Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan1
Author for correspondence: Tetsuro Okuno. Fax +81 75 753 6131. e-mail okuno{at}kais.kyoto-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brome mosaic virus (BMV), a well-studied plant RNA virus, is the type member of the genus Bromovirus in the family Bromoviridae in the alphavirus-like superfamily (Kao & Sivakumaran, 2000 ). The BMV genome is composed of three RNAs designated RNA1, RNA2 and RNA3. Subgenomic RNA4 is transcribed from the complementary strand of RNA3 (Miller et al., 1985
). The RNAs are capped with 7-methylguanylate at the 5' end, and have a tRNA-like structure at the 3' end (Kao & Sivakumaran, 2000
). RNA1 and RNA2 encode the 109 kDa 1a and 94 kDa 2a proteins, respectively, which are required for RNA replication (Kao & Sivakumaran, 2000
). The 1a protein has capping-associated activities (Ahola & Ahlquist, 1999
). RNA3 encodes the 32 kDa 3a protein and the 20 kDa coat protein, which is translated from RNA4 (Ahlquist, 1992
). The 3a protein is required for cell-to-cell movement of the virus (Schmitz & Rao, 1996
). The coat protein is required for encapsidation, cell-to-cell movement and systemic spread of the virus (Okinaka et al., 2001
; Sacher & Ahlquist, 1989
; Schmitz & Rao, 1996
).
BMV RdRp is partially purified as a complex of 1a and 2a proteins with some cellular factors, including eukaryotic elongation initiation factor 3 or a related protein (Horikoshi et al., 1988 ; Quadt & Jaspars, 1990
; Quadt et al., 1993
). In barley plants infected with BMV, 1a and 2a proteins co-localize at cytoplasmic infection-specific structures at the sites of viral RNA synthesis (Dohi et al., 2001
). In barley protoplasts and yeast cells, 1a and 2a proteins co-localize at the sites of viral RNA synthesis, which takes place in the perinuclear endoplasmic reticulum (ER) (Restrepo-Hartwig & Ahlquist, 1996
, 1999
), suggesting that BMV replicase complexes associate with the cytoplasmic ER membrane. Viral RdRp activity was initially fractionated in a membrane-bound state with 1a and 2a proteins during purification (Hardy et al., 1979
; Maekawa & Furusawa, 1984
; Horikoshi et al., 1988
; Quadt & Jaspars, 1990
).
BMV replicase proteins have been investigated by molecular genetic and biochemical approaches. The 1a protein contains a methyltransferase-like domain in the N-terminal region, and a helicase-like domain in the C-terminal region (Ahlquist, 1992 ). The methyltransferase-like domain of 1a protein is involved in the localization of the replicase complex to the ER in yeast cells (Rostrepo-Hartwig & Ahlquist, 1999
; Chen & Ahlquist, 2000
; den Boon et al., 2001
). The helicase-like domain of 1a has a globular structure, which resists protease digestion in vitro (OReilly et al., 1995
). The 2a protein has a central domain conserved in RdRps (Ahlquist, 1992
). The polymerase domain of 2a is predicted to have a structure reminiscent of a right hand by analogy with the crystal structure of poliovirus 3D polymerase (Hansen et al., 1997
; OReilly & Kao, 1998
). Inter- and intra-subunit interactions of the BMV replicase components have been investigated using proteins translated in vitro, and in a yeast two-hybrid system using 1a and 2a derivatives with various site-specific mutations and deletions. The helicase-like domain of 1a interacts directly with the N-terminal region of 2a (Kao & Ahlquist, 1992
; OReilly et al., 1995
). The N-terminal region of 1a interacts with the C-terminal region of 1a and with itself (OReilly et al., 1997
, 1998
). However, the overall structure of the BMV RdRp complex is unclear.
Monoclonal antibodies (mAbs) are powerful tools with which to gain some insight into the structural and functional properties of protein molecules. In this study, we investigated the gross structure of membrane-bound and solubilized BMV RdRp complexes using mAbs raised against 1a and 2a proteins. Immunoprecipitation experiments revealed that the intermediate region between the methyltransferase-like domain and the helicase-like domain of the 1a protein, and the N terminus region of the 2a protein, are exposed on the surface of the solubilized RdRp complex in its active state. Moreover, inhibition assays suggest that the intermediate region between the methyltransferase-like and helicase-like domains of 1a is located just at the boundary of a region buried within the membrane structure.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies.
Mouse mAbs specific for 1a and 2a proteins were prepared as described previously (Dohi et al., 2001 ). mAbs specific to maltose-binding protein (MBP) were obtained as by-products of the production of anti-2a mAbs, in which a fusion protein composed of MBP and 2a protein (MBP2a) was the immunogen. The immunoglobulin isotype of mAbs was determined using a Mouse Monoclonal Antibody Isotyping Kit (Amersham Biosciences), according to the manufacturers recommendations. Alkaline phosphatase-conjugated goat anti-mouse IgG used for ELISA and Western blotting analysis was from Bio-Rad.
Plasmids.
Plasmid pBET1, a vector expressing full-length 1a protein, and pMCB21, a vector expressing MBP-2a, have been described previously (Dohi et al., 2001 ). Plasmid pMB1amb expressing a fusion protein of MBP and 1a protein (MBP1a) was constructed as follows. pMALc2 (New England Biolabs) was cut with PstI, blunted with T4 DNA polymerase, and religated to create pMALc2d(Pst). A HindIII linker (5' AGCTTCTACCTAACTAGCTGCAGCTAGTTAGGTAGA 3'), containing a PstI site (shown underlined) and three amber codons in different frames (shown in italics), was inserted into the HindIII site of pMALc2d(Pst) to create pMALc2amb. Plasmid pBB1(Bam), which is a pBB1 (Mori et al., 1991
) derivative with a BamHI site (underlined) upstream from the 1a start codon (shown in italics) (...GGATCCAACAAAATG...), was kindly provided by Masashi Mori. The 3 kb BamHIEcoRI fragment of pBB1(Bam) containing the 1a gene was inserted into the BamHISalI site of pMALc2amb to create pMB1amb. Hereafter, all the DNA fragments with incompatible ends (e.g. the EcoRI and SalI-generated ends) were ligated after blunting with T4 DNA polymerase.
A series of plasmids encoding the MBP1a fusion protein with various lengths of C-terminal truncation was constructed as follows: pMB1amb was cut with KpnI and PstI, blunted, and re-ligated to create pMB1D1. Similarly, pMB1D2, pMB1D3, pMB1D4, pMB1D5, pMB1D6, pMB1D7, pMB1D33 and pMB1D34 were created using SmaI, NheI, Bsu36I, AatII, NruI, AflI, SphI and EcoT22I, respectively, instead of KpnI as used for pMB1D1. Plasmids pMB1D31 and pMB1D32 were created by inserting the EcoRIPsp1406I fragment of pMB1amb and the EcoRIDraI fragment of pMB1amb into the EcoRIPstI site of pMB1amb, respectively. Plasmids pMB1D8 and pMB1D9 were created as follows: pMB1amb was treated with AflII and PstI, digested with exonuclease III, blunted, and re-ligated. Plasmids pMB1D21, pMB1D22, pMB1D23, pMB1D24 and pMB1D25 were constructed as follows: a DNA fragment was amplified by PCR from pMB1amb using a sense primer (5' GAAGTGCAGACAGCCAA 3') together with antisense primers (5' AACTGCAGCACCTCAGCATCCGGGA 3'), (5' AACTGCAGTAGACTCGGAGTTGTTA 3'), (5' AACTGCAGACATTCACCATATCGTC 3'), (5' AACTGCAGGTACGTTTCATCTGCGT 3') or (5' AACTGCAGCTCCATCAACCATGGAA 3'), respectively. The amplified fragment was treated with Bsu36I and PstI, and inserted into the Bsu36IPstI site of pMB1amb, to create pMB1D21, pMB1D22, pMB1D23, pMB1D24 and pMB1D25, respectively.
A series of plasmids encoding MBP-2a fusion proteins with various lengths of C-terminal truncation was constructed as follows: pMCB21 was digested with HindIII, blunted, and ligated with a PstI linker to create pMCB21(Pst). To introduce three amber codons downstream from the 2a ORF in different frames, the PstISwaI fragment of pMALc2amb was ligated into the PstISwaI site of pMCB21(Pst) to create pMB2amb. pMB2amb was treated with StuI and PstI, digested with exonuclease III, blunted, and re-ligated to obtain pMB2D1, pMB2D3, pMB2D4 and pMB2D7. pMB2D11 was created by self-ligation of the fragment from pMB2amb digested with NheI and PstI. Similarly, pMB2D12, pMB2D13, pMB2D14, pMB2D15, pMB2D16 and pMB2D31 were created using NruI, Bsu36I, ClaI, SpeI, KpnI and AflII, respectively, to digest pMB2amb in place of NheI in the creation of pMB2D11. pMB2D32 and pMB2D33 were created by ligating the 0·13 kb AflIIAccII fragment and the 0·35 kb AflIIHincII fragment, respectively, into the AflIIPstI site of pMB2amb.
The resulting plasmids were verified by nucleotide sequencing analysis. Regions of the 1a and 2a proteins and the extra C-terminal residues encoded by each plasmid are described in Table 1.
|
The SPOTs system (Sigma Genosys) was used to determine the fine epitopes for mAbs. In accordance with the manufacturers instructions, a series of peptides with one amino acid overlapping to the neighbouring peptide was synthesized on activated cellulose membrane, and the interactions between each peptide and the mAbs were investigated.
Preparation and assay of BMV RdRp.
BMV RdRp fractions were prepared from BMV-infected barley (Hordeum vulgare L. cv. Goseshikoku) seedlings by the method of Horikoshi et al. (1987) , with some modifications. The following procedure was carried out at 4 °C. At 7 days after inoculation, about 100 g of inoculated primary leaves and systemically infected secondary leaves was homogenized in a mortar with 300 ml of 50 mM TrisHCl, pH 7·4, 10 mM KCl, 10 mM magnesium acetate, 1 mM EDTA, 10 mM dithiothreitol (DTT), 0·1 mM PMSF and 10% (v/v) glycerol. The homogenate was filtered through two layers of cheesecloth and then centrifuged at 3000 g for 10 min. The supernatant was centrifuged at 30000 g for 30 min and the resulting pellets were suspended in 200 ml of buffer A (50 mM TrisHCl, pH 8·2, 50 mM KCl, 15 mM MgCl2, 10 mM DTT, 1 mM EDTA, 20%, v/v, glycerol) to obtain the membrane-bound RdRp fraction. After addition of Nonidet P-40 (NP40) to a final concentration of 2% (v/v), the mixture was stirred for 1 h, and then centrifuged at 30000 g for 30 min to obtain the solubilized RdRp fraction. The supernatant was applied to a DEAE-Biogel (Bio-Rad) column (1·5x5 cm). Unbound materials were washed with 150 ml buffer A containing 2% NP40, and then the RdRp activity was eluted with buffer A containing 250 mM KCl and 2% (v/v) NP40 at a flow rate of 0·2 ml/min to obtain the DEAE-purified RdRp fraction.
RdRp activity was assayed by mixing 25 µl of each fraction with 38 µl of an assay mixture containing 50 mM TrisHCl, pH 8·2, 15 mM MgCl2, 10 mM KCl, 10 mM DTT, 1·3 mM each of ATP, CTP and GTP, 1 mM EDTA, 2·5 µM UTP, 2 µCi [3H]UTP (3060 Ci/mmol, Amersham Biosciences), 1 µg actinomycin D and 20 U RNasin (Promega). Template BMV RNA (2·5 µg) was also added to assay DEAE-purified RdRp activity, which is template-dependent. After incubation for 90 min at 30 °C, 50 µl was taken for each reaction, and trichloroacetic acid-insoluble radioactivity was measured by the paper-disc method (Horikoshi et al., 1987 ). In an inhibition assay, 5 µl of mAb solution was added to 25 µl of the RdRp fraction, and incubated at 25 °C for 30 min with occasional shaking, before the RdRp activity was measured. Synthetic peptides used for a competition assay were obtained from Sawady Technology Inc.
Immunoprecipitation from the DEAE-purified RdRp fraction.
DEAE-purified RdRp fraction (200 µl) was incubated with 5 µg of mAb and 50 µl of 20 % (v/v) protein ASepharose CL-4B (Amersham Biosciences) in buffer A containing 2% (v/v) NP40 overnight at 4 °C with gentle rotation. The resins were washed five times with buffer A containing 2% (v/v) NP40. The resins were suspended in 25 µl of buffer A containing 2 % (v/v) NP40, and mixed with 38 µl of the assay mixture to measure RdRp activity as described above.
Alternatively, materials bound to the gel were eluted from the beads by boiling in 60 µl of Laemmli sample buffer (Laemmli, 1970 ), and 15 µl of each sample was subjected to Western blotting analysis as described (Nagano et al., 1999
) using anti-1a mAbs B109, B118 and B137 (2 µg/ml of each) or anti-2a mAbs, B202, B218 and B223 (2 µg/ml of each) as primary antibodies. For controls, 200 ng of recombinant MBP1a and MBP2a proteins that were denatured in 4 µl of Laemmli sample buffer were diluted with buffer A containing 2% (v/v) NP40, and subjected to immunoprecipitation, as described above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epitope mapping of 1a and 2a proteins
Epitopes for the anti-1a and the anti-2a mAbs were mapped by ELISA using a series of recombinant MBP1a and MBP2a fusion proteins with various truncations at their C termini. E. coli inclusion bodies containing the recombinant proteins were solubilized with guanidine hydrochloride, and applied to 96-well plates for the ELISA. Anti-MBP mAb M211 interacted with all the MBP1a and MBP2a derivatives, but did not interact with the unfused full-length 1a protein, indicating that the experiments were well controlled (Table 1).
Anti-1a mAb B109 interacted with the unfused full-length 1a protein, the MBP1a fusion protein, and the C-terminal-truncated MBP1a containing residues 1919 of 1a, but did not interact with MBP1a containing residues 1851 of 1a, or shorter MBP1a forms (Table 1). This suggests that residues 852919 of 1a protein contain the epitope for mAb B109. Similarly, interactions between, respectively, the anti-1a and anti-2a mAbs with the MBP1a and the MBP2a derivatives were investigated, and the epitopes were mapped (Table 1
; Fig. 1A
).
|
|
Fine mapping of the epitopes located on the surface of the active RdRp complex
Epitopes for the mAbs (B110, B152, B212, B215, B217 and B223) that interacted strongly with the DEAE-purified RdRp complex were mapped finely using synthetic peptides. A series of peptides consisting of 10 amino acid residues included in a region of 1a between amino acids 514586 was synthesized with one amino acid overlapping the neighbouring peptides, using the SPOTs system (Sigma Genosys), and their abilities to interact with mAbs B110 and B152 were examined by enzyme-linked assay. mAb B110 specifically interacted with decapeptides containing TDVVP, corresponding to amino acids 544548 of 1a, and mAb B152 specifically interacted with decapeptides containing SVEVP, corresponding to amino acids 553557 of 1a (data not shown). These results indicate that B110 and B152 recognize amino acid sequences TDVVP and SVEVP, respectively (Fig. 1B).
Similarly, the epitopes for anti-2a mAbs B212, B215, B217 and B223 were mapped using a series of decapeptides that correspond to amino acids 133 in the N-terminal region of 2a. mAbs B212 and B215 interacted with decapeptides containing QWIIDQ, corresponding to amino acids 1823 of 2a protein, and mAbs B217 and B223 interacted with decapeptides containing DDFVR, corresponding to amino acids 812 of 2a (data not shown). These results indicate that B212 and B215 recognize the same amino acid sequence, QWIIDQ, and B217 and B223 recognize the same amino acid sequence, DDFVR (Fig. 1B).
Effects of anti-1a and anti-2a mAbs on the DEAE-purified RdRp activity
Anti-1a and anti-2a mAbs were tested for their ability to influence RdRp activity in the DEAE-purified RdRp fraction. Of the anti-1a mAbs, B110 and B152 reduced the RdRp activity to approximately 60% and 30% of the control, respectively (Fig. 3). Other anti-1a mAbs did not significantly affect the RdRp activity. Increasing the amount of B110 and B152 from 5 µg to 25 µg did not produce additional effects on RdRp activity (Fig. 4A
). The inhibition of RdRp activity by B110 and B152 was observed throughout the incubation, for at least 120 min (Fig. 4B
). Anti-2a mAbs B212, B215, B217 and B223, which recognize the N-terminal region of 2a protein and interact with the DEAE-purified RdRp complex, did not inhibit but rather slightly stimulated RdRp activity (Fig. 3
). The other mAbs did not affect RdRp activity (Fig. 3
).
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Locations of the epitopes for anti-1a mAbs in the BMV RdRp complex
mAbs B110 and B152, the epitopes of which are located at amino acid residues 544-TDVVP-548 and 553-SVEVP-557 of 1a protein, respectively, specifically precipitated the DEAE-purified RdRp complex in an active state (Fig. 2), and partially inhibited the enzymatic activity of RdRp (Figs 3
and 4
). This indicates that amino acid residues 544-TDVVP-548 and 553-SVEVP-557 of 1a protein are exposed on the surface of the DEAE-purified RdRp complex. Analysis of the 1a protein amino acid sequence with the PHD method using the PredictProtein server program (Rost, 1996
) predicts that the epitopes for B110 and B152 are both located in a loop region of the protein structure (OReilly et al., 1998
). The loop regions of proteins are often highly exposed on the surfaces of proteins (Kuntz, 1972; Rose et al., 1985
). Previous studies have shown that the predicted loop region that includes the epitopes for B110 and B152 is located at the hinge between the methyltransferase-like domain and the helicase-like domain (Ahlquist, 1992
; Fig. 1
), adjacent to the C-terminal protease-resistant domain (OReilly et al., 1995
). BMV mutants with a two-amino-acid insertion just on or close to the epitopes for B110 and B152 have the ability to replicate in barley protoplasts (Kroner et al., 1990
), suggesting that these mutations do not affect the structure of BMV replicase, and that these regions are not included in the catalytic sites of the replicase. mAbs B110 and B152, however, significantly inhibited RdRp activity in the DEAE-purified fraction (Figs 3
and 4
), implying that the binding of the mAbs to the RdRp complex causes a structural change that affects its enzymatic activity. As well as causing a structural change, antibody binding might lower activity by inhibiting a structural change, steric hindrance, or other mechanisms.
In contrast to this result, only B152 but not B110 affected RdRp activity in the membrane-bound enzyme fraction (Fig. 7). This suggests that the epitope for B110 was not accessible and was possibly buried in the membrane-bound RdRp complex. Moreover, treatment of the membrane-bound fraction with NP40 rendered the B110 epitope on the RdRp complex accessible to B110 (Fig. 7
), suggesting that some factor that otherwise blocks an interaction with B110 was released by the detergent treatment. The blocking factor is unlikely to be a template RNA because B110 inhibited RdRp activity on endogenous templates in the solubilized fraction. Because the epitopes for B110 and B152 are located close to the C-terminal region of the methyltransferase-like domain, which mediates the association of 1a to the ER membrane (den Boon et al., 2001
), the region that includes the epitopes for B110 and B152 may be located just on the border of the region buried within the membrane structure or with a membrane-associated material.
The mAbs with epitopes located at residues 1143, 586619 or 852919 of 1a did not interact with the DEAE-purified RdRp complex (Fig. 2), suggesting that the regions that include these epitopes are highly structured and buried inside the 1a protein. Alternatively, these regions may be buried in the highly ordered quaternary structure of the replicase complex and be involved in the formation of the complex. N-terminal residues 1515 of 1a protein are required for the 1a1a interaction in a yeast two-hybrid analysis (OReilly et al., 1998
). The helicase-like domain of 1a protein includes the regions essential for its interaction with 2a protein as well as with N-terminal residues 1515 of 1a protein, and forms a putative globular structure, which is resistant to protease treatment (Kao & Ahlquist, 1992
; OReilly et al., 1995
, 1998
). Insertion of two amino acids into the regions that include the epitopes of these mAbs abolishes the ability of the BMV mutants to replicate in barley protoplasts or in yeasts, and disrupts the predicted secondary structure of 1a protein (Kroner et al., 1990
; OReilly et al., 1998
). These data together imply that the regions that include residues 1143, 586619 and 852919 of 1a protein may play an important role(s) in the formation of functional BMV replicase.
Locations of the epitopes for anti-2a mAbs in the BMV RdRp complex
Anti-2a mAbs with epitopes located at N-terminal residues 812 or 1823 of 2a precipitated the RdRp complex in an active state without interrupting RdRp activity (Figs 2 and 3
). This indicates that these regions are located on the surface of the DEAE-purified RdRp complex and are not involved in viral RNA synthesis. A BMV mutant with a deletion of residues 324 of 2a protein replicates similarly to wild-type BMV in barley protoplasts (Traynor et al., 1991
), and 2a fused to GFP at the N terminus is functional in BMV RNA replication in yeast cells (Chen & Ahlquist, 2000
). These results strongly suggest that the region near the N terminus of 2a protein is not involved in the formation of the functional BMV replicase.
The mAbs with epitopes located in the region including residues 270310 of 2a precipitated a small amount of RdRp complex that displayed RdRp activity (Fig. 2). This indicates that the epitope region is not directly involved in RdRp activity. Moreover, most of the RdRp complex in the DEAE-purified RdRp fraction did not interact with these mAbs, suggesting that the region that includes residues 270310 of 2a protein is buried inside the RdRp complex and is involved in an interaction with some components that constitute the functional replicase complex. These epitopes are included in the RdRp unique region implicated in polymerasepolymerase interactions (OReilly & Kao, 1998
).
mAbs B202 and B201, the epitopes of which are located in the regions that include residues 5095 and 341376 of 2a, respectively, did not interact with the DEAE-purified RdRp complex (Fig. 2). This suggests that the epitopes for these mAbs are buried inside the RdRp complex. This is supported by the previous finding that the epitope for B202 occurs in the region required for the interaction of 2a with 1a to form the replicase complex (Kao & Ahlquist, 1992
; OReilly et al., 1995
). Sequence alignment of BMV 2a protein with poliovirus 3D polymerase, the crystal structure of which has been determined, shows that the region that includes the B201 epitope corresponds to the fingers subdomain of the poliovirus polymerase, which is thought to be important for polymerase activity (OReilly & Kao, 1998
).
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahlquist, P. (1992). Bromovirus RNA replication and transcription. Current Opinion in Genetics & Development 2, 71-76.[Medline]
Ahlquist, P., Wu, S.-X., Kaesberg, P., Kao, C. C., Quadt, R., DeJong, W. & Hershberger, R. (1994). Protein-protein interactions and glycerophospholipids in bromovirus and nodavirus RNA replication. Archives of Virology supplementum 9, 135145.
Ahola, T. & Ahlquist, P. (1999). Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a. Journal of Virology 73, 10061-10069.
Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., De Francesco, R. & Rey, F. A. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proceedings of the National Academy of Sciences, USA 96, 13034-13039.
Buck, K. W. (1996). Comparison of the replication of positive-stranded RNA viruses of plants and animals. Advances in Virus Research 47, 159-251.[Medline]
Chen, J. & Ahlquist, P. (2000). Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a. Journal of Virology 74, 4310-4318.
den Boon, J. A., Chen, J. & Ahlquist, P. (2001). Identification of sequences in brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. Journal of Virology 75, 12370-12381.
Dohi, K., Mori, M., Furusawa, I., Mise, K. & Okuno, T. (2001). Brome mosaic virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells. Archives of Virology 146, 1607-1615.[Medline]
Hansen, J. L., Long, A. M. & Schultz, S. C. (1997). Structure of the RNA-dependent RNA polymerase of poliovirus. Structure 5, 1109-1122.[Medline]
Hardy, S. F., German, T. L., Loesch-Fries, L. S. & Hall, T. C. (1979). Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus. Proceedings of the National Academy of Sciences, USA 76, 4956-4960.[Abstract]
Horikoshi, M., Nakayama, M., Yamaoka, N., Furusawa, I. & Shishiyama, J. (1987). Brome mosaic virus coat protein inhibits viral RNA synthesis in vivo. Virology 158, 15-19.
Horikoshi, M., Mise, K., Furusawa, I. & Shishiyama, J. (1988). Immunological analysis of brome mosaic virus replicase. Journal of General Virology 69, 3081-3087.
Kao, C. C. & Ahlquist, P. (1992). Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus. Journal of Virology 66, 7293-7302.[Abstract]
Kao, C. C. & Sivakumaran, K. (2000). Brome mosaic virus, good for an RNA virologists basic needs. Molecular Plant Pathology 1, 91-97.
Kroner, P. A., Young, B. M. & Ahlquist, P. (1990). Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis. Journal of Virology 64, 6110-6120.[Medline]
Kuntz, I. D. (1975). Protein folding. Journal of the American Chemical Society 94, 4009-4012.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F. & Weber, P. C. (1999). Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nature Structural Biology 6, 937-943.[Medline]
Maekawa, K. & Furusawa, I. (1984). Purification of RNA-dependent RNA polymerase from brome mosaic virus-infected barley leaves. Annals of the Phytopathological Society of Japan 50, 491-499.
Miller, W. A., Dreher, T. W. & Hall, T. C. (1985). Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA. Nature 313, 68-70.[Medline]
Mise, K., Mori, M., Nakayashiki, H., Koyama, T., Okuno, T. & Furusawa, I. (1994). Nucleotide sequence of a set of cDNA clones derived from the brome mosaic virus ATCC66 strain and comparison with the Russian strain genome. Annals of the Phytopathological Society of Japan 60, 454-462.
Mori, M., Mise, K., Kobayashi, K., Okuno, T. & Furusawa, I. (1991). Infectivity of plasmids containing brome mosaic virus cDNA linked to the cauliflower mosaic virus 35S RNA promoter. Journal of General Virology 72, 243-246.[Abstract]
Nagano, H., Mise, K., Okuno, T. & Furusawa, I. (1999). The cognate coat protein is required for cell-to-cell movement of a chimeric brome mosaic virus mediated by the cucumber mosaic virus movement protein. Virology 265, 226-234.[Medline]
Okinaka, Y., Mise, K., Suzuki, E., Okuno, T. & Furusawa, I. (2001). The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement. Journal of Virology 75, 5385-5390.
Okuno, T. & Furusawa, I. (1979). RNA polymerase activity and protein synthesis in brome mosaic virus-infected protoplasts. Virology 99, 218-225.
OReilly, E. K. & Kao, C. C. (1998). Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology 252, 287-303.[Medline]
OReilly, E. K., Tang, N., Ahlquist, P. & Kao, C. C. (1995). Biochemical and genetic analysis of the interaction between the helicase-like and polymerase-like proteins of brome mosaic virus. Virology 214, 59-71.[Medline]
OReilly, E. K., Paul, J. D. & Kao, C. C. (1997). Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay. Journal of Virology 71, 7526-7532.[Abstract]
OReilly, E. K., Wang, Z., French, R. & Kao, C. C. (1998). Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses. Journal of Virology 72, 7160-7169.
Quadt, R. & Jaspars, E. M. J. (1990). Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase. Virology 178, 189-194.[Medline]
Quadt, R., Kao, C. C., Browning, K., Hershberger, R. & Ahlquist, P. (1993). Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase. Proceedings of the National Academy of Sciences, USA 90, 1498-1502.[Abstract]
Restrepo-Hartwig, M. & Ahlquist, P. (1996). Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. Journal of Virology 70, 8908-8916.[Abstract]
Restrepo-Hartwig, M. & Ahlquist, P. (1999). Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum. Journal of Virology 73, 10303-10309.
Rose, G. D., Gierasch, L. M. & Smith, J. A. (1985). Turns in peptides and proteins. Advances in Protein Chemistry 37, 1-109.[Medline]
Rost, B. (1996). PHD: predicting one-dimensional protein structure by profile based neural networks. Methods in Enzymology 266, 525-539.[Medline]
Sacher, R. & Ahlquist, P. (1989). Effects of deletions in the N-terminal basic arm of brome mosaic virus coat protein on packaging and systemic infection. Journal of Virology 63, 4545-4552.[Medline]
Schmitz, I. & Rao, A. L. (1996). Molecular studies on bromovirus capsid protein. I. Characterization of cell-to-cell movement-defective RNA3 variants of brome mosaic virus. Virology 226, 281-293.[Medline]
Traynor, P., Young, B. M. & Ahlquist, P. (1991). Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread. Journal of Virology 65, 2807-2815.[Medline]
Received 14 May 2002;
accepted 3 July 2002.