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.     

cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts

Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.     

Comparison of the nucleotide sequences of cucumber mosaic virus and brome mosaic virus

Cucumber mosaic virus (CMV) and brome mosaic virus (BMV) are isometric plant viruses. Although biologically distinct, they share many common chemical properties. An analysis of the partial genomic RNA sequence available for these two viruses reveals that they are evolutionarily related. Different segments of the genome exhibit different evolutionary rates. The coat proteins, which serve as carriers of genetic material, possess little or no homology. In contrast, the 3a proteins show over 35% homology. The non-coding regions of the genome also exhibit extensive but variable homology suggesting the functional importance of the nucleic acid.     

The nucleotide sequence of cowpea mosaic virus B RNA

The complete sequence of the bottom component RNA (B RNA) of cowpea mosaic virus (CPMV) has been determined. Restriction enzyme fragments of double-stranded cDNA were cloned in M13 and the sequence of the inserts was determined by a combination of enzymatic and chemical sequencing techniques. Additional sequence information was obtained by primed synthesis on first strand cDNA. The complete sequence deduced is 5889 nucleotides long excluding the 3' poly(A), and contains an open reading frame sufficient to code for a polypeptide of mol. wt. 207 760. The coding region is flanked by a 5' leader sequence of 206 nucleotides and a 3' non-coding region of 82 residues which does not contain a polyadenylation signal.     

Genetic recombination in brome mosaic virus: effect of sequence and replication of RNA on accumulation of recombinants.

In order to facilitate the isolation of recombinants in brome mosaic virus, a series of duplication mutants with alterations in the RNA3 3' noncoding region has been engineered. The distribution of crossovers, which was observed to be dependent on the parental RNA3 sequence, supported the role of RNA structure in recombination. However, a negative correlation between replication of the parental RNA3 constructs and the accumulation of recombinant progeny confirmed the role of selection.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

Complete nucleotide sequence of alfalfa mosaic virus RNA3.

A full-length cDNA clone of alfalfa mosaic virus (AMV) RNA3 was prepared and sequenced. The 2,037 base sequence contains two open reading frames of 903 and 666 nucleotides that code for a 32,400 dalton protein (32.4K protein) and the 24,380 dalton coat protein, respectively. A 5'-noncoding sequence of 240 bases preceeding the 32.4K protein contains homologous regions that may have a function in its translation. The intercistronic junction is 49 bases long, the last 36 bases representing the 5'-end of the subgenomic RNA4. The remaining 179 bases comprise the 3'-terminal noncoding sequence.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.     

Two types of non-homologous RNA recombination in brome mosaic virus

Non-homologous RNA recombination is a process enabling the exchange of genetic material between various (related or unrelated) RNA-based viruses. Despite extensive investigations its molecular mechanism remains unclear. Studies on genetic recombination in brome mosaic virus (BMV) have shown that local hybridization between genomic RNAs induces frequent non-homologous crossovers. A detailed analysis of recombinant structures suggested that local complementary regions might be involved in two types of non-homologous recombination in BMV: site-specific and heteroduplex-mediated. To verify the above hypothesis and better recognize the mechanism of the phenomenon studied we have tested how the putative types of recombination are affected by a specific mutation in the BMV polymerase gene or by changes in RNA structure. The experiments undertaken revealed substantial differences between site-specific and heteroduplex-mediated recombination, indicating that they occur according to different mechanisms. The former can be classified as homology-assisted, and the latter as homology-independent. In addition to local RNA/RNA hybridization, short regions of homology are required for site-specific crossovers to occur. They are most efficiently mediated if one homologous sequence is located at the beginning of and the second just before a double-stranded region. At present it is difficult to state what is the mechanism of heteroduplex-mediated recombination. Earlier it was postulated that strong RNA/RNA interaction enforces template switching by the viral replicase. There are, however, several observations questioning this model and indicating that some other factors, which are still unknown, may influence heteroduplex-mediated crossovers.     

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.     

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.     

RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase in human cells reveals requirements for de novo initiation and protein-protein interaction

Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5'-GUAAA-3') identical to those from the 5' sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.     

Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [gamma-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3'-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3'-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3' end. However, only limited oligonucleotide 3' extensions can be present on active templates. In order to assess the 5' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3' (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5' to position 134 also showed high template activity. Thus, the 3'-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5' to it do not play a role in template selection.