Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3.

The genome of brome mosaic virus (BMV) is divided among messenger polarity RNA1, RNA2, and RNA3 (3.2, 2.9, and 2.1 kilobases, respectively). cis-Acting sequences required for BMV RNA amplification were investigated with RNA3. By using expressible cDNA clones, deletions were constructed throughout RNA3 and tested in barley protoplasts coinoculated with RNA1 and RNA2. In contrast to requirements for 5'- and 3'-terminal noncoding sequences, either of the two RNA3 coding regions can be deleted individually and both can be simultaneously inactivated by N-terminal frameshift mutations without significantly interfering with amplification of RNA3 or production of its subgenomic mRNA. However, simultaneous major deletions in both coding regions greatly attenuate RNA3 accumulation. RNA3 levels can be largely restored by insertion of a heterologous, nonviral sequence in such mutants, suggesting that RNA3 requires physical separation of its terminal domains or a minimum overall size for normal replication or stability. Unexpectedly, deletions in a 150-base segment of the intercistronic noncoding region drastically reduce RNA3 accumulation. This segment contains a sequence element homologous to sequences found near the 5' ends of BMV RNA1 and RNA2 and in analogous positions in the three genomic RNAs of the related cucumber mosaic virus, suggesting a possible role in plus-strand synthesis.     

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.     

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.     

Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis

Brome mosaic virus (BMV) belongs to a "superfamily" of plant and animal positive-strand RNA viruses that share, among other features, three large domains of conserved sequence in nonstructural proteins involved in RNA replication. Two of these domains reside in the 109-kDa BMV 1a protein. To examine the role of 1a, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the 1a gene. The majority of these mutations blocked BMV RNA replication in protoplasts, indicating that both intervirally conserved domains function in RNA replication. Coinoculation tests with a large number of mutant combinations failed to reveal detectable complementation between mutations in the N- and C-terminal conserved domains, implying that these two domains either function in some directly interdependent fashion or must be present in the same protein. Four widely spaced mutations with temperature-sensitive (ts) defects in RNA replication were identified, including a strongly ts insertion near the nucleotide-binding consensus of the helicaselike C-terminal domain. Temperature shift experiments with this mutant show that 1a protein is required for continued accumulation of all classes of viral RNA (positive strand, negative strand, and subgenomic) and is required for at least the first 10 h of infection. ts mutations were also identified in the 3' noncoding region of RNA1, 5' to conserved sequences previously implicated in cis for replication. Under nonpermissive conditions, the cis-acting partial inhibition of RNA1 accumulation caused by these noncoding mutations was also associated with reduced levels of the other BMV genomic RNAs. Comparison with previous BMV mutant results suggests that RNA replication is more sensitive to reductions in expression of 1a than of 2a, the other BMV-encoded protein involved in replication.     

Generation and analysis of nonhomologous RNA-RNA recombinants in brome mosaic virus: sequence complementarities at crossover sites.

All three single-stranded RNAs of the brome mosaic virus (BMV) genome contain a highly conserved, 193-base 3' noncoding region. To study the recombination between individual BMV RNA components, barley plants were infected with a mixture of in vitro-transcribed wild-type BMV RNAs 1 and 2 and an RNA3 mutant that carried a deletion near the 3' end. This generated a population of both homologous and nonhomologous 3' recombinant BMV RNA3 variants. Sequencing revealed that these recombinants were derived by either single or double crossovers with BMV RNA1 or RNA2. The primary sequences at recombinant junctions did not show any similarity. However, they could be aligned to form double-stranded heteroduplexes. This suggested that local hybridizations among BMV RNAs may support intermolecular exchanges.     

Modulation of replication, aminoacylation and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3'' end.

The genome of brome mosaic virus (BMV) is comprised of three (+) strand RNAs, each containing a similar, highly structured, 200 base long sequence at its 3' end. A 134 base subset of this sequence contains signals directing interaction of the viral RNA with BMV RNA replicase, ATP,CTP:tRNA nucleotidyl transferase and aminoacyl tRNA synthetase. A series of mutants containing deletions within this region, previously constructed and tested in vitro for the effect on replication and aminoacylation activities, has now been assayed in vitro for adenylation function and in vivo for ability to replicate in isolated protoplasts and whole plants. These tests indicate that features of viral RNA recognized by BMV replicase overlap those directing adenylation, but are distinct from those directing aminoacylation. Consequently, the lethality of a deletion preferentially inhibiting aminoacylation suggests that this function may have an essential role contributing to viral replication in vivo. An RNA3 mutant bearing a 20-base deletion yielding normal levels of aminoacylation and enhanced levels of replicase template activity and adenylation in vitro was able to replicate in protoplasts and plants; however, its accumulation in protoplasts was reduced relative to wild-type. This suggests that additional functions affecting the replication and accumulation of viral RNA reside in the conserved 3' sequence.     

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.     

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses

Summary A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.     

Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.     

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.     

Engineering of homologous recombination hotspots with AU-rich sequences in brome mosaic virus.

Previously, we observed that crossovers sites of RNA recombinants clustered within or close to AU-rich regions during genetic recombination in brome mosaic bromovirus (BMV) (P. D. Nagy and J. J. Bujarski. J. Virol. 70:415-426, 1996). To test whether AU-rich sequences can facilitate homologous recombination, AU-rich sequences were introduced into parental BMV RNAs (RNA2 and RNA3). These insertions created a homologous RNA2-RNA3 recombination hotspot. Two other AU-rich sequences also supported high-frequency homologous recombination if a common sequence with high or average G/C content was present immediately upstream of the AU-rich element. Homologous RNA recombination did not require any additional sequence motifs or RNA structures and was position nonspecific within the 3' noncoding region. These results suggest that nucleotide content (i.e., the presence of common 5' GC-rich or moderately AU-rich and 3' AU-rich regions) is the important factor that determines the sites of homologous recombination. A mechanism that involves replicase switching during synthesis of positive-sense RNA strands is presented to explain the observed results.     

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.     

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.     

Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication

The nucleotide sequences of brome mosaic virus (BMV) RNAs 1 (3234 bases) and 2 (2865 bases) have been determined, completing the primary structure of the 8200 base tripartite BMV genome. cDNA clones covering 99% of BMV RNA1 and a full-length cDNA clone of BMV RNA2 were isolated in the course of this work. Extensive sequence homology and known interaction with several proteins suggest that the 3' ends of the BMV RNAs are the major regulatory regions of the genome. Smaller regions at the 5' ends of RNAs 1 and 2 show strong homology to each other and lesser homology to RNA3. These and other features of the sequences are discussed in relation to replication, regulation and evolution of the BMV genome.     

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.