Use of Spring beauty latent virus to identify compatible interactions between bromovirus components required for virus infection Koki Fujisaki, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno Correspondence Kazuyuki Mise kmise@kais.kyoto-u.ac.jp Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received 30 November 2002 Accepted 10 February 2003 Spring beauty latent virus (SBLV) is a member of the genus Bromovirus, and is closely related to Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV). Compatible interactions between viral components are required for successful infection of plants by BMV and CCMV. To further our understanding of interactions between bromovirus components, we used SBLV to produce reassortants among the three bromoviruses. We found that SBLV RNA 2 functioned with heterologous bromovirus RNA 1 in infections of whole plants and protoplasts of Nicotiana benthamiana, although SBLV RNA 1 did not function with heterologous bromovirus RNA 2. A DNA-based transient assay for 1a and 2a proteins, which are encoded by RNAs 1 and 2, respectively further suggested that SBLV 2a protein may function in combination with heterologous bromovirus 1a protein. Moreover, analysis of the ability of reassortants to spread locally revealed that an RNA 2-mediated interaction between viral components may be required for efficient cell-to- cell movement of bromoviruses. INTRODUCTION Systemic infection of plants by viruses is established via three steps. These are genome replication in the initially infected cells, cell-to-cell movement into neighbouring cells through intercellular channels (plasmodesmata), and long- distance movement through the vasculature of host plants. In all three steps, compatible interactions between viral components and between viral and host components are necessary. Bromoviruses are a group of icosahedral plant viruses, the genomes of which are divided into three positive-sense tripartite RNAs, designated RNA 1, RNA 2 and RNA 3 (Lane, 1981). RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are both required for genomic RNA replication in protoplasts (Kroner et al., 1989, 1990). RNA 3, which is not necessary for viral RNA replication, encodes the 3a protein required for virus cell-to-cell movement (Mise et al., 1993; Schmitz & Rao, 1996) and the coat pro- tein (CP) translated from a subgenomic RNA designated RNA 4 (Sacher & Ahlquist, 1989). Some interactions between bromovirus factors necessary for successful infec- tion have been demonstrated. For instance, the 1a and 2a proteins of Brome mosaic virus (BMV) form a complex to achieve successful replication (Kao et al., 1992; O’Reilly et al., 1997). Moreover, BMV 1a protein must interact with the intercistronic region of BMV RNA 3 for efficient amplification of RNA 3 (French & Ahlquist, 1987; Sullivan & Ahlquist, 1999). Recent studies have identified sequences in the CP and viral RNAs that are required for bromo- virus encapsidation (Choi et al., 2002; Damayanti et al., 2002). However, the interactions between bromovirus components required for movement have not been exa- mined closely. Reassortment is a powerful strategy with which to study the compatibility of interactions among viral components in some viruses with divided genomes, such as bromo- viruses. For example, reassortants in which only RNAs 1 or 2 are exchanged between BMV and Cowpea chlorotic mottle virus (CCMV) do not allow virus genomes to accumulate to detectable levels in barley protoplasts, whereas reassor- tants in which RNA 3 is exchanged allow this accumula- tion (Allison et al., 1988). This observation reflects the importance of compatible combinations of RNAs 1 and 2, and therefore of the 1a and 2a proteins, for bromovirus replication. Spring beauty latent virus (SBLV) is a member of the genus Bromovirus, together with BMV and CCMV (Valverde, 1985). SBLV is closely related to BMV and CCMV, and biologically active SBLV cDNA clones have been con- structed (Fujisaki et al., 2003). To further analyse the interactions between bromovirus components required for systemic infection, we created reassortants of SBLV, BMV and CCMV and examined their infectivity in Nicotiana benthamiana, which is a common systemic host of the three bromoviruses. In this paper, we show that SBLV RNA 2 in combination with heterologous bromovirus RNA 1 0001-9020 G 2003 SGM Printed in Great Britain 1367 Journal of General Virology (2003), 84, 1367–1375 DOI 10.1099/vir.0.19020-0