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. 2022 Jul 21;11(7):817.
doi: 10.3390/pathogens11070817.

Variability among the Isolates of Broad Bean Mottle Virus and Encapsidation of Host RNAs

Nipin Shrestha  1 , Melvin R Duvall  1 , Jozef J Bujarski  1
Affiliations

Variability among the Isolates of Broad Bean Mottle Virus and Encapsidation of Host RNAs

Nipin Shrestha et al. Pathogens. .

Abstract

Broad bean mottle bromovirus infects legume plants and is transmissible by insects. Several broad bean mottle virus (BBMV) isolates have been identified, including one in England (isolate Ba) and five in the Mediterranean countries: Libya (LyV), Morocco (MV), Syria (SV), Sudan (TU) and Tunisia (TV). Previously, we analyzed the nucleotide sequence of the Ba RNA and here we report on and compare it with another five Mediterranean variants. The RNA segments in the latter ones were extensively homologous, with some SNPs, single nucleotide deletions and insertions, while the number of mutations was higher in isolate Ba. Both the 5' and 3' untranslated terminal regions (UTRs) among the corresponding RNAs are highly conserved, reflecting their functionality in virus replication. The AUG initiation codons are within suboptimal contexts, possibly to adjust/regulate translation. The proteins 1a, 2a, 3a and coat protein (CP) are almost identical among the five isolates, but in Ba they have more amino acid (aa) substitutions. Phylogenetic analysis revealed the isolates from Morocco and Syria clustering with the isolate from England, while the variants from Libya, Tunisia and Sudan created a different clade. The BBMV isolates encapsidate a high content of host (ribosomal and messenger) RNAs. Our studies present BBMV as a useful model for bromoviruses infecting legumes.

Keywords: RNA genome variability; bromoviruses; cellular RNA encapsidation; phylogeny; plant RNA viruses.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Denaturing gel of the BBMV RNAs isolated from five BBMV isolates. The images were taken prior to sequencing. L1, L2, L3, L4 and L5, which are the lanes loaded with the RNA from MV, LyV, SV, TU and TV isolates, respectively. Lane M is the size marker. The three major bands, characteristic for Bromoviruses, representing RNA1 + 2 (~2900–3100 bp), RNA3 (~2200 bp) and sgRNA4 (~800 bp) are visible in the gel (indicated on the right side).
Figure 2
Figure 2
A stacked plot showing proportion of reads that mapped to RNA1, RNA2 and RNA3 among the five BBMV isolates.
Figure 3
Figure 3
Multiple sequence alignment (MSA) of nucleotide sequences of BBMV RNAs 1, 2, and 3 (top to bottom) for five BBMV isolates as compared to Ba ones. The orange bars represent the ORF regions (with the length shown in parentheses). The gray regions signify identical nucleotides, while the nucleotide substitutions are depicted by the black vertical lines and the deletions by the horizontal black lines. The green bars illustrate the regions of identity.
Figure 4
Figure 4
The alignment of the nt sequences of the intercistronic noncoding region in the RNA3 of the BBMV variants covering the subgenomic promoter core (black bar) and the start site of the sg RNA4 transcription (see also Romero et al., 1992 [6]). The subgenomic RNA start site are marked by red bar, and the CP translation start codon ATG is indicated by the blue bar on top of the alignment.
Figure 5
Figure 5
MSA of amino acid sequences of proteins 1a, 2a, 3a and 4a (CP) for six BBMV isolates. The gray regions represent identical nucleotides, while the aa substitutions are depicted by the black vertical lines and the deletions by small gaps. The green bars illustrate the regions of identity. The numerical scale on top marks the positions of amino acids.
Figure 6
Figure 6
Maximum likelihood (ML) bootstrap consensus cladogram for five Mediterranean and the England isolates of BBMV. The sequences of RNAs 1, 2 and 3 were concatenated and aligned by MSA followed by the consensus tree reconstruction by the ML bootstrap method. The BMV RNA sequences were used as the outgroup to infer the rooted cladogram. Numbers along the branches are ML bootstrap values. The cladogram has been calculated at the Geneious default values.
Figure 7
Figure 7
The stacked plot illustration profiling the proportions of different types of encapsidated host RNAs in BBMV vs. BMV, originating from three different cellular compartments. Apparently in BBMV, more than 99% of the host RNAs were nuclear RNAs with ribosomal RNAs being present more in comparison to the mRNA transcripts, whereas in BMV over 50% of the reads mapped to the nuclear RNAs followed by the organellar RNAs (mitochondrial and plastid). The proportion of the mRNAs being mapped dominantly in comparison to the rRNAs also differed between BBMV and BMV.
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