-140, 139-129, 128-118, and another DI RNA (DlmD) with a deletion at nucleotides (nts) 112-102, a region which did not affect RNA-protein interactions, were transfected into MHV-JHM-infected 17CL-1 cells to assay the effects of these mutations on DI RNA replication. All of these mutations had an adverse effect on DI RNA replication. However, analysis of negative strand mutant DI RNAs revealed that two mutants (DlmC and DlmD) carrying deletions having little or no effect on RNA-protein interaction in our RNA-protein binding assays maintained their mutant sequences. In contrast, the other two mutants (DlmA and DlmB) containing deletions that dramatically decreased RNA-protein binding activity did not maintain their mutations; wild-type sequences were restored in the majority of the progeny negative strand molecules. These data indicate that the 26-nucleotide sequence at positions 154-129 from the 3' end of viral genome is important to both RNA-protein binding and viral replication. This protein binding element contains an 1 I-nt sequence (UGAGAGAAGUU, positions 139-129) very similar to a more 3' sequence (UGAAUGAAGUU) previously implicated in host protein binding and viral RNA replication (Yu and Leibowitz, 1995a and 1995b). (C) 1997 Academic Press.
