Mutual interference between genomic RNA replication and subgenomic mRNA transcription in brome mosaic virus

Abstract

Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle. Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication. Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis. We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positive-strand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components. Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5'-untranslated region. Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors. In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2.

FIG. 1.

Schematic illustration of DNA-based expression, genomic RNA replication, and sgRNA transcription of BMV RNA3 in yeast. “DNA” indicates a plasmid with RNA3 sequences (5′-UTR, 3a ORF, intergenic UTR with RE [black box], CP ORF, and 3′-UTR) flanked by the yeast GAL1 promoter and hepatitis δ virus ribozyme (Rz). Upon galactose induction, yeast RNA polymerase II synthesizes positive-strand RNA3 transcripts that, upon transport to the cytoplasm, serve as the templates for 1a- and 2a-dependent RNA3 replication and sgRNA transcription, which occur in 1a-induced ER membrane-associated replication compartments. Small white, black, and gray boxes indicate negative-strand, positive-strand, and sgRNA promoters, respectively.

FIG. 2.

Inactivation of sgRNA transcription stimulates RNA3 replication. (A) Schematic diagram of expression cassettes for RNA3 derivatives with wt or mutated sgRNA promoter. Dashed boxes represent a translationally inactive CP ORF due to a frameshift mutation (in B3CPfs) or sgRNA transcription block. SG indicates wt RNA3 sequence complementary to the core sgRNA promoter. +1, -2, and -13 indicate mutated nucleotide positions within the sgRNA promoter relative to the +1 sgRNA transcription start site. (B) Northern blot analysis of BMV-specific RNA products for RNA3 derivatives with wt or mutated sgRNA promoters. Total RNA was extracted from yeast expressing BMV 1a, 2a, and RNA3 derivatives (indicated at the top; “empty” is the yeast plasmid without RNA3 expression cassette), and 1.5 μg of total RNA was analyzed by Northern blotting using specific probes to detect RNA3 positive or negative strands as indicated on the left. Triplicate samples represent three independent transformants for each plasmid combination. The asterisk indicates a minor RNA band, discussed in the text. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (C) Relative accumulation of positive-strand RNA3, sgRNA, and negative-strand RNA3 from the RNA3 derivatives in panel B. The histogram compares positive-strand (black bars) and negative-strand (gray bars) accumulation levels for tested RNA3 derivatives, measured using a Molecular Dynamics PhosphorImager. (D) Time course analysis of positive-strand RNA3 (left panel) and negative-strand RNA3 (right panel) accumulation for RNA3 derivatives B3CPfs and B3ΔSG. Yeast cells with 1a, 2a, and RNA3 expression plasmids were first grown in raffinose medium to prevent GAL1 promoter induction. To induce the GAL1 promoter, yeast cells were transferred into galactose-containing medium (zero hours p.i.), and cell aliquots were collected at the indicated time points. Northern blot analysis was performed as for panel B, and the relative accumulation of positive- and negative-strand RNA3 was measured.

FIG. 3.

Effect of CP on sgRNA transcription-mediated inhibition of RNA3 replication. (A) Schematic diagram of expression cassettesfor RNA3 derivatives and CP mRNA used in this experiment and not introduced in Fig. 2. A solid box used for the CP ORF indicates a translationally active wt CP ORF; a dashed box represents a translationally inactive CP ORF due to a frameshift mutation. CP-trans mRNA has the BMV CP ORF and yeast 5′- and 3′-UTRs and is expressed under control of the GAL1 promoter and ADH1 polyadenylation sequences. (B) Northern blot and Western blot analyses of BMV RNA3-specific products and CP, respectively. Yeast expressing BMV 1a, 2a, and RNA3 derivatives plus empty plasmid or CP-trans plasmid were grown in galactose medium, collected, and divided for RNA (as for Fig. 2) or protein analysis. CP expression was analyzed by Western blotting using total cell lysate and polyclonal anti-CP serum. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (C) Effect of CP expression on the relative accumulation of positive- and negative-strand RNA3 derivatives. The histogram compares accumulation of the positive strand (black bars) and negative strand (gray bars) for the RNA3 derivatives shown in panel B.

FIG. 4.

Neither sgRNA transcription nor sgRNA itself inhibits replication of exogenous RNA3 replicons. (A) Effect of trans-expressed sgRNA on the replication of an RNA3-derived replicon. The upper diagram represents expression cassettes for B3ΔSG and DNA-derived BMV sgRNAs with wt [B4(WT-sgRNA)] and frameshifted CP ORF [B4(CPfs)]. The lower panel shows a Northern blot analysis of total RNA from yeast expressing B3ΔSG and sgRNA (with or without translationally active CP ORF) alone or in combination with BMV 1a and 2a replication factors. The asterisk indicates a positive-strandRNA whose appearance is associated with the inactivation of sgRNA transcription. (B) Effect of sgRNA transcription on the replication of an exogenous RNA3-derived replicon. The upper diagram depicts the RNA3 derivatives used in this experiment. The lower panel shows Northern blot analyses of total RNA from yeast expressing 1a, 2a, and the indicated RNA3 cassettes to compare the replication level of B3ΔSGΔCP to that of B3ΔCP. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control.

FIG. 5.

Mutations in the sgRNA promoter do not alter the 1a-dependent accumulation of DNA-derived RNA3 transcripts. (A) Effects of core sgRNA promoter mutations (B3ΔSG and B3SG[-13]) on 1a-dependent RNA3 accumulation. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (B) Primer extension analysis of the 5′ ends of RNA3 species in yeast expressing BMV replication factors 1a and 2a and either B3CPfs, B3ΔSG, or B3SG[-13]. Total RNA from yeast was used in primer extension reactions containing a 5′ ³²P-labeled oligonucleotide primer complementary to RNA3 bases 30 to 44. A sequencing ladder, shown on the left, was prepared by extending the same labeled primer using pB3CPfs as template, and the sequence corresponding to the 5′-end of the positive-strand RNA3 is shown. As demonstrated previously, two major primer extension bands were detected due to the cap-dependent incorporation of an additional nucleotide by reverse transcriptase (22, 24). Longer (shown next to the sequencing ladder) and shorter (right panel) exposures of the same gel are presented to better visualize the relative accumulation of the indicated RNA3 species.

FIG. 6.

A box B mutation inactivating the template recruitment element does not exacerbate the negative effect of sgRNA transcription on RNA3 replication. (A) Distribution of RNA3 and sgRNA in yeast cells. Yeast cells expressing replication factors 1a and 2a and either B3CPfs, B3ΔSG, or B3SG[-13] were spheroplasted and lysed osmotically to yield a total RNA fraction (T). A portion of the lysate then was centrifuged at 20,000 × g to yield a membrane-enrichedpellet (P) fraction and a supernatant (S) fraction. RNA was isolated from each fraction and analyzed by Northern blotting to detect positive-strand and negative-strand products of viral replication. Substantial amounts of sgRNA produced by B3CPfs were detected in the pellet fraction. The asterisk indicates an RNA band, discussed in the text. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (B) Schematic diagram of wt box B (Box B^WT) and mutated box B (Box B*). A 3-nt replacement in the box B motif of the RE template recruitment element was designed to destabilize functionally important base-pairing at the top of the RE stem-loop structure. (C) Effect of the box B mutation on the interaction of RNA3 replication and sgRNA transcription. Northern blot analysis of total RNA was performed for yeast expressing 1a, 2a, and RNA3-derived replicons containing the Box B* mutation and either a wt or mutated sgRNA promoter. (D) The histogram compares accumulation of positive-strand (black bars) and negative-strand (gray bars) RNAs for RNA3 derivatives with the Box B* mutation.

FIG. 7.

Subgenomic RNA transcription does not inhibit negative-strand RNA3 accumulation. (A) Schematic diagram of expression cassettes for B3CPfs and RNA3 derivatives in which the viral 5′-UTR was replaced with the GAL1 mRNA 5′-UTR. (B) Northern blot analysis of BMV-specific RNA products. Total RNA was extracted from yeast expressing RNA3 alone or in combination with 1a or both 1a and 2a (indicated at the top). Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (C) GAL1 5′-UTR replacement strongly stimulates sgRNA transcription efficiency. The histogram compares negative-strand RNA3 synthesis efficiency (measured as the ratio of negative-strand RNA3 to positive-strand RNA3) and sgRNA transcription efficiency (measured as the ratio of sgRNA to negative-strand RNA3) for B3CPfs (gray boxes) and B3GAL1-CPfs (black boxes).

FIG. 8.

Inhibition of positive-strand RNA3 synthesis stimulates sgRNA transcription. (A) Point mutations (bold letters in MUT1 to MUT5) were introduced into the putative positive-strand RNA3 promoter of BMV expression cassettes. Shown in the table are the sequences of the wt and mutant cDNAs in the region corresponding to the 5′-end of wt RNA3. (B) Northern blot analysis of BMV-specific RNA products. Total RNA was extracted from yeast expressing BMV 1a, 2a, and RNA3 derivatives (indicated at the top) and analyzed by Northern blotting using specific probes to detect positive-strand RNA3 and sgRNA or negative-strand RNA3. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (C) The histogram compares the relative sgRNA transcription efficiency (measured as a ratio of the sgRNA to negative-strand RNA3) for B3CPfs and the 5′-UTR mutants.

FIG. 9.

Generation of RNA3 derivatives with additional, nonviral 5′ nucleotides. (A) Primer extension analysis of the 5′ ends of RNA3 species from yeast expressing BMV replication factors 1a and 2a and either B3CPfs or its derivatives containing mutations in the 5′-UTR as described in the legend for Fig. 8B. Primer extension was performed on total yeast RNA as for Fig. 5B. Arrows indicate the natural site of initiation for positive-strand RNA3 (WT) and novel positive-strand RNA3 initiation sites at position −10 (I) or −13 (II). Two major primer extension bands for each initiation position were detected due to cap-dependent incorporation of an additional nucleotide by reverse transcriptase (22, 24). (B) Alignment of the sequences of wt RNA3 and derivatives A and B produced as a result of upstream initiation at the positions indicated in panel A. (C) Alignment of the wt sequences of wt RNA1, wt RNA2, and wt RNA3.

FIG. 10.

Effect of inactivating sgRNA transcription on RNA3 accumulation in barley protoplasts inoculated with all three BMV genomic RNAs. (A) Northern blot analysis of BMV-specific RNA products in barley protoplasts. Total RNA was extracted from barley protoplasts 20 h p.i. with BMV wt RNA1, wt RNA2, and one of the indicated RNA3 derivatives. Ten micrograms of total RNA was analyzed by Northern blotting using specific probes to detect positive- or negative-strand BMV RNA or 18S rRNA as indicated on the left. Representative results are shown. (B) Relative accumulation of positive-strand (dark bars) and negative-strand (light bars) RNA3 derivatives. The histogram summarizes results from nine independent transfections for each RNA3 derivative.