A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases

Abstract

A systematic bioinformatic approach to identifying the evolutionarily conserved regions of proteins has verified the universality of a newly described conserved motif in RNA-dependent RNA polymerases (motif F). In combination with structural comparisons, this approach has defined two regions that may be involved in unwinding double-stranded RNA (dsRNA) for transcription. One of these is the N-terminal portion of motif F and the second is a large insertion in motif F present in the RNA-dependent RNA polymerases of some dsRNA viruses.

Figure 1

Similarity peaks in four groups of RdRps. These are PLOTSIMILARITY outputs of four PILEUP (23) alignments of the RdRps of Picornaviridae-related RdRps, Totiviridae RdRps, Togaviridae-related RdRps and Bunyaviridae-related RdRps. Picornaviridae and Togaviridae are positive strand RNA viruses; Bunyaviridae are negative strand RNA viruses; and Totiviridae are dsRNA viruses. The Picornaviridae-related RdRps include broad bean wilt virus (Comoviridae, AF144234) (U.Lee, unpublished); porcine enterovirus (Picornaviridae, AJ011380) (55); cowpea mosaic virus, (ComoviridaeX00206) (56); echovirus 23 (Picornaviridae, AJ005695) (57); grapevine fanleaf virus (Comoviridae, D00915) (58); hepatitis A virus, (Picornaviridae, M59809) (59); maize chlorotic dwarf virus (Sequiviridae, U67839) (60); poliovirus type 3, (Picornaviridae, A03900) (24); rice tungro spherical virus, (Sequiviridae, X98396) (61); sacbrood virus, (Picornaviridae, AF092924) (62); Satsuma dwarf virus, (Comoviridae, D45026) (63); and tomato ringspot virus, (Comoviridae, AF135410) (A.Wang, unpublished). The Togaviridae-related RdRps are of tobacco mosaic virus (Tobamovirus, Z29370) (64), sindbis virus (Togaviridae, M69205) (65), Sagiyama virus (Togaviridae, AB032553) (66), rape mosaic virus (Tobamovirus, U309204) (67), grapevine leafroll virus 2 (Closterovirus, AF039204) (68), grapevine leafroll virus 1 (Closterovirus, Y14131) (N.Abou-Ghanem, unpublished), Citrus tristeza virus (Closterovirus, AF260651) (69), beet yellows closterovirus (Closterovirus, AF056585) (70), and alfalfa mosaic virus (Alfalfa mosaic virus group, K2702) (71). The Totiviridae RdRps are those of ScVL1, ScVLa, TvV1, TvV2, TvV5, LrV1-1, LrV2-1, GaVl1, SsV1, SsV2, Hv190sv, EbV1, UmVP1H1, GlV and Abv1L1. This set was chosen to exclude any RdRps with more than ∼50% identity with any other Totiviridae RdRp. The CcV sequence is excluded because it is only a partial sequence. The accession numbers and literature references for all of these sequences are in Table 2. The Bunyaviridae-related RdRps were from rice stripe virus (Tenuivirus, D31879) (72), Uukuniemi virus (Bunyaviridae, D10759) (73), Bunyamwera virus (Bunyaviridae, X14383) (74), Tomato spotted wilt virus (Bunyaviridae, D10066) (75), Hantaan virus (Bunyaviridae, X55901) (76) and La Crosse virus (Bunyaviridae, U12396) (77). PLOTSIMILARITY was run with a window of 10 amino acids. PILEUP was run with a similarity matrix of Blosum30 for the Totiviridae and the Bunyaviridae and Blosum62 for both the Picornaviridae- and Togaviridae-related RdRps. Gap weight was set at 5 and gap length weight at 1 for the Bunyaviridae, gap weight at 8 and gap length weight at 1 for the Totiviridae, and at the default values otherwise (gap weight at 8 and gap length weight at 2 for Blosum62 and at 15 and 5 for Blosum30). The horizontal dashed lines indicate levels of similarity that could arise by chance.

Figure 2

Structures of the Phi6, HCV and RHDV RdRps in the region including conserved motifs A to D. These are MOLSCRIPT(22) figures derived from the PDB files for the structures of the Phi6 (1HHS) (11), HCV (1QUV) (13) and RHDV (1KHV) (16) RdRps in the designated region. The conserved motifs are all shown in dark blue and labeled.

Figure 3

Similarity peaks in the Cystoviridae and Flaviviridae RdRps. The Cystoviridae sequences were of Phi6 (M17461) (34), Phi8 (AF226851) (35) and Phi13 (AF261668) (36); the Flaviviridae sequences were of HCV (D84264) (78), hepatitis G virus (AB021287) (30), hepatitis GB virus (U94421) (31), and bovine viral diarrhea virus (M31182) (79). PLOTSIMILARITY was run with a window of 10 amino acids. PILEUP was run with a similarity matrix of Blosum30 for both groups of viruses. Gap weight was set at 5 and gap length weight at 1 for the Flaviviridae and at the default values for the Cystoviridae.

Figure 4

Structures of the Phi6, HCV and RHDV RdRps in the N-terminal region, including conserved motif F. As in Figure 2, but regions F1, F2 and F3 are shown in red and labeled. The residues in the Phi6 RdRp that may function as a helicase are shown (K247, D248 and R251). For comparison with Figure 2, the regions shown include conserved motif A but end just prior to conserved motif B.