INTRODUCTION

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While the sequences of many viral replication proteins have been identified, we have only a minimal understanding of the higher-order interactions between them. The interactions of the replicase subunits of the negative-strand influenza virus have been elucidated at the biochemical level (12, 31). However, the interactions of the subunits of the replicases of positive-strand RNA viruses, including those of the well-characterized coliphage Q, are poorly understood (5). We have focused on dissecting the interactions between the replication proteins of the monocot-infecting brome mosaic virus (BMV). These studies are essential for the eventual comparison and understanding of the three-dimensional structure and function of viral RNA replicases.

The BMV genome is composed of three genomic positive-strand RNAs designated RNAs 1, 2, and 3. The genomic RNAs serve dual functions as mRNAs for translation and as templates for the synthesis of the complementary negative-strand RNAs. To complete the replication cycle, the negative-strand RNAs then serve as templates for positive-strand RNA synthesis. RNAs 1 and 2 encode the replication proteins 1a (109 kDa) and 2a (96 kDa), which are sufficient for BMV RNA replication in protoplasts (16). The two proteins have evolved to work specifically with each other, because heterologous combinations of 1a and 2a from the closely related BMV and cowpea chlorotic mottle virus (CCMV) exhibit RNA synthesis defects (7).

Several domains of the 1a and 2a proteins have been identified based on sequence similarities. The 2a protein contains two nonconserved regions flanking a centrally conserved domain which shares sequence motifs with many polymerases, including the presence of the Mg²⁺-binding GDD motif (3). The N terminus of 1a resembles the nsP1 protein of Sindbis virus, indicating its possible involvement in RNA capping functions (10, 20, 27). The 1a C terminus has sequence homology to many viral and cellular helicases (8). Mutations in 1a and 2a have been shown to abolish or greatly reduce RNA replication levels (17, 32).

We have previously shown that BMV 1a and 2a interact both in vitro and in the yeast two-hybrid system (15, 21). Furthermore, we used a set of well-characterized 1a mutants (PK mutants [17]) to show an absolute correlation between the in vivo phenotypes of these mutants and their ability to interact with 2a in the two-hybrid system (22). In this study, we use the PK mutants to demonstrate a perfect correlation between their in vivo phenotypes and their ability to interact with 1a in the two-hybrid system. We also map the 1a-1a interaction to the N-terminal putative capping domain (previously referred to as the methyltransferase-like domain) of both BMV and cucumber mosaic virus (CMV) and show the interaction to be species specific. These results, along with those from previous mapping data, are placed in the context of the predicted secondary structure of the 1a protein. Finally, we describe a novel interaction between the putative capping and helicase-like domains of the 1a protein.

	MATERIALS AND METHODS

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Strains, reagents, and two-hybrid procedures. Saccharomyces cerevisiae strains, plasmids, growth conditions, and reporter protein assays have been described previously (22). In our previous work characterizing the 1a-2a interaction, DNA coding for each PK mutant was individually fused to the LexA DNA binding domain in plasmid pBTM116 (22). For our current analysis, we created fusions of the PK mutants to the GAL4 transcription activation domain in plasmid pGAD424 by using the previously described primers and cloning procedures (Table 1) (22). In general, viral DNA fragments flanked by appropriate restriction sites were produced by PCR and then cloned into the above-mentioned two-hybrid plasmids (kind gifts of Stan Fields). pGBT9 (Clonetech Laboratories, Inc.), which contains a GAL4 DNA binding domain, was used in some experiments. The fusions were then tested for interaction with the appropriate wild-type 1a (wt1a) fusion construct by their ability to activate the -galactosidase and/or HIS3 reporter genes.

Western blot analysis. Yeast extracts were prepared by scraping three-day-old yeast colonies (100 to 200 mg) from plates and suspending them in 3.0 ml of YPD broth. After growth for 2 h at 30°C, the protein synthesis inhibitor cycloheximide (50 µg/ml) was added followed by another hour of incubation. The cells were then washed with ice-cold water and suspended in 200 µl of cracking buffer (40 mM Tris-HCl [pH 6.8], 0.1 mM EDTA, 5% [wt/vol] sodium dodecyl sulfate (SDS), 8 M urea, 0.05 M -mercaptoethanol, 0.4 mg bromophenol blue/ml, and the protease inhibitors pepstatin A [1 µg/ml], leupeptin [3 µM], benzamidine [14.5 mM], aprotinin [37 µg/ml], and phenylmethylsulfonyl fluoride [33.4 mg/ml]). The cell suspension was heated at 70°C for 10 min, and then the cells were lysed by vortexing in the presence of glass beads as previously described (22). The extracts were then separated on a SDS-8% polyacrylamide gel, which was then blotted onto polyvinylidene difluoride membrane (Millipore) at 31 V for 5 h in Western transfer buffer (39 mM glycine, 48 mM Tris-HCl [pH 8.3], 0.0037% SDS, 20% methanol). After the transfer, exhausted gels were stained with Coomassie blue to ensure even transfer. The blots were then probed with a polyclonal LexA-specific antibody (kindly provided by Barak Cohen) and a secondary antibody linked to horseradish peroxidase. Bands were visualized by chemiluminescence (U.S. Biochemical Corp.). Quantitation was performed with a densitometer (ImageQuant; Molecular Dynamics). The abundance of each mutant protein was determined relative to that of wt1a. A cross-reactive cellular band of approximately 40 kDa was used as an internal control to normalize for the amount of protein loaded. Each number shown is an average of two independent experiments.

Construction of deletion series. The deletion series of 1a was made by Wang and French, using pGAD424 and pGBT9 (Clonetech Laboratories, Inc.). A DNA segment encoding the entire BMV 1a reading frame was generated by PCR with primers to introduce BamHI restriction sites (upstream primer B1a, 5'-CAACAGGATCCCAAGTTCTA-3' [native BMV sequences are underlined]; downstream primer B1aRev, 5'-AGACAGGATCCTCACTTAAC-3'). After amplification, the PCR fragments were liberated with BamHI and cloned in frame with the GAL4 transcriptional activation domain in pGAD424 to generate pGAD1a. The BamHI fragment from pGAD1a was then cloned into the BamHI site of pGBT9 to generate pGB1a. The orientation of the insert was determined by restriction analysis, and the junctions of the fusions were sequenced to ensure that the coding frame was correct.

Secondary structure analysis. Predictions of secondary structure were generated by the method of Rost and Sander (24, 25), which is more than 70% accurate. The 1a-like proteins from three related plant virus families were analyzed: bromoviruses, cucumoviruses, and tobamoviruses. The specific sequences evaluated can be found in the GenBank database. Bromovirus sequences used were BMV (strain Japanese), CCMV, and broad bean mottle virus (strain BA). Cucumovirus sequences used were CMV (strain fny), peanut stunt virus (strain J), and tomato aspermy virus. Tobamovirus sequences used were tobacco mosaic virus (TMV) (strain Korean), pepper mild mottle virus (strain Spain), tobacco mild green virus (strain U2), and cucumber green mottle virus (strain watermelon).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Effects of two- to three-amino-acid (aa) insertions on BMV 1a-1a interaction. We have previously established that the two-hybrid system is a convenient and suitable tool for the dissection of the BMV replicase structure. Using this tool, we discovered a novel interaction between two 1a proteins of BMV and those of CMV and CCMV (22). Furthermore, the interaction of the 1a proteins of BMV, CCMV, and CMV occurred in a species-specific manner (22).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   (A) Locations of insertion mutations and their replication phenotypes in protoplasts. The box represents the 1a open reading frame. The lightly shaded N-terminal portion contains sequences putatively involved in capping, while the darkly shaded C-terminal portion contains helicase-like sequences. The vertical bars represent the positions of the 2- to 3-aa insertions made and characterized by Kroner and colleagues (17). Bars pointing up represent viable mutants, while bars pointing down represent mutants unable to replicate in barley protoplasts. PK1, -4, and -19 were found to be temperature sensitive for replication at 35°C. The ability (+) or inability () of each PK mutant to interact with wt1a in the two-hybrid system is indicated. (B) -Galactosidase activity detected when each of the PK mutants is coexpressed with wt1a in yeast strain Y835. The results from two independent experiments (Expt) are shown. These activities are presented as fold activity over that of wt1a in the DNA binding domain plasmid. -Galactosidase activity is shown as micromoles of O-nitrophenyl galactoside hydrolyzed per minute per milligram of protein. (C) -Galactosidase activity detected for strains grown at 24°C. PK1, a replication-competent mutant in protoplasts which did not interact with wt1a as a LexA fusion at 30°C, did interact at 24°C. PK9, a replicating mutant, and PK6, a nonreplicating mutant, serve as positive and negative controls, respectively. N.D., not determined.

All of the replicating mutants can interact with themselves and each other. The replication-competent PK mutants were tested for interaction with themselves and with each other. Self-interactions were expected, since the original replication results of Kroner et al. (17) were obtained in protoplast transformations which had no source of wt1a. All of the replicating mutants retained interaction with themselves (i.e., PK9-PK9), as detected by the ability of the double transformants to turn blue in the presence of X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) and to grow on plates lacking histidine (data not shown). This is consistent with the hypothesis that 1a-1a interaction is necessary for replication. The PK1-PK1 interaction was observed at 24 but not at 30°C, consistent with our previous results showing GAL4-PK1 to be temperature sensitive (Fig. 1B and data not shown). Additionally, all replicating mutants retained the ability to interact with each other (i.e., PK9-PK21), while PK11 (a nonreplicating mutant) could not interact with itself or any other PK mutant (data not shown). Therefore, all of our results are consistent and the PK mutants which allowed for replication in plant cells also allowed for the 1a-1a interaction between themselves.

Stability of the mutant proteins in vivo. A lack of interaction in the two-hybrid system can simply be due to an unstable fusion protein. To determine if some of the PK mutants were unstable, we checked the abundance of each LexA fusion protein by Western blot analysis. All of the replicating mutants had abundances from 48 to 118% of that of wt1a (Fig. 2). The two nonreplicating mutants which retained the ability to interact with wt1a, PK3 and PK15, were 58 and 53% as abundant as wt1a (Fig. 2). Of the nonreplicating, noninteracting mutants, only PK18 and -20 were somewhat reduced in abundance, present at only 33 and 31% of the abundance of wt1a, respectively (Fig. 2). The observation that many of the noninteracting mutants were as abundant as wt1a indicates that a lack of interaction was not due to a gross reduction in overall protein stability. Thus, the presence of a protein alone is not sufficient for protein-protein interaction.

View larger version (72K): [in this window] [in a new window]   FIG. 2.   Relative abundance of wt and mutant 1a proteins as observed in Western blots. (A) The basic diagram is the same as that in Fig. 1A. The numbers above or below each PK mutant indicate the percent abundance of each protein relative to that of wt1a. The results shown are an average of the quantitations from two independent Western blots. Quantitation was performed by laser densitometry (Molecular Dynamics). The abundance of PK17 (*) was somewhat inconsistent between trials, being 150 and 35% that of wt1a. N.D., not determined. (B) Scan of a Western blot showing the abundance of the ca. 140-kDa LexA-PK mutant fusions. The presumed full-length protein is indicated along with the presumed helicase and capping fusions. For unknown reasons the migration of the capping domain was reproducibly faster than expected based on its predicted mass. Lower-molecular-mass bands are presumed to be degradation products. A ca. 40-kDa cellular protein (**) cross-reacted with the antibodies and was used to normalize the amount of protein loaded.

Mapping of the 1a-1a binding domain with deletion analysis. To further identify the region(s) needed for 1a-1a interaction, we tested the abilities of 1a deletions fused to the GAL4 activation domain to interact with wt1a fused to either the LexA or the GAL4 DNA binding domain. Deletions of C-terminal residues 738 to 961, 622 to 961, 564 to 961, and 516 to 961, removing the entire helicase-like domain, all retained interaction with wt1a when tested in yeast strain Y835 (Fig. 3). An additional deletion of 37 residues, producing 1a-480-961, resulted in a loss of interaction with wt1a, indicating that the C-terminal boundary lies between aa 480 and 515 (Fig. 3). At the N terminus of 1a, removal of only 45 residues abolished interaction with full-length 1a. Thus, the region containing putative capping sequences is required for 1a-1a interaction. The same results were obtained when the deletion series was tested by the filter lift assay for the ability to interact with wt1a fused to the GAL4 DNA binding domain (in pGBT9) in strain Y187 (Fig. 3). Negative controls for these assays included the DNA binding domain plasmid in the absence of a partner plasmid and each fusion in combination with a partner plasmid lacking 1a sequences. All of the negative controls resulted in relative specific activities at or below background levels (data not shown). Due to a lack of GAL4-specific antibody, we did not examine the stability of these N- and C-terminal deletions of 1a.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Mapping the 1a-1a binding domain by deletion analysis. The domains of the 1a protein are depicted along with various deletions in either the N or C terminus. Fold -galactosidase levels detected when each of the deletions was expressed with wt1a from two independent experiments are shown relative to those of wt1a alone. For strain Y187 (Clonetech Laboratories, Inc.), only the results of the qualitative filter assays are shown. 516-966 retains the ability to interact with both itself and wt1a. B, blue colonies; W, normal yeast colony color. AA, amino acid.

Mapping the 1a-1a interaction in CMV. We have previously shown that the 1a proteins of both CCMV and CMV interact with themselves in a species-specific manner (22). To determine if the 1a-1a interaction occurred in the putative capping regions of other viruses, we tested for such an interaction in CMV, which is more distantly related to BMV than CCMV, thus providing a more rigorous test of the biological relevance of this interaction. Sequence alignments generated by CLUSTAL W (30) indicated that CMV residues 1 to 533 were homologous to BMV residues 1 to 515. Therefore, PCR was used to generate the CMV DNA fragment flanked by the appropriate restriction enzymes. The fragment containing the putative capping sequences of CMV 1a was then cloned into pBTM116 and pGAD424 to create pLex-C_MT and pG-C_MT.

View larger version (52K): [in this window] [in a new window]   FIG. 4.   Assay for induction of the HIS3 gene in homologous or heterologous pairings of BMV and/or CMV 1a methyltransferase fusions. The plate on the left is supplemented with histidine, while the one on the right is not. Each doubly transformed strain was written onto plates to indicate the identity of the 1a fusion proteins. The LexA fusion is indicated above the slash, and the GAL4 fusion is indicated below the slash (i.e., LexA/GAL4).

Secondary structure analysis of BMV 1a and related proteins. Currently, the only structural analysis of BMV 1a comes from protease digestion studies, which indicate that a globular domain exists from aa 556 to 961 in the helicase-like portion of the protein which is needed for interaction with the N terminus of 2a both in vitro and in the two-hybrid system (21) (Fig. 5A). Further analysis would be greatly enhanced by secondary structure predictions which are at least 70% accurate (24, 25). This analysis is also necessitated by the discrepancy in the boundaries encoding the various activities of the putative capping and helicase-like domains (compare the figures in references 1 and 22 with those in references 4, 26, and 29). The discrepancy is based on the interpretations of sequence comparisons of the 1a-like proteins from a number of plant-infecting members of the alphavirus-like superfamily. When compared to Semliki Forest virus (SFV) and Sindbis virus, the highly conserved sequences (H, DxxR, and Y, where x is any amino acid) required for capping in BMV 1a are located from residues 1 to 263 (26) (Fig. 5A). Mutation of the conserved histidine in SFV specifically abolished guanylyltransferase activities, while the homologous mutation in Sindbis virus affected both methyltransferase and guanylyltransferase functions (2, 33). Mutation of the other three conserved residues affected both guanylyltransferase and methyltransferase functions in both SFV and Sindbis virus (2, 33). Comparisons of these and other conserved residues in alfalfa mosaic virus, TMV, BMV, CMV, and tobacco rattle virus revealed a large relatively nonconserved region of >400 aa residing between the putative capping and helicase-like sequences (4, 29).

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Summary of the predicted secondary structures of BMV and related 1a proteins. Boxes, -helices; arrows, -sheets; solid shapes, >80% accuracy; open shapes, >50% accuracy. (A) Diagram of the BMV 1a protein and its predicted secondary structures. The previously identified sequences involved in capping and helicase function are indicated. Consistent with Fig. 1A, the putative capping domain required for 1a-1a interaction is lightly shaded while the protease-resistant helicase-like domain required for 1a-2a interaction is darkly shaded. The locations of the PK mutants in the predicted secondary structures are indicated, with the replication-competent mutants circled. The secondary structures which correspond to those found in DNA methyltransferases are numbered according to the system of Schluckebier et al. (28). We also labeled helices  and  to facilitate the flow of the text. (B) Expanded picture of the predicted secondary structure of the putative capping domain of the bromo-, cucumo-, and tobamovirus families compared to that of the DNA methyltransferase HhaI. 3 of HhaI, shown as an unshaded arrow, was not present in the originally solved crystal structure and was not predicted by PHD analysis (6). This strand was later added upon comparison of the structure HhaI to other methyltransferase crystal structures (19, 28). Also, there is a 4-aa -sheet (4') following 4 which we have not included in our diagram for simplicity (6). (C) Functional residues involved in SAM binding in HhaI and their putative functional homologs in the SAM binding capping proteins of RNA viruses. Residues conserved between the DNA and RNA SAM binding proteins, including the G-loop, are shown in bold letters. The underlined residues of SFV nsP1 have been mutated to alanine and all, except D180A, have effects on methyltransferase and guanylyltransferase functions. Asterisks indicate identical amino acids, and periods indicate similar amino acids.

Correlation of predicted domain borders with function. The predicted secondary structures of the putative capping domain agree remarkably well with our functional analysis of protein-protein interaction. Our deletion analysis places the end of the putative capping domain at aa 515 (Fig. 3). Only the truncations which retain all of the predicted capping domain intact can interact with 1a. The minimally functional truncation, aa 1 to 515, which removes 2 aa of , does not grossly disturb its structure (Fig. 5A). Removal of an additional 34 aa completely removes and is not functional for 1a-1a interaction. An N-terminal truncation of 45 residues in GAL4-1-45 removes the first two -helices and is unable to interact with 1a. Our mapping and structural prediction data suggest that BMV residues 1 through 517 exist as a functional domain.

Effect of 2- to 3-aa insertions on predicted secondary structures. We noted that two of the replicating mutants, PK21 and -14, are present in the newly positioned hinge region and, as expected, have no affect on the predicted secondary structure (data not shown). We wondered if similar correlations could be drawn from other PK mutants. Four of the eight replicating mutants, PK9, -1, -21, and -14, are located in predicted loops and have no affect on predicted -helices or -sheets (Fig. 5A). The other four replicating mutants, PK5, -4, -2, and -19, are located within predicted -helices or -sheets, but their insertions do not disrupt the predicted structures. All of the nonreplicating mutants had predicted adverse effects on the elements they occupied and/or nearby elements (Fig. 5A and data not shown). Thus, a perfect correlation exists between the maintenance of the predicted secondary structure and the ability to replicate in protoplasts. Since we have shown that all PK mutants are somewhat stable (Fig. 2), it is likely that the perturbations of secondary structure are affecting function.

Comparisons of predicted elements to those found in animal virus and DNA methyltransferases. Portions of the predicted secondary structures in the putative capping domain of the plant-infecting viruses appear to resemble structures important for S-adenosyl-L-methionine (SAM) binding found in DNA methyltransferases (Fig. 5). The resemblance between RNA and DNA methyltransferases was first observed when the structure of the VP39 protein of vaccinia virus was solved (11). The bromo-, cucumo-, and tobamovirus 1a-like proteins all contain the alternating --- structures universally found in SAM-dependent methyltransferases (19, 28) (Fig. 5B). We used a group  methyltransferase, HhaI, to elaborate this comparison because its crystal structure complexed with SAM has been solved (6, 19). All of HhaI's known elements were predicted accurately by the PHD program, including the absence of 3, which was not present in the originally reported crystal structure (6) (Fig. 5B). However, this strand was later added based on comparisons to other methyltransferase crystal structures (6a, 19, 28). The bromo- and cucumovirus 1a proteins retain the secondary structure elements in the same order as HhaI with a single insertion of an -helix between E and 6 (Fig. 5B). The TMV-like proteins have several additional -helices between B and 3, E and 6, and 6 and 7 (Fig. 5B). It is interesting to speculate that these and the other extra -helical TMV elements shown in Fig. 5B may be related to the effects of this domain on viral pathogenesis proposed for TMV p126 (4, 29).

An interaction between the helicase- and methyltransferase-like portions of the 1a protein. In our previous work we found that none of the nonreplicating mutants could interact with 2a (22). This is surprising for PK mutants 6, 7, 10, and 11 because their insertions are in the putative capping domain while the helicase-like domain has been demonstrated to be sufficient for interacting with 2a. Further, Western blotting results show that these mutants are as stable as wt1a (Fig. 3), indicating that there may be an additional requirement for protein-protein interaction. One hypothesis consistent with these observations is that the N- and C-terminal halves of the 1a protein may interact. Thus, mutations in the 1a N terminus affect interactions that occur in the 1a C terminus. To test this possibility, we used pEO500, a LexA fusion retaining the entire hinge and helicase-like domain (21), for interaction with pG-B_MT, which retains the putative capping domain fused to GAL4. The product of pEO500, LexA-B_Hel, was able to interact with GAL4-B_MT with a relative activity of greater than 25-fold over background (Table 3). We subcloned sequence coding for 1a helicase into pGAD424, generating pG-B_Hel. When tested, GAL4-B_Hel interacted with LexA-B_MT with a relative activity of threefold over background. Although many helicases function as multimers (18), we found no evidence for a helicase-helicase interaction (Table 3). Negative controls for these experiments all gave relative activities at or below background (Table 3).

Model for replicase assembly. Our observations to date suggest a working model for the assembly of the 1a-2a complex (Fig. 6). The presence of the putative intramolecular interaction in 1a may serve to prevent the formation of the 1a-2a complex until the appropriate 1a-1a complex has formed. It is also possible that each step is influenced by host factors and/or RNA interactions.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Working model for the assembly of the 1a-2a complex. The putative intramolecular interaction in 1a prevents the formation of the 1a-2a complex until the intermolecular 1a-1a interaction has occurred. The 2a protein interacts with the helicase-like domain of 1a through its N terminus.

Summary. In this work we have (i) shown a perfect correlation between replication and the ability of the PK mutants to interact with wt1a, (ii) mapped the species-specific intermolecular interaction to the N-terminal putative capping domains of both the BMV and CMV 1a proteins, (iii) demonstrated a high degree of conservation in the predicted secondary structures among members of the plant alphavirus-like superfamily and structures associated with SAM binding in DNA methyltransferases, and (iv) demonstrated a novel interaction between the N- and C-terminal halves of the 1a proteins of both BMV and CMV that is likely to be intramolecular in nature. The predicted secondary structures correlate well with the effects of deletions and insertions on protein-protein interactions and on RNA replication in barley protoplasts. These results predict new borders for the functional domains of the putative capping, helicase-like, and hinge regions of 1a. The figures in this paper reflect these new borders. Additionally, this analysis predicts the positions of functional residues involved in BMV 1a SAM binding. These results contribute further to an understanding of the architecture and assembly of replicase complexes from positive-strand RNA viruses.