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Although RNA viruses evolved the mechanisms of specific encapsidation, miss-packaging of cellular RNAs has been reported in such RNA virus systems as flock house virus or cucumber necrosis virus. To find out if brome mosaic virus (BMV), a tripartite RNA virus, can package cellular RNAs, BMV was propagated in barley and in Nicotiana benthamiana hosts, purified by cesium chloride (CsCl) gradient ultracentrifugation followed by nuclease treatment to remove any contaminating cellular (host) RNAs. The extracted virion RNA was then sequenced by using next-generation sequencing (NGS RNA-Seq) with the Illumina protocol. Bioinformatic analysis revealed the content of host RNAs ranging from 0.07% for BMV extracted from barley to 0.10% for the virus extracted from N. benthamiana. The viruses from two sources appeared to co-encapsidate different patterns of host-RNAs, including ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) but also mitochondrial and plastid RNAs and, interestingly, transposable elements, both transposons and retrotransposons. Our data reveal that BMV virions can carry host RNAs, having a potential to mediate horizontal gene transfer (HGT) in plants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Two viral proteins, 1a and 2a, direct replication of brome mosaic bromovirus (BMV) RNAs as well as they participate in BMV RNA recombination. To study the relationship between replication and recombination, double BMV variants that carried mutations in 1a and 2a genes were tested. The observed effects revealed that the 1a helicase and 2a N-terminal or core domains were functionally linked during both processes in vivo. The use of a series of mutant BMV replicase (RdRp) preparations demonstrated in vitro the participation of the 1a and 2a domains in BMV RNA copying and in template switching during minus-strand synthesis. The observed effects support previous observations that the characteristics of homologous and nonhomologous recombination can be modified separately by mutations at different sites on BMV replicase proteins., (Copyright 2001 Academic Press.)

The mechanism of homologous recombination has been studied previously in brome mosaic virus (BMV), a tricomponent, positive-stranded RNA virus of plants, by using artificial sequences (reviewed by J. J. BujarskiP. D. Nagy (1996). Semin. Virol. 7, 363-372). Here we extend these studies over BMV-derived sequences to obtain clues on prediction of homologous recombination hot spots. First, mismatch mutations, which reduced the AU content, were introduced into the common 60-nt recombination hot-spot sequence, either in the RNA2 or in both RNA2RNA3 components. This decreased the frequency of targeted homologous RNA2/RNA3 recombinationchanged the distribution of junction sites. Second, several short BMV RNA1- or RNA2-derived sequences were introduced into the RNA3 component, homologous recombination activity of these sequences was compared with that observed for previously characterized artificial sequences. Third, sequences at homologous recombinant junctions were compared among a large number of targetednontargeted recombinants. All these studies revealed several factors important for homologous recombination including the length of sequence identity, the extent of sequence identity, the AU content of the common sequences, the relative position of the AU-rich segment vs a GC-rich segment,the presence of GC-rich sequences. Based on this novel model, we suggest that recombination hot spots can be predicted by means of RNA sequence analysis. In addition, we show that recombination can occur between positivenegative strands of BMV RNAs. This provides further clues toward the mechanism of recombination processes in BMV., (Copyright 1999 Academic Press.)

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799-3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar "homologous recombination silencing" effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.

Broad bean mottle bromovirus (BBMV) was shown to accumulate RNA2-derived defective interfering (DI) RNAs [Romero et al., Virology 194, 576-584 (1993); Pogany et al., Virology 212, 574-586 (1995)]. In this work, we utilize three sets of BBMV RNA2-derived artificial DI RNA constructs to determine factors that affect the accumulation of defective RNAs in planta. One set of deletion constructs was used to localize sequences required for efficient accumulation within the 5' 883 nt and the 3' 387 nt of the DI RNAs. The second set had a gradually increasing size of 3' nested deletions to determine the minimal length required for efficient DI RNA accumulation. The smallest DI RNA still accumulating in plants was found to be 1712 nt long. The third set consisted of frameshift mutants which showed that at least 64.4% of BBMV DI RNA sequences must have the 5' portion of the 2a open reading frame to ensure efficient accumulation. The importance of these factors in the selection of DI RNAs is discussed.

Broad been mottle virus (BBMV) is the only member of the bromoviruses that is known to accumulate defective-interfering (DI) RNAs (Romero et al., Virology 194, 576-584, 1993). De novo generation of DI-like RNAs was demonstrated during serial passages of BBMV in broad bean using either DI RNA-free virion RNA preparations or transcribed genomic RNA inocula. As for previously described DI RNAs, all but one of the characterized de novo generated DI-like RNAs were derived by a single in-frame deletion from the RNA2 component. The sole exception was derived by two shorter in-frame deletions from RNA2. The maintenance of an open reading frame by all DI-like RNAs suggests the importance of coding capacity and/or the shortened 2a protein in the accumulation of these RNAs during infection. The deletion junction sites were between nucleotides 1152 and 2366, suggesting that the retained regions are essential for the efficient accumulation of BBMV DI-like RNAs in planta. Short regions of sequence similarity and/or complementarity were revealed at the 5' and 3' junction borders. We speculate that these regions can facilitate DI (DI-like) RNA formation. In addition to DI-like RNAs, the full-length nucleotide sequences of RNA2 components of the Type and Morocco strains of BBMV are presented.

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3' noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc. Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation codon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3' marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.

The coat protein (CP) open reading frame (ORF) of brome mosaic virus (BMV) has been mutated to study host-related CP functions in barley, a systemic host, and in Chenopodium hybridum L. which supports both local lesion formation and systemic spread of BMV. To test the role of the N-terminal region of CP, mutants C1 to C3, which synthesized the CP lacking first seven amino acids, and mutant D1, which had Trp 22 and Thr 23 replaced with Phe-Gly-Ser, were generated. C1 to C3 inhibited virus systemic spread in C. hybridum but not in barley while D1 only reduced virus accumulation in noninoculated leaves of C. hybridum. More internal CP regions were tested by mutation of Lys 63 to Leu (mutant SP3) and Lys 129 to Arg (mutant SP1). SP1 behaved similarly to C1 to C3 while SP3 similarly to D1. In addition, SP3 reduced concentrations of RNA3 and RNA4 in both hosts. Apparently, various CP regions differentially affect, either directly or indirectly, virus translocation in different hosts, suggesting both the CP and host factors to be important for virus spread. Larger deletions in the CP ORF (mutants BB4 and SX1) or a decrease of CP production by using a frameshift mutant C, inhibited virus systemic spread in both hosts, and delayed the appearance of smaller local lesions on C. hybridum. Thus, the CP is not required for cell-to-cell movement but is required for systemic translocation of BMV.

Full-length complementary DNA (cDNA) copies of genomic RNA1, RNA2, and RNA3 segments of cowpea chlorotic mottle virus (CCMV) strains D, N, and S were synthesized using polymerase chain reaction and were cloned downstream of a T7 RNA polymerase promoter. Mixtures of the homologous in vitro-transcribed RNAs produced typical CCMV symptoms when inoculated on soybean (cv. Bragg) and cowpea (cv. California Blackeye) plants. Using either gel-purified or in vitro-transcribed CCMV RNA components, the pseudorecombinants SSD, SSN, DDS, and NNS were constructed. The properties of these pseudorecombinants were tested by comparing the induced symptoms, virus concentration, and systemic spread. These studies revealed a direct involvement of RNA3 genetic information in necrotic lesion formation and in breakage of CCMV resistance in soybean. Exchanges between RNA1 or RNA2 components further affected the biological features of reassorted CCMV variants. The true nature of the pseudorecombinants was confirmed by reconstitution of the parental strains from the corresponding pseudorecombinant RNA components.

Several strains of the broad bean mottle virus (BBMV), an icosahedral tripartite plant RNA virus, which show distinct reactions on certain plant hosts have been described (K. M. Makkouk et al., Neth. J. Plant Pathol. 94, 195-212, 1988). Here we report defective interfering (DI) RNAs encapsidated in two BBMV strains from Morocco and Tunisia. While not effective in some plants, these DI RNAs exacerbated the severity of symptoms in others. The most dramatic, lethal effect of DI RNAs has been found on pea (Pisum sativum, cv. Rondo) seedlings. Sequence analysis has revealed that the DI RNAs were derived by single in-frame central deletions of 448 to 537 nt in the corresponding genomic RNA2 components. A comparison of the intensities of full-length RNA2 bands from DI molecule-containing and DI molecule-deficient virion RNA preparations revealed that the DI RNAs decreased the level of RNA2 components in total RNA preparations. The differences between corresponding virion RNAs were much smaller. This suggests an interference with RNA replication. In vitro assays and an analysis of the polyribosomal RNA fractions confirmed the translational activity of DI RNAs. This paper reports the first description of natural DI RNAs in tripartite isometric plant RNA viruses.

Complete nucleotide sequences of broad bean mottle virus (BBMV) genomic RNAs 2 and 3 were determined. They consist of 2811 and 2293 nucleotides, respectively. Both RNAs are caped and, unlike in other tricornaviruses, both initiate with an A residue. BBMV RNA2 is monocistronic and encodes an 815 amino acid 2a protein, whereas RNA3 is dicistronic, encoding for a 295 amino acid 3a protein and for the 190 amino acid coat protein. A central, 423 amino acid 2a protein core region is highly homologous among the three bromoviruses, whereas both N- and C-termini are more heterologous. Most of the homologies among 3a proteins are concentrated within the N-termini two-thirds of the molecule that is predominantly hydrophobic, whereas the C-terminal one-third contains a large number of charged amino acids. The homologies among coat proteins are clustered within several mostly hydrophobic, or neutral, domains. The 5' noncoding region of the RNA2 has 110 nucleotides, whereas that of RNA3 contains 330 nucleotides. As in cowpea chlorotic mottle virus, but unlike in Brome mosaic virus, the 5' noncoding region includes subgenomic promoter-like sequences. The BBMV RNA3 intercistronic region also has subgenomic promoter sequences and contains a long poly(A) stretch. At the 3' end, BBMV RNAs 2 and 3 have 257 and 236 noncoding nucleotides, respectively.

The complete nucleotide sequences of the RNA-1 segments in broad bean mottle virus (BBMV) and cowpea chlorotic mottle virus (CCMV) were determined. BBMV RNA-1 consists of 3158 nucleotides and CCMV RNA-1 has 3171 nucleotides. Both BBMV and CCMV RNA-1 are capped at the 5' end but, unlike in other tricornaviruses, BBMV RNA-1 initiates with an A residue. Both BBMV and CCMV RNA-1 are monocistronic encoding for highly homologous 1a proteins of 966 and 958 amino acids, respectively. The highest homologies are clustered within two domains: the N-domain that aligns with the nsP1 Sindbis virus protein, a putative methyl transferase, and the C-domain which has a conserved nucleotide binding motif. Previous sequence comparisons suggest that the C-terminal domain may function as an NTP-dependent RNA helicase. In addition, we find that the C-domain has patterns similar to those of the reovirus and vaccinia virus guanylyl transferases. All this implies that 1a protein is a multifunctional polypeptide involved in both RNA capping and RNA polymerization processes.

3' terminal fragments of BMV RNA as short as 153 bases in length serve as efficient templates in vitro for BMV-specific RNA polymerase. Template activity of such fragments or of native BMV RNA is abolished when cDNA fragments as short as 39 bases are hybridized to their 3' termini. Hybridization of cDNa fragments to regions of BMV RNA 200 or more bases distal to the 3' end has no discernible effect on initiation and little effect on elongation. We conclude that BMV RNA polymerase initiates binding with an RNA template through a mechanism mediated by the tRNA-like 3' end of BMV RNA, requiring at least some of the last 39, but no more than the last 153 bases.

The activity and specificity of RNA-dependent RNA polymerase (replicase) isolated from brome mosaic virus-infected barley was enhanced by extraction with the nonionic detergent dodecyl-beta-d-maltoside. The enzyme was stable for at least 8 weeks when stored at -70 degrees . A further 100-fold purification was obtained by centrifugation through sucrose in the presence of detergent. The polymerase activity was associated with the pellet fraction; the template dependence and specificity were similar to those of the enzyme before sucrose purification. SDS-PAGE analysis revealed a 110-kd protein in the purified pellet fraction from infected leaves that was absent from a similar fraction from healthy leaves. The protein had an identical electrophoretic mobility to that of protein la, the product of brome mosaic virus RNA 1 translation in vitro, and the profile of its tryptic polypeptides was very similar to that of protein 1a. These results support data obtained by inoculation of protoplasts with separated BMV RNA components (Kiberstis, et al. (1981), Virology 112, 804-808) that are consistent with the notion that RNA 1 codes for the viral replicase, or a subunit thereof.

The method of immunoadsorptional purification of Bean Yellow Mosaic Virus has been worked out. Immunosorbents were obtained by coupling the antibody (IgG) fraction isolated from anti-BYMV and anti-pea leaf protein antisera with CNBr-activated 1% agarose beads. Conditions for preparation of immunosorbents, for BYMV adsorption and elution as well as the method of plant protein separation from BYMV were pointed out. The purity of BYMV was checked by double immunodiffusion as well as by SDS-acrylamide gel electrophoresis. Also biological activity was determined. TMV was used as the model virus for further BYMV studies.

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.