Bromovirus movement protein genes play a crucial role in host specificity.

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small.     

Use of bromovirus RNA2 hybrids to map cis- and trans-acting functions in a conserved RNA replication gene.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with tripartite genomes. RNA replication by either virus requires genomic RNAs 1 and 2, which encode protein 1a and the polymeraselike, 94-kilodalton 2a protein, respectively. Proteins 1a and 2a share extensive sequence similarity with proteins encoded by a wide range of other positive-strand RNA viruses of animals and plants. Heterologous combinations of BMV and CCMV RNAs 1 and 2 do not support viral RNA replication, and although BMV RNA2 is amplified in CCMV-infected cells, CCMV RNA2 is not amplified by BMV. Construction of hybrids by precise exchange of segments between BMV and CCMV RNA2 has now allowed preliminary mapping of such virus-specific replication functions in RNA2 and the 2a protein. The ability to support replication in trans with BMV RNA1 segregated with a 5' BMV RNA2 fragment encoding the first 358 2a gene amino acids, while a 5' fragment extending over 281 BMV 2a codons transferred only cis-acting competence for RNA2 amplification in cells coinfected with wild-type BMV. Successful trans-acting function with CCMV RNA1 segregated with a CCMV RNA2 3' fragment that included the last 206 2a gene codons. Thus, the less conserved N- and C-terminal 2a segments appear to be involved in required interaction(s) of this polymeraselike protein with the 1a protein or RNA1 or both. Moreover, when individual hybrid RNA2 molecules that function with either BMV or CCMV RNA1 were tested, BMV- and CCMV-specific differences in recognition and amplification of RNA3 templates appeared to segregate with RNA1.     

Use of bromovirus RNA3 hybrids to study template specificity in viral RNA amplification.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2 encode the viral RNA replication factors, which share extensive conservation with proteins encoded by the animal alphaviruses and diverse plant viruses. In barley protoplasts, CCMV RNAs 1 and 2 support high but distinguishable amplification of either BMV RNA3 (B3) or CCMV RNA3 (C3), while BMV RNAs 1 and 2 show even greater discrimination, amplifying C3 poorly relative to B3. To identify the cis-acting determinants of these template-specific and virus-specific differences in RNA3 accumulation, we constructed and tested a series of B3/C3 hybrids that exchange in turn the 5',3', and intercistronic noncoding regions, which contain all sequences required in cis for efficient B3 and C3 amplification. Despite suggestive prior in vitro results, the 3' noncoding regions were not the major determinant of the differences in amplification of B3 and C3 in vivo. Rather, 3' exchanges had relatively modest effects and did not transfer the distinctive asymmetry of amplification between B3 and C3. Intercistronic exchanges produced larger effects on RNA3 accumulation and transferred some of the polarized characteristics of the wild-type B3 and C3 behaviors. 5' exchanges revealed context-specific effects showing that the contribution of the B3 5' region to RNA3 amplification is dependent on some other B3 segment or segments. Together with previous results implicating the BMV and CCMV 1a genes in trans-acting discrimination between B3 and C3 (P. Traynor and P. Ahlquist, J. Virol. 64:69-77, 1990), these observations should help to guide studies of protein-RNA interactions governing template specificity in bromovirus RNA replication.     

Genotypic and phenotypic analysis of bromovirus adaptive mutants derived from a single plant

Eight adaptive mutant clones have been made from the total RNA extracted from uninoculated upper leaves of a single cowpea plant exhibiting systemic infection after inoculation with a hybrid cowpea chlorotic mottle bromovirus (CCMV) with the 3a movement protein gene of CCMV replaced by that of cowpea-nonadapted brome mosaic bromovirus (BMV). Sequence and mutational analyses of these clones showed genotypic and phenotypic diversity of the cloned virus population, but all examined clones had the adaptive mutation, A to C at position 776 within the BMV 3a gene, required for the systemic infection of cowpea. The data support the quasispecies model for RNA virus population, and suggest that the maintenance of the adaptive mutation may be due to powerful selection pressure in an infection process.     

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.     

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.     

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.     

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.     

Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies

Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.     

Repair of the tRNA-like CCA sequence in a multipartite positive-strand RNA virus

The 3' portions of plus-strand brome mosaic virus (BMV) RNAs mimic cellular tRNAs. Nucleotide substitutions or deletions in the 3'CCA of the tRNA-like sequence (TLS) affect minus-strand initiation unless repaired. We observed that 2-nucleotide deletions involving the CCA 3' sequence in one or all BMV RNAs still allowed RNA accumulation in barley protoplasts at significant levels. Alterations of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at wild-type levels. However, substitutions in all three BMV RNAs severely reduced RNA accumulation, demonstrating that substitutions have different repair requirements than do small deletions. Furthermore, wild-type BMV RNA1 was required for the repair and replication of RNAs with nucleotide substitutions. Results from sequencing of progeny viral RNA from mutant input RNAs demonstrated that RNA1 did not contribute its sequence to the mutant RNAs. Instead, the repaired ends were heterogeneous, with one-third having a restored CCA and others having sequences with the only commonality being the restoration of one cytidylate. The role of BMV RNA1 in increased repair was examined.     

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.     

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.