Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.     

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.     

Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo

Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3′ terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.     

Brome mosaic virus RNA syntheses in vitro and in barley protoplasts

The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.     

Genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase requires a sequence that is complementary to the binding site of the BMV helicase-like protein

Initiation of genomic plus-strand RNA synthesis by the brome mosaic virus (BMV) replicase in vitro requires a 26-nucleotide (nt) RNA sequence at the 3' end of the minus-strand RNA and a nontemplated nucleotide 3' of the initiation cytidylate [ Sivakumaran, K. and Kao, C.C. (1999) J. Virol. 64 , 6415–6423]. At the 5' end of this RNA is a 9-nt sequence called the cB box, the complement of the previously defined B box. The cB box can not be functionally replaced by the B box and has specific positional and sequence requirements. The portion of the cB box that is required for RNA synthesis in vitro is well-conserved in species in the Bromoviridae family. An equivalent RNA from Cucumber mosaic virus was unable to direct efficient RNA synthesis by the BMV replicase until the cB box was positioned at the same site relative to the BMV RNA and guanylates were present at positions +6 and +7 from the initiation cytidylate. These results further define the elements required for the recognition and initiation of viral genomic plus-strand RNA synthesis and suggest that a sequence important for minus-strand RNA synthesis is also required for plus-strand RNA synthesis.     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

Replicase-binding sites on plus- and minus-strand brome mosaic virus RNAs and their roles in RNA replication in plant cells

The cis-acting elements for Brome mosaic virus (BMV) RNA synthesis have been characterized primarily for RNA3. To identify additional replicase-binding elements, nested fragments of all three of the BMV RNAs, both plus- and minus-sense fragments, were constructed and tested for binding enriched BMV replicase in a template competition assay. Ten RNA fragments containing replicase-binding sites were identified; eight were characterized further because they were more effective competitors. All eight mapped to noncoding regions of BMV RNAs, and the positions of seven localized to sequences containing previously characterized core promoter elements (C. C. Kao, Mol. Plant Pathol. 3:55-62, 2001), thus suggesting the identities of the replicase-binding sites. Three contained the tRNA-like structures that direct minus-strand RNA synthesis, three were within the 3' region of each minus-strand RNA that contained the core promoter for genomic plus-strand initiation, and one was in the core subgenomic promoter. Single-nucleotide mutations known previously to abolish RNA synthesis in vitro prevented replicase binding. When tested in the context of the respective full-length RNAs, the same mutations abolished BMV RNA synthesis in transfected barley protoplasts. The eighth site was within the intercistronic region (ICR) of plus-strand RNA3. Further mapping showed that a sequence of 22 consecutive adenylates was responsible for binding the replicase, with 16 being the minimal required length. Deletion of the poly(A) sequence was previously shown to severely debilitate BMV RNA replication in plants (E. Smirnyagina, Y. H. Hsu, N. Chua, and P. Ahlquist, Virology 198:427-436, 1994). Interestingly, the B box motif in the ICR of RNA3, which has previously been determined to bind the 1a protein, does not bind the replicase. These results identify the replicase-binding sites in all of the BMV RNAs and suggest that the recognition of RNA3 is different from that of RNA1 and RNA2.     

Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity

An RNA-dependent RNA polymerase (replicase) activity that specifically copies brome mosaic virus (BMV) RNAs in vitro can be prepared from BMV-infected barley leaves. The signals directing complementary (minus) strand synthesis reside within the 3' 134-nucleotide-long tRNA-like structure that is common to each of the virion RNAs. By studying the influence of minus strand synthesis of numerous mutations introduced throughout this region of the RNA, we have mapped in detail the sequence and structural elements necessary for minus strand promoter activity. Sequence alterations (either substitutions or small, structurally discrete deletions) in most parts of the tRNA-like structure resulted in decreased minus strand synthesis. This suggests that BMV replicase is a large enzyme, possibly composed of several subunits. The lowest activities, 5 to 8% of wild type, were observed for mutants with substitutions at three separate loci, identifying one structural and two sequence-specific elements essential for optimal promoter activity. (1) Destabilization of the pseudoknot structure in the aminoacyl acceptor stem resulted in low promoter activity, demonstrating the importance of a tRNA-like conformation. (2) Substitution of the C residue adjacent to the 3' terminus resulted in low promoter activity, probably by interfering with strand initiation. (3) The low activities resulting from substitutions and a small deletion in arm C suggest this region of the RNA to be a major feature involved in replicase binding. In particular, nucleotides within the loop of arm C appear to be involved in a sequence-specific interaction with the replicase.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

Completion of RNA synthesis by viral RNA replicases

How the 5′-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA–DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5′-terminus and the C2′-hydroxyl of a ribose at the third nucleotide from the 5′-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.     

The AU-rich RNA recombination hot spot sequence of Brome mosaic virus is functional in tombusviruses: implications for the mechanism of RNA recombination

RNA recombination can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase "landing" during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.     

RNA Binding by the Brome Mosaic Virus Capsid Protein and the Regulation of Viral RNA Accumulation

Viral capsid proteins (CPs) can regulate gene expression and encapsulate viral RNAs. Low-level expression of the brome mosaic virus (BMV) CP was found to stimulate viral RNA accumulation, while higher levels inhibited translation and BMV RNA replication. Regulation of translation acts through an RNA element named the B box, which is also critical for the replicase assembly. The BMV CP has also been shown to preferentially bind to an RNA element named SLC that contains the core promoter for genomic minus-strand RNA synthesis. To further elucidate CP interaction with RNA, we used a reversible cross-linking-peptide fingerprinting assay to identify peptides in the capsid that contact the SLC, the B-box RNA, and the encapsidated RNA. Transient expression of three mutations made in residues within or close by the cross-linked peptides partially released the normal inhibition of viral RNA accumulation in agroinfiltrated Nicotiana benthamiana. Interestingly, two of the mutants, R142A and D148A, were found to retain the ability to down-regulate reporter RNA translation. These two mutants formed viral particles in inoculated leaves, but only R142A was able to move systemically in the inoculated plant. The R142A CP was found to have higher affinities for SLC and the B box compared with those of wild-type CP and to alter contacts to the RNA in the virion. These results better define how the BMV CP can interact with RNA and regulate different viral processes.     

Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.     

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.