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Journal of Virology, May 2004, p. 5528-5530, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5528-5530.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

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


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LETTER
 
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. 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. 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. 1 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).



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  		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; @, other consensus hydrophobic positions (A, V, L, I, M, F, or Y); aa, amino acids (in the variable-length linker region between the motifs); amino acid positions are numbered for the first and last residue shown for each virus. Virus: mORV, mammalian orthoreovirus; GCRV, grass carp reovirus; GIRV, golden ide reovirus; GSRV, golden shiner reovirus; BmCPV, Bombyx mori cypovirus; DpCPV, Dendrolimus punctatus cypovirus; LdCPV, Lymantria dyspar cypovirus; FDV, Fiji disease virus; MRCV, Mal de Río Cuarto virus; NLRV, Nilaparvata lugens reovirus; OSDV, oat sterile dwarf virus; RBSDV, rice black-streaked dwarf virus; RRSV, rice ragged stunt virus; CTFV, Colorado tick fever virus; EYAV, Eyach virus. Protein names are as listed in the genpept files; hypoth., hypothetical protein. The findings for another virus tentatively assigned to this family, RArV in the proposed genus Mycoreovirus, is included at the bottom.

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 {lambda}1 and RNA-dependent RNA polymerase (RdRp) protein {lambda}3, 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. 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. 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. 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. 1 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. 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.


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ADDENDUM IN PROOF
 
The International Committee on Taxonomy of Viruses has approved the names of two new genera, Mycoreovirus (see Fig. 1) and Seadornavirus, in the family Reoviridae. This brings the total to 11 recognized genera in this family. For more information, see the recent article by P. Mertens (P. Mertens, Virus Res. 101:3-13, 2004).


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Max L. Nibert*
Jonghwa Kim
Harvard Medical School
Department of Microbiology and Molecular Genetics
200 Longwood Ave.
Boston, MA 02115

* Phone: (617) 645-3680, Fax: (617) 738-7664, E-mail: mnibert{at}hms.harvard.edu

Journal of Virology, May 2004, p. 5528-5530, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5528-5530.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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