Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosagedependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5 -untranslated region (5 UTR); BMV RNA3 that lacks a B box in its 5 UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.Viruses must produce their gene products in the proper amounts and with the appropriate timing. Failure to do so can lead to innate defense responses from the host that may act on RNA replication intermediates (47). A number of mechanisms can contribute to the differential expression of viral products, such as ribosome shunting, leaky scanning, frameshifting, functional recoding, and reinitiation (11, 12). For Sindbis virus and Tobacco mosaic virus, with single genomic RNAs, the expression of the RNA-dependent RNA polymerase is decreased by readthrough of leaky termination codons (27,37,44,45). Translational control also plays an important role in regulating viral gene expression in negative-sense RNA viruses and retroviruses (19,28,31,41,49). Regulation of protein production from segmented plus-strand RNA viruses is less well understood.Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of RNA viruses, is a model segmented positivestrand RNA virus. The BMV genome consists of three capped, messenger-sense genomic RNAs which have a tRNA-like structure within the 3Ј-untranslated region (3Ј UTR). Genomic RNA1 and RNA2 encode nonstructural proteins 1a and 2a, respectively, which direct RNA replication (33). Genomic RNA3 is a bicistronic RNA encoding the cell-to-cell movement protein (MP) and the coat protein (CP). The MP is translated from RNA3, whereas the 3Ј-proximal coat protein is translated from a subgenomic RNA4 that is made using minus-strand RNA3 as the template (33). In addition to replication in various plant species, BMV can replicate and transcribe its genome in Saccharomyces cerevisiae (20).The multifunctional 1a protein contains two domains separated by a proline-rich sequence. The N-terminal domain contains activities for 7-methyl-guanosine methyltransferase and covalent GTP binding required for viral RNA capping (2, 23). The C-terminal domain contains all of the motifs of the DEAD box RNA helicase domain, with ATPase and GTPase activities (24, 48). 1a is the primary viral protein determinant for the subcellular localization of the BMV RNA replication complex (9, 39, ...