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

Abstract

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.

FIG. 1.

Schematic representation of the deleted regions in BMV RNA3. Nucleotide deletions (69 and 66 nt) from 3a ORF (open box) are indicated by a horizontal line with nucleotide numbers at their 5′ and 3′ ends. Wild-type RNA3 is also shown at the top of this figure. All BR3D-RNAs (BR3D-200, BR3D-374, BR3D-584, BR3D-797, BR3D-866, and BR3D-935) were constructed by in vitro mutagenesis to create in-frame deletions.

FIG. 2.

Northern blot analysis of the virion RNA fraction extracted from barley protoplasts at 24 h after inoculation with a mixture of in vitro transcripts of BMV RNAs 1, 2, and 3 or BR3D-RNAs or wt RNA3. The virion RNA fraction was prepared from 1.25 × 10⁴ protoplasts. (A) Northern blot results from an experiment. The complete set of inocula is shown above the Northern blot photograph with individual BR3D-RNAs and RNA3 designated lane by lane. Lane M, inoculated with water only. Positions of RNAs 1, 2, 3, and 4 and BR3D are indicated on the right. (B) Relative values for the accumulation of RNA3s. RNAs 1 and 2 (RNAs 1+2) was used as an internal standard. The mean values for RNA3 and its derivatives (shaded columns) and for RNA4 (white columns) with standard deviations (thin vertical lines) were calculated from three independent experiments.

FIG. 3.

Northern blot analysis of total RNA extracted from barley protoplasts at 24 h after inoculation with a mixture of in vitro transcripts of BMV RNAs 1 and 2 and BR3D(CPfs) RNAs. Total RNA was extracted from 3.0 × 10⁴ protoplasts. (A) Northern blot results from an experiment. (B) Relative values for the accumulation. For others, refer to the Fig. 2 legend.

FIG. 4.

Computer-predicted stem-loop structures in the 3′-proximal region of the BMV 3a ORF. Fourteen-base sequences in SL-I, SL-II, and SL-III and in the sequence between SL-II and SL-III (bases are shaded), were replaced with synthetic oligonucleotides of identical length (bases bracketed in a box) to produce SL1 M, SL2 M, SL3 M, and CM cDNA clones, respectively.

FIG. 5.

Northern blot analysis of the virion RNA or total RNA fraction extracted from barley protoplasts at 24 h after inoculation with a mixture of in vitro transcripts of BMV RNAs 1 and 2 and RNA3 mutants of SLMs and CM or their CP frameshift derivatives. Virion RNA fraction was extracted from 1.25 × 10⁴ protoplasts and total RNA was extracted from 3.0 × 10⁴ protoplasts. (A) Northern blot results of the virion RNA fraction from an experiment using SLMs, CM, or wt RNA3; (B) Northern blot results of the total RNA fraction from an experiment using CP frameshift mutants. The complete set of inocula is shown at the top. Positions of RNAs 1, 2, 3, and 4 and RNA3 mutants are indicated on the right. Relative values for the accumulation of RNA3s are presented below Northern blot photographs. Coinoculated RNA1 + RNA2 was used as an internal standard. The mean values (shaded columns) and standard deviations (thin vertical lines) were calculated from three independent experiments.

FIG. 6.

Accumulation of chimeric BMV RNA3s and their CP frameshift mutants in virion or total RNA fraction from infected barley protoplasts. Schematic representations of chimeric BMV RNA3s (A) and their CP frameshift mutants (D) with the corresponding abbreviations. Wild-type BMV RNA3 (B3) and CMV RNA3 (C3) are also shown. The ORFs encoded by B3 and C3 are shown as solid and open boxes, respectively, and the noncoding regions of BMV and CMV are represented as thick and thin lines, respectively. Northern blot results from the virion RNA fraction (B) and from the total RNA fraction (C) extracted from barley protoplasts inoculated with chimeric BMV RNA3s or wt BMV RNA3 together with RNAs 1 and 2. Northern blot results from total RNA fraction (E) extracted from barley protoplasts inoculated with CP frameshift mutants of chimeric BMV RNA3s or wt BMV RNA3 together with RNAs 1 and 2. The complete set of inocula is shown above the photograph, with the designations of individual chimeric RNAs given lane by lane. Relative values for the accumulation of RNA3s are presented below the Northern blot photographs. For others, refer to the legend of Fig. 5.

FIG. 7.

Effect of disruption of stem-loop structure II in chimeric BMV RNA3 (B3CmpBR3SL) on RNA-packaging efficiency. Virion RNA fractions were extracted from barley protoplasts at 24 h after inoculation with a mixture of in vitro transcripts of BMV RNAs 1 and 2 and chimeric RNAs. B3CmpBR3SLSL2 M is a derivative of B3CmpBR3SL in which SL-II is disrupted. The names of the RNA3 mutants are shown above the photograph. Positions of RNAs 1, 2, 4, and chimeric RNA3s (3 M) are indicated on the right. Relative values for the accumulation of RNA3s are presented on the right of the Northern blot photograph. For others, refer to the legend of Fig. 5.