INTRODUCTION

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The alphavirus-like superfamily comprises a large number of animal and plant viruses sharing the properties of a capped, positive-stranded RNA genome and homologies in their RNA replication proteins. Despite homologies between the guanylytransferase/methyltransferase-like (MT), helicase-like (HEL), and polymerase-like (POL) domains, they may be expressed as parts of a single protein or as separate entities distributed over two or three proteins. Little is known about the nature of the interactions between these polypeptides and the ratio in which they are present in the viral replication complex, although purified RNA-dependent RNA polymerases (RdRp) have been used extensively to study viral replication in vitro. We have studied the interactions between the replicase proteins of Alfalfa mosaic virus (AMV) and their in situ localization to gain insight in the assembly of the replication complex.

AMV is the type species of the genus Alfamovirus. It belongs to the Bromoviridae family of plant viruses, all having a tripartite RNA genome of messenger-sense polarity. RNA 3 encodes the movement protein (MP) and, via a subgenomic mRNA 4, the coat protein (CP). RNAs 1 and 2 encode the P1 and P2 replicase proteins, respectively. The N terminus of P1 contains a domain with sequence homology to the domains involved in RNA capping of the alphavirus Semliki Forest virus (SFV) nsP1 protein and the more closely related Brome mosaic virus (BMV) 1a protein (1, 2, 41). The C-terminal end of P1 has homology to the alphavirus-like supergroup of HEL domains (17, 22), including the SFV nsP2 protein, for which helicase activity was recently shown in vitro (16). The P2 protein contains a central domain with sequence motifs conserved among many polymerases, including the presence of the Mg²⁺-binding GDD motif (3, 26). The N terminus of P2 does not contain conserved sequence elements. The corresponding sequence of the BMV 2a polymerase protein has been shown to interact with the HEL domain of the BMV 1a protein (23).

In addition to P1 and P2, CP is involved in AMV replication (reviewed in reference 4). CP in the inoculum is required in a step prior to viral minus-strand RNA synthesis, possibly translation of the inoculum RNAs (31, 32). Subsequently, CP expressed from RNA 3 is required for viral plus-strand RNA accumulation in vivo (31, 48) and in vitro (11).

The replication complexes of all eukaryotic positive-stranded RNA viruses studied so far are associated with intracellular membranes. Alphavirus RNA replicase proteins are localized on the cytoplasmic surface of endosomes and lysosomes, probably connected with the 50-nm membrane-associated vesicles that occupy these organelles (14). Endoplasmic reticulum (ER)-derived 50- to 100-nm cytoplasmic vesicles detected in bromovirus-infected plants localize with the site of replication of BMV (39). Replication complexes of other plant viruses are localized in vesicular structures derived from chloroplast membranes or believed to be associated with vesicles on the tonoplast, i.e., the membrane surrounding the vacuoles (19, 29).

In this paper we provide in vitro and in vivo evidence for the interaction between the AMV P1 and P2 replication proteins and show that the HEL domain of P1 and the nonconserved N terminus of P2 are involved in this interaction. In addition, we found a colocalization of P1 and P2 at the tonoplast of infected cowpea protoplasts.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Construction of two-hybrid plasmids. AMV P1 and P2 genes were fused to the binding and activation domains of GAL4 encoded by the yeast expression vectors pGBT9, pGAD424, pAS2-1, and pACT2 (Clontech) as follows. The P1 coding sequence plus 3' untranslated region (3'-UTR) and nos-terminator were excised from pCa17T-Nco (52) with NcoI and PvuII. This sequence was ligated into a NcoI-SmaI digested pUC21 plasmid to give pUC-P1. The P1 sequence was removed using XhoI and PstI and ligated into SalI-PstI digested pGBT9, giving clone pBP1 (1-1126). Similarly, a XhoI-BamHI fragment was cloned in pGAD424 digested with SalI and BglII, to yield plasmid pAP1(1-1126). The resulting fusion proteins contain a 14-amino-acid (aa) linker peptide at the junction with P1.

Construction of other plasmids. pCaHA17T gives Cauliflower mosaic virus 35S promoter-driven expression of the P1 protein with an N-terminal fusion to a hemagglutinin (HA) tag. Plasmid pCa17T-Nco (52) was digested with NcoI. The HA sequence was inserted by ligation of a synthetic DNA fragment, obtained by hybridization of two oligonucleotides, CATGTACCCATACGATGTTCCAGATTACGC and CATGGCGTAATCTGGAACATCGTATGGGTA.

Antibodies. The P1-specific rabbit polyclonal antibody was directed against the C-terminal aa 1100 to 1120 of P1 (51). For detection of CP, we used a rabbit polyclonal CP-specific antibody. An N-terminal fragment of P2 was overexpressed as a GST fusion protein from pGEX-NP2 in Escherichia coli strain BL21, cleaved with thrombin to remove the GST sequence, and used to immunize rabbits for the production of anti-P2 polyclonal antiserum (Eurogentec). The antibody against HA was a monoclonal mouse antibody (12CA5; Roche). The fluorescein isothiocyanate (FITC)-labeled secondary antibodies were from Jackson Laboratories; the Alexa-568- labeled secondary antibodies were from Molecular Probes.

In vitro RNA transcription and translation. AMV P1, P2, MP, and CP were translated from RNA transcripts of plasmids pT7L4P1, pT7L4P2, pAL3, and pT72-42, respectively. Transcription of linearized plasmids with T7 RNA polymerase was done as described elsewhere (31, 47). In vitro translation was done in a rabbit reticulocyte lysate according to the protocol provided by the manufacturer (Promega), with minor modifications.

Immunoprecipitation of in vitro translated proteins. Precipitation was performed with 10 µl of in vitro-translated protein, in the presence of 400 µl of NET-gel buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.2% Igepal CA630, 1 mM EDTA, 0.25% teleostean gelatin) and 2.5 µl of antibody. The sample was incubated at 4°C under continuous rotation. After 60 min, 50 µl of a 50% protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) solution in NET-gel was added, and incubation was continued for another 60 min. The beads were washed three times with 1-ml portions of NET-gel buffer and washed once with NET buffer (which contains no gelatin). The beads were resuspended in 30 µl of protein loading buffer (27), and the samples were boiled for 3 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel was fixed, dried, and exposed to X-ray film.

Immunoprecipitation of RdRp complexes. Proteins were precipitated from 50 µl of RdRp glycerol fractions by 5 µl of antibody in the presence of 800 µl of NET-gel buffer. The samples were incubated and washed as described above. After electrophoresis, the gel was analyzed by the Western blot technique (45).

Protoplast infection and immunofluorescent labeling. Cowpea (Vigna unguiculata cv. Blackeye) mesophyl protoplasts were isolated and transfected with plasmid DNA as described elsewhere (46). The same protocol was used for infection of 5 × 10⁵ protoplasts with 10 µg of phenol-extracted AMV RNA. Protoplasts were incubated for 42 h at 25°C under continuous illumination. Fixation and antibody incubations were performed as described previously (50). Primary antibodies were diluted 1:100 (HA and P2 antibodies) or 1:1,000 (CP antibody) in blocking buffer, which is phosphate-buffered saline containing 0.1% acetylated bovine serum albumin (Aurion) and 0.02% teleostean gelatin (Sigma). Goat anti-mouse or goat anti-rabbit antibodies conjugated to FITC or Alexa-568 were diluted 1:40 in blocking buffer. For nuclear staining, cells were incubated for 15 min with a 5-ng/ml solution of propidium iodide in phosphate-buffered saline-before the last washing step.

Confocal laser scanning microscopy (CLSM). Immunofluorescence images were obtained with a Bio-Rad MRC-1024 ES confocal microscope. Images with FITC-labeled antibodies were acquired with an excitation wavelength of 488 nm and a 522 DF 32-nm band-pass filter. The Alexa-568 antibody and propidium iodide nuclear stain were detected with a 568-nm laserline and 585-nm long-pass filter. Images were acquired using one laserline at a time to minimize background and merged later using the Confocal Assistant 4.02 software. Each experiment was performed at least three times to ensure the reproducibility of the results.

Yeast two-hybrid system. All yeast experiments were performed with Saccharomyces cerevisiae strain PJ69-4A (21), which carries the adenine (A) and histidine (H) reporter genes. Yeast cells were grown on minimal synthetic medium plates containing methionine and uracil (MU plates) plus other selective components if required. Binding domain (pB) and activation domain (pA) plasmids carry tryptophan (T) and leucine (L) auxotrophic markers, respectively. Yeast transformations were performed as described before (15). All pB and pA constructs were separately transformed to yeast and plated on MUAHL or MUAHT medium. Single transformants were maintained on these media, and colonies were replated to medium lacking A and H to test for activation of the reporter genes. Combinations of pB and pA constructs were cotransformed and plated on MUAH medium. After 2 to 3 days on MUAH plates, single colonies were transferred to both MUAH and MU plates to test for interaction. Growth on selective medium was monitored over 6 days. Double transformations were performed at least three times.

RdRp isolation and activity assay. The RdRp was purified from Nicotiana benthamiana plants infected with AMV strain 425 as described previously (37), omitting the DEAE ion-exchange chromatography step. The glycerol gradient was subdivided into 16 fractions that were assayed for in vitro RdRp activity (37).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Interaction of P1 and P2 in the yeast two-hybrid system. Viral sequences were fused to the C terminus of the activation domain or DNA binding domain of GAL4. When the N-terminal 352 aa of P2 were fused to the binding domain, the construct showed activation of the ADE and HIS3 genes in the absence of a P1 construct. Therefore, most of the P1 derivatives were fused to the binding domain (pBP1 constructs), whereas the P2 derivatives were mainly fused to the activation domain (pAP2 constructs). In addition, we tested a few constructs with P1 or P2 sequences fused to the activation or DNA binding domain, respectively (pAP1 and pBP2 constructs). Figure 1 shows schematic representations of the full-length P1 and P2 proteins and a selection of pBP1 and pAP2 constructs that were used in two-hybrid assays. Table 1 summarizes all P1-P2 interactions tested. None of the constructs shown in Fig. 1 or Table 1 was able to self-activate the ADE and HIS3 selectable marker genes. Western blot analysis revealed stable expression in yeast of all fusion proteins shown in Fig. 1 (data not shown). Growth of yeast on nonselective (MUAH) or selective (MU) medium is shown in Fig. 1. No growth was observed using full-length P1 and P2 (Fig. 1, row 2). However, cotransformation with several constructs encoding C-terminal P1 and N-terminal P2 sequences resulted in growth of the yeast cells. Growth of colonies within 1 to 2 days was found with the C-terminal 618 aa of P1 [pBP1 (509-1126)] and the N-terminal 352 aa of P2 [pAP2 (1-352)] (Fig. 1, row 6; Table 1). N-terminal extension of this P1 sequence in pBP1(340-1126) did not affect this growth rate (Fig. 1, row 5), but truncation to a sequence corresponding to the C-terminal 509 aa of P1 [pBP1(618-1126)] strongly reduced the growth rate (Fig. 1, row 7). When the C terminus of the P1 sequence in pBP1(509-1126) was truncated by deleting 129 aa [pBP1(509-997)], the growth of yeast colonies was abolished (Table 1). When the P2 sequence in pAP2(1-352) was reduced to a sequence corresponding to the N-terminal 241 aa of P2 [pAP2(1-241)], growth was reduced (Fig. 1, compare rows 6 and 11). Further deletions at the N terminus [pAP2(75-242) and pAP2(75-351)] or C terminus [pAP2(1-157)] of this P2 sequence abolished growth (Table 1). With the N-terminal sequence of P1 (aa 1 to 509 or 1 to 686), no interaction with any P2 sequence was detectable (Table 1). Similarly, no interaction was found between C-terminal domains of P2 and any P1 sequence tested (Table 1).

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Examples of interactions between AMV proteins in the yeast two-hybrid system, 4 days after plating. Yeast cells were grown on medium that does not select for interaction (MUAH) and medium that selects for adenine and histidine auxotrophy (MU). In the schematic representations of P1 and P2 segments fused to the GAL4 binding domain (B) or activation domain (A) that were used to transform yeast cells, GAL4 sequences are represented by black bars, MT, HEL, and POL, domains and the proline cluster (PC) are hatched, and deletions are shown as dotted lines. Typical interactions are shown along with positive and negative controls (rows 12 and 1, respectively). Growth rates of yeast cells are indicated by plus and minus signs as explained in the footnote to Table 1.

Coimmunoprecipitation of in vitro-translated P1 and P2. To confirm the interaction observed in the two-hybrid system, P1 and P2 were translated in a cell-free system and interaction between the translation products was analyzed by coimmunoprecipitation. This assay was also used to analyze a possible interaction of P1 or P2 with other AMV proteins. Figure 2A shows the products obtained by translation of the AMV proteins P1, P2, MP, and CP, along with luciferase as a control: in Fig. 2B, the radiolabeled translation products were mixed with unlabeled P2 and subjected to precipitation with a P2 antiserum. Labeled P1 coprecipitated with P2 (Fig. 2B, lane 2), but MP, CP, and luciferase did not. The P2 antiserum did not precipitate P1 in the absence of P2 (Fig. 2B, lane 1). Addition of RNase A (0.1 µg ml¹) to the translated proteins before binding had no affect on precipitation (data not shown). These results corroborate the interactions observed in the two-hybrid system between P1 and P2 and the finding that an interaction between replicase proteins and CP was not detectable. The fact that interaction between full-length P1 and P2 was detectable by coimmunoprecipitation but not in the two-hybrid system may be due to steric hindrance caused by the activation or DNA binding domains in the yeast system or impaired nuclear import of these large fusion proteins.

View larger version (128K): [in this window] [in a new window]   FIG. 2.   Immunoprecipitation of in vitro-translated proteins. AMV P1, P2, MP, and CP and luciferase (Luc.) were translated in a rabbit reticulocyte lysate in the presence of [35S]methionine, and samples of the translation mixtures were analyzed by SDS-PAGE (A). The remaining parts of the translation mixtures were incubated with nonlabeled P2 (except the lane 1, to which no P2 was added) (B) and subjected to immunoprecipitation with a polyclonal anti-P2 serum followed by SDS-PAGE. The labeled proteins in the translation mixtures are indicated above the lanes. Positions of molecular size markers (in kilodaltons) are indicated between the panels.

Coimmunoprecipitation of P1 and P2 from purified RdRp. AMV RdRp was solubilized from membrane structures sedimenting at 30,000 × g and centrifuged in a glycerol gradient (37). The gradient was subdivided into 16 fractions, each of which was analyzed for polymerase activity by an in vitro RdRp assay and for the presence of P1, P2, and CP by Western blotting. Over 80% of the total RdRp activity was present in fractions 8 to 11, with a peak of 37% in fraction 9 (Fig. 3A). The sedimentation pattern of P1 (Fig. 3B) closely corresponded to the distribution of the RdRp activity. P2 was present in all fractions that showed RdRp activity, but the majority of P2 was found in the top fractions of the gradient that showed little or no RdRp activity (Fig. 3C). The antiserum against CP detected CP monomers as well as CP dimers. CP monomers were found in all fractions of the gradient, particularly in the top and botton fractions, with relatively little CP in the fractions that contained RdRp activity (Fig. 3E). CP dimers were found exclusively in the top fractions and did not cosediment with RdRp activity. We do not know why these dimers do not dissociate in the presence of SDS and reducing agents in the gel loading buffer.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Analysis of AMV RdRp by glycerol gradient centrifugation. Samples from each gradient fraction were assayed for RdRp activity (A) and analyzed by Western blotting (B to E). RdRp activity was measured in vitro, using plus-strand AMV RNA 3 as the template; a gel run with the radiolabeled minus-strand RNA 3 products is shown. Western blots were analyzed using polyclonal antibodies against AMV proteins P1 (B), P2 (C), and CP (D and E). The antiserum against CP detected CP dimers and monomers, which are separately shown in panels D and E, respectively. Sedimentation is from right to left.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Immunoprecipitation of RdRp complexes. RdRp purified by glycerol gradient centrifugation (Fig. 3, fraction 9) was subjected to immunoprecipitation with a polyclonal antiserum against P1, P2, or CP or with preimmune (P.I.) serum, as indicated. The immunoprecipitates were analyzed by Western blotting using antiserum against P1 (A) or CP (B). The positions of P1 and CP are indicated at the right.

Localization of P1 and P2 in infected cowpea protoplasts. A possible colocalization of P1 and P2 in AMV-infected cowpea protoplasts was analyzed by immunofluorescence CLSM. Using antibodies against CP, it was determined by immunofluorescence that 10 to 50% of the protoplasts were infected. The P1 antibody did not give a specific signal in any of the cells (results not shown). The P2 antibody gave a faint signal throughout the cytoplasmic part of the cell and distinct labeling of the tonoplast (Fig. 5A). Labeling was not concentrated around or near the nucleus (propidium iodide staining shown in blue).

View larger version (56K): [in this window] [in a new window]   FIG. 5.   In situ localization of P1 and P2 in AMV-infected cowpea protoplasts by immunofluorescence microscopy. Each column of images shows the analysis of a single protoplast, stained with a specific antiserum (left) and with propidium iodide to reveal the nucleus (middle); merge of the two stained images are shown at the right (A) Two different protoplasts infected with wild-type (wt) AMV that were analyzed with a polyclonal antiserum against P2. (B) Two different protoplasts infected with AMV that was engineered to express HA-tagged P1. The protoplasts were analyzed with an antiserum against the HA epitope. (C) Protoplast transfected with a plasmid expressing -TIP fused to an HA epitope. The protoplast was analyzed with an antiserum against the HA epitope. Bar = 10 µm.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Analysis of the colocalization of P1, P2, and CP in AMV-infected cowpea protoplasts by immunofluorescence microscopy. Each column of images shows the analysis of a single protoplast: left and middle, protoplasts analyzed with specific antisera; right, the left and middle images merged. (A) Two protoplasts infected with AMV that was engineered to express HA-tagged P1. The protoplasts were analyzed with antisera against the HA epitope (left) and P2 (middle). (B) Protoplast infected with AMV that was engineered to express HA-tagged P1. The protoplast was analyzed with antisera against the HA epitope (left) and CP (middle). (C) Two protoplasts transfected with a plasmid expressing -TIP fused to an HA epitope. The protoplasts were analyzed with antisera against HA (left) and P2 (middle). (D) Healthy protoplasts analyzed with antisera against the HA epitope (left) and P2 (middle). (E) White-field picture of the lower protoplast shown in panel C. Bar = 10 µm.

Colocalization with -TIP. When transfected to cowpea protoplasts, plasmid pMON-HA-SITIP expressed -TIP fused N-terminally to the HA tag. The HA antibody detected the protein exclusively at the tonoplast and sometimes around small vesicles (Fig. 5C). Cowpea protoplasts cotransfected with wild-type AMV RNAs and pMON-HA-SITIP were double labeled with antibodies against HA and P2. The -TIP and P2 proteins were found to colocalize at the tonoplast (Fig. 6C, merged pictures). The white-field picture shown in Fig. 6E corresponds to the protoplast analyzed in the lower panels of Fig. 6C. These results confirm that the colocalization of P1 and P2 (Fig. 6A) occurs at the tonoplast.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

P1 and P2 replication proteins interact in vitro and in vivo. The AMV P1 and P2 proteins show similarities with their BMV 1a and 2a counterparts in amino acid sequence and in the organization of the MT, HEL, and POL domains. Thus, it was expected that AMV P1 and P2 would interact in the same way as reported for BMV 1a and 2a (23, 24). The AMV P1-P2 interaction was demonstrated by co immunoprecipitation of full-length in vitro-translated proteins and of RdRp complexes purified from infected plants. Application of the yeast two-hybrid system permitted the identification of C-terminal sequences of P1 and N-terminal sequences of P2 that are involved in the P1-P2 interaction.

Replication proteins localize at the tonoplast. Infection of plant cells by many positive-strand RNA viruses is accompanied with the appearance of vesicular structures that originate from various cellular membranes. For a few viruses it has been shown that the viral RdRp and synthesis of viral RNA are associated with these structures; for others, the accumulation of these structures has been observed only by electron microscopy, and their role in RNA replication is unproven (reviewed in references 10 and 28). Induction of vesicular structures by Cowpea mosaic virus (CPMV) is due to proliferation of the ER membrane and requires de novo biosynthesis of lipids (6). TMV also replicates at the ER membrane, but replication is not dependent on de novo lipid synthesis and the membrane aggregates appear to be derived from preexisting ER (6, 38). Replication of Turnip yellow mosaic virus is associated with small virus-induced invaginations of the chloroplast membrane (29). Similar 50- to 70-nm vesicles are abundant at the tonoplast of umbravirus-infected cells (12, 30).