Sequence and structural elements at the 3 ' terminus of bovine viral diarrhea virus genomic RNA: Functional role during RNA replication

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a si ngle open reading frame and untranslated regions (UTRs) at the 5' and 3' en ds, Computer modeling suggested the 3' UTR comprised single-stranded region s as well as stem-loop structures--features that were suspected of being es sentially implicated in the viral RNA replication pathway. Employing a subg enomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA repl icon (S.-E. Behrens, C, W. Grassmann, H. J, Thiel, G. Meyers, and N, Tautz, J, Virol, 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investig ate the significance of defined RNA motifs for the RNA replication pathway, Enzymatic and chemical structure probing revealed mainly the conserved ter minal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motif s, i.e, a stable stem-loop, SL I, near the RNA 3' terminus and a considerab ly less stable stem-loop, SL II, that forms the 5' portion of 3'C, SL I and SL II are separated by a long single-stranded intervening sequence, denote d SS, The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e,g,, with ups tream DI9c RNA sequences, were not detected under the experimental conditio ns used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS moti fs do indeed play essential roles during RNA replication, Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as subs titution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate, Conve rsely, SL I stems comprising compensatory base exchanges turned out to supp ort replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previ ously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c, In summ ary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acti ng element of the replication process. The data further point at possible i nteraction sites of host and/or viral proteins and thus provide valuable in formation for future experiments intended to identify and characterize thes e factors.