Insertion of large amino acid repeats and point mutations contribute to a high degree of sequence diversity in the X4 protein of tomato ringspot virus (genus Nepovirus)

Abstract

Tomato ringspot virus (ToRSV) is a serious pathogen of small fruits and fruit trees in North America [14]. Several ToRSV isolates have been described that differ in natural host range and intensity of symptoms in herbaceous hosts [3, 17]. Tomato ringspot virus is a species of the genus Nepovirus [15], which has recently been reassigned to the proposed new family ‘‘Secoviridae’’ (subfamily ‘‘Comovirinae’’) within the order Picornavirales [16]. Nepoviruses have a bipartite positive-strand RNA genome. Each RNA encodes a polyprotein that is cleaved by the viral proteinase (Pro) at specific cleavage sites. RNA1 encodes replication proteins, while RNA2 codes for the coat protein (CP), movement protein (MP) and other protein(s) of less defined function. Using in vitro processing assays, three proteinase cleavage sites have been identified in the RNA2encoded polyprotein of ToRSV (a subgroup C nepovirus), allowing the definition of four protein domains (X3, X4, MP and CP) [4, 5]. In contrast, only three protein domains are present in the polyprotein of subgroup A and B nepoviruses (2a, MP and CP; see Fig. 1a). The ToRSV X3 protein shares conserved sequence motifs with the 2a protein of subgroup A and B nepoviruses [12]. The 2a protein of grapevine fanleaf virus has been shown to play a role in the replication of RNA2 [7]. The ToRSV X4 protein does not have significant sequence identity with proteins available in the database and does not have a functional equivalent in the genome of nepoviruses of subgroups A and B (Fig. 1a). The only other nepovirus of subgroup C for which the entire nucleotide sequence is available is blackcurrant reversion virus (BRV). The extent of sequence identity between ToRSV and BRV is very low in the deduced amino acid (a.a.) sequence of the X4 protein (8%) but higher in the deduced a.a. sequence of the CP (29%), VPg (viral protein linked to the genome), Pro and polymerase (Pol) (36%). In this study, we examined the extent of sequence diversity in the X4 protein among closely related ToRSV isolates. We show that there is a high degree of sequence diversity in the X4 protein, which is due in part to the insertion of multiple copies of two types of large amino acid repeats.