Birnavirus VP1 Proteins Form a Distinct Subgroup of RNA-Dependent RNA Polymerases Lacking a GDD Motif1

Virology 296, 241–250 (2002) doi:10.1006/viro.2001.1334

Philip S. Shwed,* Peter Dobos,* Lynne A. Cameron,* Vikram N. Vakharia,† and Roy Duncan‡,2

*Department of Microbiology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; †Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742;and ‡Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received June 27, 2001;returned to author for revision August 22, 2001;accepted November 28, 2001

We have cloned and characterized the Drosophila X virus (DXV) genome segment B and its encoded VP1, the putative RNA-dependent RNA polymerase (RdRp) present in the virion. The 2991-bp open reading frame encodes the largest

birnavirus VP1 at 977 aa, with a calculated Mr of 112.8 kDa. As with the VP1 proteins of the type species of the other two genera in the family Birnaviridae, namely, infectious pancreatic necrosis virus (genus Aquabirnavirus) and infectious bursal disease virus (genus Avibirnavirus), the DXV (genus Entomobirnavirus) VP1 protein contains a consensus GTP-binding site and appears to possess self-guanylylation activity. All of the birnavirus VP1 proteins contain conserved RdRp motifs that reside in the catalytic “palm” domain of all classes of polymerases. However, the birnavirus RdRps lackthe highly conserved Gly-Asp-Asp (GDD) sequence, a component of the proposed catalytic site of this enzyme family that exists in the conserved motif VI of the palm domain of other RdRps. All three birnavirus RdRps do contain downstream DD motifs that could function as part of the catalytic triad. These motifs are, however, located in spatially distinct regions of the various birnavirus VP1 proteins. These results suggest that the VP1 proteins of birnaviruses form a defined subgroup of polymerases that either are lacking the conserved RdRp motif VI or have repositioned this motif to different structural regions. © 2002 Elsevier Science (USA)

INTRODUCTION as RNA polymerization, nucleotide triphosphate binding, and template and product binding. In particular, a Gly- All RNA viruses, irrespective of replication and coding strategies, encode an RNA-dependent RNA polymerase Asp-Asp (GDD) sequence found in motif VI is conserved (RdRp) (EC 2.7.7.48), which carries out multiple functions in almost all polymerases. In a few cases, such as with from replication to capping activities (Spies et al., 1987; the RdRps of infectious bronchitis virus and the dsRNA ␾ Ishihama and Nagata, 1988). The genes encoding RdRps phage 6, the glycine residue is substituted in this motif are among the most conserved of all viral genes and for but the di-aspartate sequence is conserved (Boursnell et this reason have been used extensively for phylogenetic al., 1987; Mindich et al., 1988). These two aspartate analysis (Koonin and Dolja, 1993). Crystal structures and residues, in conjunction with a third conserved aspartate sequence-predicted structural analysis of RdRps, as well in motif IV, are believed to represent the catalytic site of as those of DNA-dependent DNA polymerases and re- the polymerase by functioning to coordinate the position- verse transcriptases, suggest a conserved polymerase ing of two metal ions, water, the free 3Ј-hydroxyl of the tertiary structure resembling a right hand with finger, nascent strand, and the incoming ribonucleoside thumb, and palm domains (Ollis et al., 1985; Kohlstaedt et triphosphate (Kamer and Argos, 1984; Argos, 1988; Gor- al., 1992; Hansen et al., 1997; O’Reilly and Kao, 1998; balenya and Koonin, 1988; Steitz, 1993, 1998). Lesburg et al., 1999; Butcher et al., 2001). Amino acid In view of the highly conserved nature of the RdRp sequence analysis has also highlighted eight conserved GDD motif and its postulated role as the catalytic site of RdRp motifs, four of which (denoted IV–VII or A–D) reside this enzyme family, it is surprising that the presumptive in the catalytic palm domain (Poch et al., 1989; Koonin, RdRps of the dsRNA virus family Birnaviridae apparently 1991; O’Reilly and Kao, 1998). Although the functions of lack the GDD motif (Morgan et al., 1988; Duncan et al., these motifs are not entirely understood, they are be- 1991). The bisegmented dsRNA genomes of the mem- lieved to correspond to RNA polymerase functions such bers of the Birnaviridae family are found in non-envel- oped icosohedral nucleocapsids of 60 nm in diameter (reviewed in Dobos, 1995a). Both genome segments of 1 The nucleotide sequence data reported in this paper have been genus Aquabirnavirus (type species IPNV; Duncan and submitted to the GenBank nucleotide sequence database and have Dobos, 1986; Duncan et al., 1991) and genus Avibirnavi- been assigned Accession No. AF196645. 2 To whom reprint requests should be addressed. Fax: (902)494- rus (type species IBDV; Azad et al., 1985; Morgan et al., 6770. E-mail: [email protected]. 1988; Kibenge et al., 1990; Kibenge and Qian, 1994) have

0042-6822/02 $35.00 241 © 2002 Elsevier Science (USA) All rights reserved. 242 SHWED ET AL. been cloned and sequenced. The larger one, segment A, encodes the major structural proteins of the viral particle and a proteolyticenzyme involved in precursorprotein processing. Genome segment A of Drosophila X virus (DXV), the type species of the third and final genus of the Birnaviridae (genus Entomobirnavirus), has also been sequenced and shows a genetic organization similar to that of the other birnaviruses (Chung et al., 1996). The smaller segment B of IPNV and IBDV encodes a minor FIG. 1. Conserved terminal sequences of DXV, IPNV, and IBDV ge- Ј Ј structural protein, VP1, the putative RdRp. To date, no nome segments. The 5 - and 3 -terminal deoxynucleotide sequences of individual birnavirus genome segment cDNAs are shown (DXV, IBDV, sequence information has been available for DXV ge- and IPNV segments A and B). The conserved terminal trinucleotides nome segment B. are indicated in boldface type. The birnavirus VP1 protein has a low copy number and features several conserved motifs found in the RdRps of other viruses (Bruenn, 1991; Koonin, 1991, 1992; Ribas putative RdRp, exhibits self-guanylylation activity and like and Wickner, 1992). Birnavirus RdRps, however, possess IPNV, it lacks a GDD sequence. Sequence comparisons a number of attributes that distinguish them from most and secondary structure predictions reveal that the RdRp RdRps. Similar to the reverse transcriptase of hepatitis B sequences of the three genera of Birnaviridae are more virus, they are found in both free and genome-linked similar to one another than to other viral RdRps. Further- forms (Revet and Delain, 1982; Nagy and Dobos, 1984a,b; more, the sequence analysis suggests that motif VI ei- Calvert et al., 1991; Weber et al., 1994). Rather than being ther is absent altogether or has been repositioned to primer-independent or using the free hydroxyl groups of distinct locations in each of the birnavirus VP1 proteins. RNA as primers for initiation of RNA synthesis, birnavirus RdRps use hydroxyl groups of amino acid residues RESULTS AND DISCUSSION within the protein itself. This situation is similar to the Cloning and sequence analysis of DXV segment B example of the reverse transcriptase of the hepadnavirus that primes RNA-directed DNA synthesis from an internal In order to more fully evaluate the relationship be- site of the polypeptide (Wang and Seeger, 1992). In con- tween the GDD motif and birnavirus RdRp function, we trast to reovirus and similar to the dsRNA phage ␾6, the cloned and characterized the DXV segment B and its in vitro synthesis of IPNV mRNA appears to follow a encoded VP1. Two overlapping cDNA clones that hybrid- semiconservative strand displacement mechanism ized to a segment B RNA probe were identified (data not (Mertens et al., 1982; Dobos, 1995b). More importantly, shown). The nucleotide sequences of these clones, as the RdRps of IPNV and IBDV lack the GDD sequence, the well as separate amplifications of the 5Ј- and 3Ј-ends of hallmark feature of this enzyme family. In the case of the segment, were compiled to generate the contiguous IBDV, VP1 contains a LDD motif in place of the GDD in segment B sequence (Accession No. AF196645). There motif VI, and the flanking amino acid residues are hydro- were no sequence discrepancies in the 464-nucleotide philic, not hydrophobic as with most RdRps (Morgan et overlap region of these two cDNA clones or with the al., 1988; Duncan et al., 1991). For IPNV, the GDD motif is sequence of the 5Ј-terminal 650 nucleotides and the completely absent from the region corresponding to the sequence from nucleotides 1300 to 1900 of a third inde- presumptive motif VI of IBDV (Duncan et al., 1991). One pendent cDNA clone, suggesting that the presented se- possible explanation for the absence of the GDD motif in quence is a reasonable estimate of the quasispecies. the IPNV RdRp sequence is that a downstream LDD The DXV full-length cDNA sequence is 3243 nucleo- motif (residues 653–655) might function as the catalytic tides long with a percentage GC content that is low site, possibly reflecting a repositioning of motif VI (Koo- relative to other birnavirus genome segment B se- nin, 1992). Based on the VP1 sequences obtained from quences (44.7% versus 53.9% for IPNV jasper and 53% for two of the three genera, it appears that birnaviruses IBDVser2). Terminal noncoding regions of 104 and 148 contain a novel catalytic site and represent a new class bp flank a single continuous open reading frame of 2991 of RdRps or that the birnavirus XDD motif is spatially bp that encodes the largest birnavirus VP1 at 977 aa with Ј rearranged relative to the conserved motifs found in a calculated M r of 112.8 kDa. The 5 UTR sequence of other RdRps. DXV segment B shares a high degree of nucleotide In order to clarify the relationship between the GDD identity with segment A (83.3% over the first 40 nucleo- motif, the birnavirus polymerase, and other RdRps, we tides). Furthermore, a 5Ј-terminal GGA triplet and a 3Ј- cloned and sequenced the VP1-encoding genome seg- terminal CCC triplet that are conserved in all of the ment B of DXV, the type species of the only remaining birnavirus genome segments previously sequenced are genus of birnaviruses, the Entomobirnaviruses. Our re- also present in the DXV genome segment B sequence sults indicate that the 113-kDa DXV VP1 protein, the (Fig. 1). The presence of the conserved birnavirus termi- BIRNAVIRUS VP1 PROTEINS 243 nal trinucleotides indicates that the reported sequence bos, 1993), the GMP associated with the DXV VP1 protein represents the entire cDNA sequence of DXV segment B, following in vitro guanylylation is linked to a tyrosine thereby completing the sequence analysis of this ento- residue. mobirnavirus genome. Although no tyrosine in the DXV VP1 protein aligns with a serine in the IPNV sequence to suggest a con- In vitro self-guanylylation of DXV VP1 served guanylylation site, we noted the close proximity of Tyr48 in DXV with Ser 44 of IPNV and Ser 46 of IBDV (Fig. A consensus NTP-binding motif, G/AXXXXGKS/T 2). Since the specific site of serine guanylylation in the (Walker et al., 1982), is present in ras-type GTP-binding IPNV VP1 protein is unknown, the significance of this proteins (Argos and Leberman, 1985) and in viral pro- observation is not apparent. If the guanylylation site is teins with a role in RNA replication (Kaariainen et al., spatially conserved in the birnavirus VP1 proteins, this 1987). A GXXXXGR/KT motif is present in the IPNV and represents a potential site for GMP addition. However, it IBDV VP1 sequences (Fig. 2) and may represent a po- is also conceivable that the extensive sequence diver- tential GTP-binding site involved in the self-guanylylation gence of the birnavirus VP1 proteins may contribute to activity associated with these birnavirus VP1s (Dobos, altered protein folding and the localization of a tyrosine 1993). Self-guanylylation of the IPNV and IBDV VP1 pro- residue from the DXV VP1 protein in a similar position as teins results in the formation of a VP1pG complex linked a serine residue of IPNV in the tertiary structure of the Ј Ј by a serine–5 GMP phosphodiester bond to the 5 -end of birnavirus VP1 proteins. Regardless of the actual site of both genome segments (Dobos, 1993). The VP1pG com- VP1 guanylylation, our results suggest that the DXV VP1 plex acts as a primer during RNA synthesis both in vitro protein also possesses self-guanylylating activity, con- (Dobos, 1995a) and in vivo (Magyar et al., 1998), and the sistent with its presumptive designation as the RdRp of Ј VP1 polypeptide remains covalently linked to the 5 -end the virus and suggesting that it likely serves a similar of the RNA and thus becomes the genome-linked protein role in priming RNA replication, as occurs with the other VPg. birnaviruses. The region of the DXV VP1 sequence that corresponds to the putative GTP-binding site present in the IPNV and Identification of conserved RdRp motifs in the DXV IBDV VP1 proteins contains a GXXXXGKK sequence (res- VP1 protein idues 256–263 in Fig. 2) that could function as a GTP- binding site. We therefore determined whether the VP1 The DXV VP1 protein was used as the query in a polypeptide encoded by DXV genome segment B is gua- BLASTP search of the nonredundant Swiss-Protein da- nylylated in vitro. As shown in Fig. 3, VP1 present in tabase (version 39.0, May 2000). Only other birnavirus purified DXV virions was labeled in the presence of VP1 genes produced biologically relevant alignments. A [␣-32P]GTP. None of the other viral proteins were labeled comparison of the aligned birnavirus VP1 proteins re- with [␣-32P]GTP (data not shown). The identification of vealed significant sequence divergence with only 25% the radiolabeled protein as VP1 was determined by sequence identity between the VP1 protein of DXV and staining the blot; no other DXV structural protein that those of IPNV and IBDV (Fig. 2). The sequence hetero- stains with Coomassie blue migrates with an electro- geneity was most apparent in the amino- and carboxy- phoreticmobility similar to that of VP1 (VP1 is 113 kDa; terminal 250–300 amino acids of the birnavirus VP1 pro- the next largest detectable DXV structural protein is VP2, teins. In particular, the carboxy-proximal one-third of the with a molecular mass of approximately 55 kDa). The proteins display almost no sequence identity between all labeling was specific for GTP since VP1 was not labeled three proteins and contain numerous insertions/dele- by similar incubations with [␣-32P]UTP, [␣-32P]CTP, or tions required to maintain the alignment. Extensive [␣-32P]ATP (data not shown). amino acid identity between the three birnavirus VP1 The radiolabeled VP1 protein obtained from multiple proteins was observed in the central one-third of the lanes of a preparative gel (Fig. 3A) was subjected to proteins (residues 338–618 in the DXV sequence from phosphoamino acid analysis. The radiolabeled moieties Fig. 2). This central region of VP1 contains the consen- detected by thin-layer chromatography following partial sus GTP-binding motif discussed above. Several of the acid hydrolysis of the [␣-32P]GTP-labeled VP1 protein conserved motifs found in the catalytic palm domain of corresponded to liberated free phosphate (Pi), partial other polymerases were also identified in this central hydrolysis products (spots below the phosphotyrosine), region (Poch et al., 1989; Bruenn, 1991; Koonin, 1991, and phosphotyrosine (P-Tyr). There was no evidence of 1992; Koonin and Dolja, 1993). radiolabel associated with phosphoserine or phospho- The conserved RdRp motifs from several RNA viruses threonine, which were clearly resolved from phosphoty- were aligned with similar motifs present in the birnavirus rosine by chromatography, as indicated by the migration VP1 proteins (Fig. 4). Obvious sequence similarity and of phosphoamino acid markers (shown on the right in conserved predicted secondary structures and spatial Fig. 3B). Therefore, in contrast to other birnaviruses (Do- arrangements were observed between the organization 244 SHWED ET AL.

FIG. 2. Conserved amino acid sequences in the aligned VP1 proteins of birnaviruses. A gapped alignment of the VP1 sequences from the three genera of birnaviruses is shown (IPNV-Ja, Accession No. M58756; IBDV, Accession No. 19502; DXV, Accession No. AF196645). Amino acid sequences were aligned using CLUSTAL W (Thompson et al., 1994), using the “blosum” weight matrix, a gap penalty of 20, and a gap extension penalty of 0.1. Identical residues present in all three proteins are shaded black; identical or conserved residues present in two proteins and conserved residues present in all three proteins are shaded gray by Boxshade 3.21 (www.ch.embnet.org). Numbers refer to amino acid positions for each sequence. The locations of the conserved RdRp motifs (numbered IV–VII) and of the consensus GTP-binding site are indicated above the sequences, as are the locations of the five downstream DD sequences (marked with asterisks) that could serve as alternates to motif VI. BIRNAVIRUS VP1 PROTEINS 245

Motif V of the RdRps is located approximately 50 residues downstream of motif IV and contains a highly

conserved SGX3TX3N sequence. This conserved sequence is located in the appropriate position in the DXV VP1 protein and the VP1 proteins of the other birnaviruses (Figs. 2 and 4). Secondary structure predictions for this region of most RdRps suggest an ␣-helical conformation over the carboxy-proximal portion of the motif, which is also the predicted conformation for motif V of DXV VP1 and the VP1 proteins of the other birnaviruses (Fig. 5). Although the specific role of the conserved sequence is unclear, this motif is also believed to be involved in discriminating ribose and deoxyribose sugars (O’Reilly and Kao, 1998). The conserved GTP-binding motif and the guanylylated nature of the DXV VP1 protein, in conjunction with the clear presence of the RdRp motifs IV and V, strongly support the designation of the DXV VP1 protein as the RdRp of the virus. There is also tentative evidence supporting the presence of RdRp motif VII in the birnavirus VP1 proteins. Motif VII is the last of four conserved RdRp motifs present in the palm core structure. It is less well defined FIG. 3. In vitro guanylylation of DXV VP1 through a phosphotyrosine both structurally and functionally than the other con- linkage. (A) Demonstration of self-guanylylation of VP1. Purified DXV served palm motifs, although a positively charged resi- ␣ 32 was labeled with [ - P]GTP in vitro and subjected to SDS–PAGE and due (usually lysine) is conserved in most RdRps (O’Reilly electroblotting. The membrane was stained with Coomassie blue to locate the virion polypeptides and the dried membrane was subjected and Kao, 1998). The last region of extensive sequence to autoradiography. The arrowhead on the right indicates the position of conservation between all three birnavirus VP1 proteins VP1 in multiple samples from a preparative gel. (B) Phosphoamino acid (residues 602–611 in the DXV sequence, Fig. 2) may analysis. The labeled VP1 samples from the preparative gel in (A) were represent motif VII; it is located an appropriate distance excised from the membrane, pooled, and subjected to acid hydrolysis, downstream of motif VI and all of the birnavirus se- thin-layer chromatography, and autoradiography, as described under Materials and Methods. The positions of the origin (ori), phosphoamino quences feature a conserved arginine residue in this acid markers, and inorganic phosphate (Pi) are indicated on the right. region (Fig. 2).

DD motifs are located in spatially distinct regions of of conserved motifs present in other RNA virus RdRps the birnavirus VP1 proteins and the presumptive RdRp of DXV and the other birnaviruses, with some notable exceptions (Figs. 4 and 5). The birnavirus VP1 proteins clearly contain the RdRp The aligned RdRp sequences clearly revealed the palm motifs IV and V and also possess a potential motif presence of the conserved RdRp motifs IV and V (Figs. 2 VII. The fourth conserved palm motif, motif VI (also re- and 4). Motif IV of the RdRps contains two highly con- ferred to as motif C), contains the highly conserved GDD served aspartate residues spaced five residues apart. motif that is believed to be an essential component of the The first of these, which is conserved in the birnavirus catalytic site. In the majority of RdRps, motif VI contains VP1 proteins, is believed to be part of a catalytic triad predominantly apolar residues and is predicted to form a involved in metal ion coordination during the nucleotidyl ␤-strand, turn, ␤-strand structure (Duncan et al., 1991; transfer reaction (Steitz, 1998). The second conserved Hansen et al., 1997; O’Reilly and Kao, 1998). This con- aspartate may be involved in the discrimination between served structure serves to position the two aspartate nucleotides and deoxynucleotides. Interestingly, conver- residues close to the conserved aspartate in motif IV, sion of this second aspartate to the larger glutamate forming a catalytic triad that coordinates two divalent residue in the encephalomyocarditis virus 3Dpol almost cations for the nucleophilic attack of the 3Ј-hydroxyl and eliminates polymerase activity (Sankar and Porter, 1992), stabilization of the pyrophosphate product (Steitz, 1993, yet this natural substitution exists in the VP1 proteins of 1998). Although the requirement for a glycine residue is all three genera of birnaviruses (Fig. 4). Secondary struc- somewhat flexible, any changes to the aspartate resi- ture predictions of the core of motif IV present in the dues are poorly tolerated (O’Reilly and Kao, 1998). birnavirus RdRps suggest a ␤-strand, turn, ␣-helix con- Most interestingly, all of the presumed birnavirus formation, the same conformation predicted for motif IV RdRps lack the highly conserved GDD motif. Motif VI of other RdRps (Fig. 5). generally resides approximately 15–30 residues down- 246 SHWED ET AL.

FIG. 4. Sequence alignments of the conserved RdRp motifs. The aligned amino acid sequences from conserved domains IV–VI of representative plus-stranded animal RNA viruses, plus-stranded plant RNA viruses, and double-stranded RNA viruses are shown. The generalized consensus sequence, displayed at the top of the figure, is expressed with the following groups: J, apolar (LIVMFYWCAGP); O, polar (STNQH); B, apolar or polar (J ϩ O); Z, polar or charged (O ϩ KDER); U, aromatic (FYW); (—), any amino acid. Uppercase letters indicate that the amino acid conforms to the generalized consensus sequence. Highly conserved residues in the consensus are indicated with an asterisk, and residues conforming to the highly conserved consensus residues are indicated in boldface type. Arabic numbers represent the number of amino acids upstream or between the motifs. The virus abbreviations are as follows: POLIO, poliovirus type 1 (Accession No. P03299); EMC, encephalomyocarditis virus (Accession No. GNNYE); FMDV, foot and mouth disease virus (Accession No. P03305); SBV, Sindbis virus (Accession No. AAA96974); YFV, yellow fever virus (Accession No. POLG_TEFV2); IBV, infectious bronchitis virus (Accession No. CAC39112); TMV, tobacco mosaic virus (Accession No. RRPO_TOML); BMV, brome mosaic virus (Accession No. P2BVA); TEV, tobacco etch virus (Accession No. GNBVEV); CNV, cucumber necrosis virus (Accession No. RRVGCN); MCMV, maize chlorotic mottle virus (Accession No. CAB55589); BTV, bluetongue virus serotype 10 (Accession No. RRXRBT); ROTA, simian rotavirus SA11 (Accession No. P1XRSR); REO, mammalian reovirus strain Dearing (Accession No. MWXR33); PHI6, bacteriophage ␾6 (Accession No. VP2_BPPH6); IBDV, infectious bursal disease virus serotype P2 (Accession No. AF240687); IPNV, infectious pancreatic necrosis virus serotype jasper (Accession No. M58756); DXV, Drosophila X virus (Accession No. AF196645). stream of motif V in most RdRps (Fig. 4). This region of this region in all three birnavirus VP1 proteins is pre- the IBDV VP1 protein contains an IDD sequence, similar dicted to exist as an extended ␣-helix (Fig. 5). The con- to the SDD sequence present in the ␾6 RdRp, that could served structural prediction of clearly homologous pro- serve as the functional equivalent of the GDD motif. IBDV teins containing sequence heterogeneity lends added motif VI, however, contains an unusual preponderance of confidence to the predicted ␣-helical conformation of charged residues compared to the more hydrophobic this region in all three birnavirus VP1 proteins. The only environment surrounding the GDD motif in most other other downstream DD sequence (PDD) exists at position RdRps (Fig. 4). Secondary structure predictions for RdRp 761 in the IBDV VP1 protein. This di-aspartate sequence motif VI of IBDV also suggest potential differences in the exists in a region of limited sequence similarity to the structure of this region compared to motif VI of other other birnavirus VP1 proteins (Fig. 2), it is surrounded by RdRps. A preferred ␣-helical conformation is predicted charged residues, and secondary structure predictions for the region encompassing the IBDV IDD motif, unlike show high scores for an extended loop region (data not the predicted ␤-strand, turn, ␤-strand structure sug- shown). Therefore, if IBDV VP1 does contain a homolog gested for motif VI of most RdRps (Fig. 5). However, a of RdRp motif VI, then the first DD motif is more likely to similar ␣-helical structure is predicted for motif VI of ␾6, serve this function. yet the crystal structure of ␾6 VP2 indicates that this Striking alterations were noted when comparing the motif exists in a ␤-strand, turn, ␤-strand conformation, sequences of the DXV and IPNV regions corresponding the same conformation observed for motif VI in the crys- to the tentative motif VI of IBDV. This region in both the tal structures of hepatitis C virus and poliovirus RdRps DXV and the IPNV VP1 proteins lacks any DD sequence (Hansen et al., 1997; Lesburg et al., 1999; Butcher et al., and displays more extensive sequence heterogeneity 2001). The ␣-helical prediction for this region of IBDV than observed for the conserved motifs IV and V present must, therefore, be interpreted with caution. However, in the birnavirus VP1 proteins (Fig. 2). The absence of a BIRNAVIRUS VP1 PROTEINS 247

FIG. 5. Predicted secondary structures in the conserved region of the RdRp core palm structure (motifs IV, V, and VI). The palm motifs of poliovirus (POLIO) and hepatitis C virus (HCV) are presented along with the same motifs identified in RdRps derived from a selection of dsRNA viruses (REO, mammalian reovirus strain Dearing; PHI6, bacteriophage ␾6; IBDV, infectious bursal disease virus serotype P2; IPNV, infectious pancreatic necrosis virus serotype jasper; DXV, Drosophila X virus). The predicted consensus secondary structures were derived using the PHD method of Rost and Sander (1994) using PredictProtein software (www.embl-heidelberg.de/predictprotein/). h, alpha helix; b, beta strand; dot, no prediction. The locations of the highly conserved residues characteristic of each motif are indicated with an asterisk and the corresponding residues are shown in boldface type.

DD sequence in the DXV and IPNV sequences indicates present in the other birnavirus RdRps, and they occur in that the tentative motif VI of IBDV is not conserved in a regions of extensive birnavirus VP1 sequence heteroge- similar location in these related birnavirus RdRps. neity (Fig. 2). The last two DXV DD sequences occur in We examined the IPNV and DXV VP1 sequences for predicted loop regions (data not shown), while the first the presence of downstream DD motifs that might rep- downstream DD sequence (residues 755/56) of DXV re- resent the missing motif VI. In the case of IPNV, it has sides in a region with high-probability ␣-helix scores, been suggested that a downstream LDD sequence, res- similar to the predicted secondary structures of the ten- idues 653–655 (Fig. 2), may represent a repositioned tative motif VI in IBDV and the downstream DD sequence motif VI (Koonin, 1992). However, the alignment of the in IPNV (Fig. 5). As a result, all of the birnavirus VP1 three birnavirus VP1 proteins indicates that there is no proteins contain a DD sequence that could function in a conservation of aspartate residues in the region corre- similar fashion as the GDD motif present in the majority sponding to the LDD sequence of IPNV as might be of RdRps. However, the downstream DD motifs in the expected if a structural rearrangement of the birnavirus birnavirus VP1 proteins all exist in predicted ␣-helical or RdRp resulted in repositioning of motif VI (Fig. 2). There loop conformations, these sequences are located in spa- are no other downstream DD sequences in the IPNV VP1 tially distinct regions of the birnavirus RdRps, and they protein that could serve a similar catalytic function as the occur in regions of extensive sequence heterogeneity. GDD sequence present in other RdRps. If the down- The absence of significant sequence similarity be- stream DD sequence in IPNV VP1 does represent the tween the RdRps of the dsRNA totiviruses and other RNA missing motif VI, then the event leading to repositioning viruses, and the identification of additional conserved of this motif did not occur in a similar fashion with the motifs present in the RdRps of different totiviruses, led to other birnavirus RdRps. the suggestion that totivirus RdRps represent a distinct A similar situation exists for DXV, which also lacks a subgroup within this enzyme family (Bruenn, 1993; DD sequence in the region corresponding to the tenta- Routhier and Bruenn, 1998). Our results suggest that the tive motif VI of IBDV. DXV VP1 does contain three down- VP1 proteins of the birnaviruses form a second defined stream DD sequences (residues 755/56, 895/96, and subgroup of RdRps with significant differences in the 957/958) that could function as homologs of motif VI (Fig. arrangement of the predicted catalytic domain of this 2). As with IPNV, however, the downstream DD residues enzyme family. Based on the clear conservation of motifs of DXV do not align with any of the DD sequences IV and V in the birnavirus VP1 proteins, it seems reason- 248 SHWED ET AL. able to assume that these RdRps function in a manner polymerase (Gibco BRL). The reaction was incubated for similar to that predicted for all other RdRps. If so, then the 10 min at 37°C. The RNA was then extracted with phe- birnavirus RdRps should be dependent on the formation nol–chloroform and recovered by ethanol precipitation. of a catalytic triad of aspartate residues. The only way to Polyadenylated RNA was resuspended in 10 ␮l of dieth- envision such a conserved catalytic activity requires ac- ylpyrocarbonate-treated water. cepting that the birnavirus RdRps have repositioned mo- First-strand synthesis was carried out by denaturing tif VI to three separate downstream locations in regions 20 ␮g of virion RNA consisting of segment A and B in 9 of extensive sequence and predicted structural hetero- ␮l of 10 mM Tris–HCl, pH 7.5, by incubation at 100°C for geneity. Conversely, the only other possibility is that a 2 min, followed by quick freezing in a dry ice–ethanol homolog of motif VI and its highly conserved GDD motif bath. The thawed suspension was then made 10 mM may not function as the catalytic core of the birnavirus with respect to methyl mercury hydroxide. Denaturation polymerases. The absence or repositioning of motif VI to proceeded for 45–60 min at 23°C in the presence of 10 U separate locations in the birnavirus VP1 proteins is, as of RNasin (Promega, Canada). An additional 10 U of far as we are aware, a unique situation for a viral RdRp. RNasin was added with ␤-mercaptoethanol to a concen- Regardless of the actual catalytic mechanism of the tration of 187 mM and the reaction was incubated at birnavirus RdRps, it seems likely that the structures of 23°C for another 15 min. Viral cDNA was synthesized by the birnavirus RdRps may exhibit significant variation reverse-transcribing denatured dsRNA using random from other RdRps. The potential differences in the ar- primers as described by Gubler and Hoffman (1983). rangement of catalytic motifs and the structure of this Double-stranded cDNA was tailed with dCTP using subgroup of RdRps may account for the distinct at- TdT, cloned into the dGTP tailed PstI site of pTZ18R tributes of birnavirus RdRps, namely, their role as both (Mead et al., 1986), and then subcloned into competent E. polymerase and primer for semiconservative genome coli HB101 or DH5 cells according to Sambrook et al. replication (Mertens et al., 1982; Nagy and Dobos (1989). Two positive recombinants were identified by 1984a,b; Calvert et al., 1991; Dobos, 1995a). More de- restriction endonuclease mapping, as described by tailed structural and mutational analyses of the birnavi- Cameron (1991). rus RdRp proteins should reveal important insights into the conformation, precise location, and features of the Amplification of 5Ј and 3Ј noncoding regions catalytic site of this important subgroup of viral poly- The nucleotide sequences of the 5Ј and 3Ј noncoding merases. regions of DXV segment B were determined by the methods described for IBDV (Mundt and Muller, 1995) and IPNV MATERIALS AND METHODS (Yao and Vakharia, 1998). To determine the 3Ј-termini of both strands of segments A and B, the viral RNA was Cells and virus polyadenylated and reverse transcribed with a poly(dT) DXV was propagated in Schneider’s Drosophila line 1 primer containing a NotI site, and resulting cDNAs were cells, WR strain (Friesen and Rueckert, 1982) (WR strain amplified by PCR with virus-specific primers DXV5ЈR kindly provided by Dr. Dasgupta). Plaque-purified virus (5Ј-GTTGGTATGTGCAGGCCAGAG-3Ј (nucleotides 410– was used to infect cell cultures at a multiplicity of infec- 430, antisense orientation) and DXV3ЈF(5Ј-GCAGCACCCT- tion of 0.01 plaque-forming units per cell. Purification of GTTGTCAAAC-3Ј at nucleotides 2828–2846, sense orienta- DXV was carried out by freon extraction and sucrose and tion). The reverse transcription products were separated by CsCl gradient centrifugation in TNE buffer (0.1 M Tris, 0.1 agarose gel electrophoresis, purified by a QIAquick gel M NaCl, 1 mM EDTA, pH 7.3) as described previously extraction kit (Qiagen Inc.), and directly sequenced by the (Dobos and Rowe, 1977). The final purified virus band dideoxy chain termination method. The 5Ј terminus of seg- from CsCl gradients was dialyzed against 10 mM Tris– ment B was determined by rapid amplification of cDNA

HCl, pH 8.0, 0.3 mM MgCl2 and stored in aliquots at ends using the 5Ј RACE System (Life Technologies Inc.). Ϫ80°C at a concentration of approximately 1 mg mlϪ1. Sequence analysis and secondary structure Cloning strategy prediction cDNA clones of segment B of DXV were constructed The sequence of DXV genome segment B was ob- by denaturing genomicRNA and poly(A) tailing it with tained from two independent clones representing the 5Ј- Escherichia coli poly(A) polymerase (Cashdollar et al., and 3Ј-proximal regions of the genome segment. Both 1982). Briefly, 20 ␮g of virion RNA in 2.5 ␮l of water was clones were sequenced in their entirety in both direc- incubated with 22.5 ␮l of dimethyl sulfoxide for 45 min at tions by automated sequencing and compiled using 60°C. The polyadenylation reaction mixture consisted of Generunner version 3.0 software. The clones overlapped

50 mM Tris, pH 7.9, 10 mM MgCl2, 250 mM NaCl, 2.5 mM by 464 nucleotides, which allowed a contiguous se-

MnCl2, 0.25 mM ATP, 250 g BSA, and1Uofpoly(A) quence to be obtained. The clones were truncated by 19 BIRNAVIRUS VP1 PROTEINS 249

nucleotides at the 5Ј-end and by 193 nucleotides at the ACKNOWLEDGMENTS Ј 3 -end. The terminal sequences were obtained by sep- This research was supported by the Natural Sciences and Engineer- arate amplification of the 5Ј- and 3Ј-termini, as described ing Research Council of Canada (P.D.), and by a grant from the Cana- above, to generate the complete DXV genome segment B dian Institutes of Health Research (CIHR) to R.D. cDNA sequence. There were no sequence discrepan- REFERENCES cies in the 464-nucleotide overlap region of the two independent clones, suggesting that the presented se- Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: A new quence is a reasonable estimate of the quasispecies. generation of protein database search programs. Nucleic Acids Res. This was further confirmed by sequencing of the 5Ј- 25, 3389–3402. terminal 650 nucleotides and an additional 600 nucleo- Argos, P. (1988). 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