Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine-and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.Viral-coded replicase proteins are essential for replication of tombusviruses, similar to other positive-strand RNA viruses (20,32,45). Tomato bushy stunt virus (TBSV), the prototypical tombusvirus, codes for p33 and p92 replicase proteins (reviewed in reference 43), which are essential for TBSV replication (32, 45). Based on the presence of the signature motifs for RNA-dependent RNA polymerase (RdRp) within the Cterminal portion of p92, the predicted function of the p92 is to synthesize viral RNA progenies (29,45). The function(s) of p33 in TBSV replication is currently unknown.An interesting feature of tombusviruses is that the larger replicase protein is expressed from the genomic RNA via a ribosomal readthrough mechanism of the p33 termination codon (43,45). Due to the expression strategy, the N-terminal portion of p92 overlaps with p33. Both p33 and p92 have been proposed to be part of the TBSV replication complexes (45). Accordingly, Western blot analysis of the partially purified TBSV RdRp preparation revealed the presence of both p33 and p92 in the transcriptionally active RdRp fractions (J. Pogany and P. D. Nagy, unpublished data).Both replicase proteins are localized to membranous structures in infected cells-the putative sites of tombusvirus replication (8, 45). The membrane localization domains in the replicase proteins are present within the N-t...