Conserved Sequence Motifs for Nucleoside Triphosphate Binding Unique to Turreted Reoviridae Members and Coltiviruses

The family Reoviridae of double-stranded RNA viruses is recognized to comprise nine genera (31), with at least two others recently proposed (1, 24). Members of five recognized genera—Aquareovirus, Cypovirus, Fijivirus, Orthoreovirus, and Oryzavirus—are distinctive in having pentameric turrets that sit atop the capsid around each fivefold axis in the “inner-capsid particle” or “core” (13, 29, 36). In aquareoviruses, cypoviruses, and orthoreoviruses, these turrets are known or strongly suggested to mediate the guanylyltransferase and methyltransferase reactions in 5′ capping of the viral plus-strand RNA transcripts (6, 10, 27, 29). The cores of these viruses are also distinctive in having either 120 or 150 copies of a nodule protein that sit atop the capsid and contribute to its stability (13, 17, 29, 34, 36). In light of these features, Hill et al. (13) have proposed that the turreted viruses constitute an evolutionarily related subgroup. We now report that the recognized genera of turreted Reoviridae have another distinctive feature: conserved motifs for nucleoside triphosphate (NTP) binding in proteins of similar size to the μ2 protein of mammalian orthoreoviruses (18, 23) (Fig. ​(Fig.1).1). The motifs are related to ones in other NTP-binding proteins (19, 20, 32) but have distinguishing elements. In addition to five other positions occupied by hydrophobic residues, the motifs can be summarized as KgsgKs and dSDxyG, where uppercase letters indicate wholly conserved residues and lowercase letters indicate partially conserved residues (Fig. ​(Fig.1).1). We have recently shown that one or both lysines in motif A are essential for the triphosphatase activities of μ2 (18) (see below). Although previous authors have noted similarities to NTP-binding motifs in many of these sequences (8, 12, 15, 18), none have observed that the specific motifs shown in Fig. ​Fig.11 are conserved among all genera of turreted Reoviridae. Upon comparing the full-length protein sequences, we found that similarities outside the motif regions are less striking, with pairwise identities of ≤25% between even the two most closely related genera, Orthoreovirus and Aquareovirus (2). FIG. 1. Conserved NTP-binding motifs in 60- to 85-kDa proteins from Reoviridae members. Genpept, accession number from the translated GenBank database. Motifs: uppercase red, conserved in all of the sequences; lowercase, conserved in at least half of the sequences; ... The μ2 protein is a minor component of orthoreovirus cores (∼20 copies per particle) (7). It resides inside the core in association with both capsid protein λ1 and RNA-dependent RNA polymerase (RdRp) protein λ3, constituting the transcriptase complexes (9, 37). Genetically, μ2 determines strain differences in the transcriptase and nucleoside triphosphatase (NTPase) activities of cores (23, 35), and purified μ2 functions as both an NTPase and an RNA 5′ triphosphatase (RTPase) (18). The μ2 protein also has RNA- and microtubule-binding activities (4, 25). Less is known about the proteins with μ2-like NTP-binding motifs from other turreted Reoviridae. Like μ2, cypovirus VSP4/VP4 and fijivirus P-S8/P9/73.5KD are minor components of their respective cores (12, 22). Based on homologies to μ2 over the lengths of both proteins, aquareovirus VP5 has been proposed to be a minor core component as well (2). Oryzavirus Pns7 is reported to be a nonstructural protein (30), but given the limited work on this genus, we consider this assignment to be tentative. Instead, we postulate that the proteins with μ2-like NTP-binding motifs reside inside the cores of all turreted Reoviridae members and mediate NTPase-related functions similar to those of μ2. Upon searching the protein databases with a consensus defined by the μ2-like NTP-binding motifs from turreted Reoviridae, we found that Colorado tick fever and Eyach viruses, from the Coltivirus genus of nonturreted Reoviridae (3), also contain these motifs in a protein of similar size to that of μ2 (Fig. ​(Fig.1).1). However, viruses from the other three recognized genera of nonturreted Reoviridae—Orbivirus, Phytoreovirus, and Rotavirus—do not. In fact, using a search pattern with up to 500 residues between the two motifs, we identified only the proteins shown in Fig. ​Fig.1,1, i.e., no other viral or cellular proteins. Attoui et al. (3) have noted that VP10 contains a nucleotide-binding motif as well as broader similarities to protein kinases, the fijivirus 73.5KD protein, and oryzavirus Pns7. Whether coltivirus VP10 is a core protein has not been reported, but we now predict that it is. Considering the new findings, we propose, similarly to Hill et al. (13), that Reoviridae members exhibit at least two distinct organizational strategies for their RNA synthesis components in particles. For the genera shown in Fig. ​Fig.1,1, we propose a conserved class of transcriptase complex, including an RdRp and a μ2-like NTPase anchored beneath the capsid near each fivefold axis. This type of transcriptase is usually accompanied atop the capsid by pentameric turrets that mediate the guanylyltransferase and methyltransferase reactions in RNA capping. The μ2-like NTPase may be the RTPase that mediates the first reaction in capping or may perform another function in RNA synthesis. This organization contrasts with that of the nonturreted rotaviruses and orbiviruses, which have a guanylyltransferase protein that associates with the RdRp beneath the capsid near each fivefold axis (11, 26). In orbiviruses, this protein has been shown to mediate the RTPase reaction as well (28), but the rotavirus equivalent has not (5, 21). Orbiviruses contain a third internal protein, which mediates NTPase and RNA helicase activities in vitro (16) and contains NTP-binding motifs that do not match the Fig. ​Fig.11 consensus. However, no equivalent to this protein has been found in rotavirus particles, suggesting that the nonturreted viruses may be a more diversified group than the turreted ones. The findings for coltiviruses spark additional interest and suggest several explanations. The simplest may be that coltiviruses are turreted and that this feature has simply been missed in the limited studies to date. This idea is supported by recent findings for putative Reoviridae members that infect fungi. These agents, Rosellinia necatrix antirot virus (RArV) and Cryphonectria parasitica 9B21 virus, show strong sequence similarities to coltiviruses (14, 24, 33), and our inspections of the available RArV sequences revealed μ2-like NTP-binding motifs in protein P6 (Fig. ​(Fig.1).1). Furthermore, we were impressed to see that RArV cores contain turrets (33). Another interesting result is that the RdRp sequences of coltiviruses cluster with those of the turreted viruses in phylogenetic comparisons (2). Thus, we speculate that the coltiviruses are also turreted and have transcriptase components and strategies closely related to those of the other turreted Reoviridae.