Red clover mottle virus (RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other comoviruses, Cowpea mosaic virus (CPMV) and Bean pod mottle virus (BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.Red clover mottle virus (RCMV) is a member of the comoviruses, a group of plant viruses in the picornavirus superfamily with nonenveloped, icosahedral capsids and bipartite, singlestranded, positive-sense RNA genomes. As with other comoviruses, the two RNA molecules (termed RNA 1 and RNA 2) are encapsidated separately in isometric particles made up of 60 copies each of a large (L) and a small (S) coat protein (31,34,47). A number of strains of RCMV have been characterized (reference 28 and references therein), and the complete nucleotide sequence of the genome of one of them, RCMV strain S (34), has been determined (41,43,44). Inspection of the RNA sequence together with in vitro translation data (42,45) indicates that RCMV has a mode of gene expression similar to that of the type member of the group, Cowpea mosaic virus (CPMV). Comparison of the RNA 2 sequences of RCMV strain S with those of CPMV, Bean pod mottle virus (BPMV) and Cowpea severe mosaic virus (CPSMV) appears to divide comoviruses into two subgroups, with RCMV and CPMV forming one subgroup and CPSMV and BPMV forming the other (9).One notable feature of RCMV is the ability of different strains of the virus to form viable pseudorecombinants (35). Pseudorecombinants between strains S and O have proved useful in mapping symptom and host range determinants (11,36). The viability of the pseudorecombinants indicates that the coat proteins encoded by the RNA 2 of one strain can be processed correctly by the RNA 1-encoded 24K proteinase of another and are able to efficiently encapsidate the RNA 1 of the heterologous strain. This implies that any differences in the amino acid sequences of the coat proteins from the different strains do not dramatically alter their structure and assembly characteristics.Our basic knowledge of comovirus structure has been derived from crystallographic studies of CPMV and BPMV (10, 31, 48; T. Lin, Z. Chen, R. Usha, C. V. Stauffaacher, J.-B. Dai, T. Schmidt, and J. E. Johnson, submitted for publication). In both cases the structures reveal architectures typical of Pϭ3 plant and animal viruses, with the asymmetric unit consisting of three eight-stranded antiparallel -sandwich domains (38) (Fig. 1). The A domain is located around the fivefold axes and comprises the S subunit of either 24 kDa (CPMV) or 22 kDa ...