The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication.In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRpassociated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vitro. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage QP RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.Brome mosaic virus (BMV) and many other plant and animal (+)-strand RNA viruses share remarkable similarities in RNA replication proteins and other features of genome expression and replication. Based on these similarities these viruses have been grouped into the alphavirus-like superfamily of(+)-strand RNA viruses (1, 2). BMV is the type member of the bromoviruses, a group of (+)-strand RNA plant viruses. The BMV genome is composed of three messengersense RNAs, of which RNA1 and RNA2 encode the transacting RNA replication proteins la (115 kDa) and 2a (100 kDa), respectively (3). The la protein contains domains sharing amino acid similarity with viral and cellular helicases (4) and with the nsPl protein of Sindbis virus, which has methyltransferase activity thought to be involved in capping of viral RNA (5). The 2a protein shares similarity with many RNA virus replication proteins, including the 3D protein of poliovirus and the ,B subunit of bacteriophage Qf replicase, which are RNA-dependent RNA polymerases (RdRps; ref. 6). Based on these similarities, la and 2a are referred to as the helicase-like and the polymerase-like RNA replication proteins, respectively.In contrast to accumulating data on the involvement of viral proteins in RNA replication, little is known about the role of host proteins in this process. Among (+)-strand RNA viruses, bacteriophage Q13 RdRp has served as the paradigm in this regard. Qf RdRp is a complex of the virus-encoded polymerase p subunit and three cellular proteins: ribosomal protein S1 and elongation factors EF-Tu and EF-Ts...

In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins la and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing la and 2a but lacking RNA3 contained normal amounts of la and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5', 3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing la, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable ofviral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.Brome mosaic virus (BMV), a plant-infecting virus, is a representative member of the alphavirus-like superfamily of (+)-strand RNA viruses of animals and plants. The BMV genome consists of three separately encapsidated RNAs, designated RNA1-3. Monocistronic RNA1 and RNA2, respectively, encode proteins la (109 kDa) and 2a (94 kDa). la and 2a are essential mutually interacting components of the RNA-dependent RNA polymerase (RdRp) involved in BMV RNA replication (1, 2). This RdRp, whose functions have been studied in vivo and in vitro, is a complex of la, 2a, and host proteins (2-6). RNA3 contains two genes separated by an -250-nt intercistronic region. The 5'-proximal gene, encoding the 3a infection movement protein (7,8), is translated directly from RNA3. The 3'-proximal coat protein gene is expressed via a subgenomic mRNA, RNA4. The 3a and coat proteins are dispensable for RNA replication but are required for infection spread in plants (8).BMV-directed replication of RNA3 in vivo depends on cis-acting sequences in three regions of RNA3 (see Fig. 3A): the 3' and 5' noncoding regions (NCRs) and the intercistronic NCR (9). The last 160-200 nt of the 3' NCR contain signals for initiation of (-)-strand RNA synthesis in vitro and areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U....

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Brome mosaic virus (BMV) is a positive-strand RNA virus that encodes two RNA replication proteins, the helicaselike la and the polymeraselike 2a. la and 2a share extensive sequence similarities with proteins encoded by many other members of the alphaviruslike superfamily. While further purifying enzymatically active RNA-dependent RNA polymerase from plants infected by BMV, we observed that la, 2a, and the polymerase activity all cofractionated through multiple independent purification steps. Moreover, using immunoprecipitation, we found that BMV la and 2a proteins synthesized in rabbit reticulocyte lysates or insect cells can form a specific complex in vitro. Complex formation was more efficient when la and 2a were cotranslated than when they were mixed after independent synthesis. In an antibody-independent assay, in vitro-translated la protein was also found to bind to 2a protein fixed on a nylon membrane. A three-amino-acid insertion in la that blocks BMV RNA replication in vivo also blocked in vitro interaction with 2a, while another two-amino-acid insertion that renders the la protein temperature sensitive for RNA replication interacted in vitro with 2a at 24°C but not at 32°C. These results and previous genetic data suggest that the la-2a interaction observed in vitro is required for BMV RNA replication and may have direct implications for other members of the alphaviruslike superfamily.