Characterization of a novel 5' subgenomic RNA3a derived from RNA3 of Brome mosaic bromovirus

Abstract

The synthesis of 3' subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5' sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3' oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5' sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.

FIG. 1.

Northern blot analysis showing accumulation of RNA3-related products during BMV infection. (A and B) Accumulation of positive strands in barley plants inoculated in two separate experiments. Leaves were infected with BMV, and the RNA was extracted 2, 4, 6, 8 and 10 dpi. After being separated by electrophoresis in an agarose gel, the RNA material was blotted onto a nylon membrane and probed with a radioactive RNA probe complementary to an internal RNA3 sequence between nucleotides 962 and 1111. Lanes M1 and M2 carry the 1-to-1221-nt sequence and the full-length BMV RNA3 size standard transcripts, respectively. Line 0 represents RNA extracted from a healthy plant. (B) Accumulation of RNA3 and sgRNA3a was quantified by using Image Quant software in a Phosphoimager. (C) Accumulation of minus strands. RNA was extracted from plants infected with BMV at 2 (lane 2) and 10 (lanes 3 and 4; two separate extractions with lane 4 overexposed) days postinoculation. The probe represents the internal positive-strand RNA3 sequence between nucleotides 401 and 510. Lane 0, uninfected barley; lane 5, BMV RNA minus strands in total RNA from infected barley after 10 dpi, detected with BMV 3′-end positive-strand probe.

FIG. 2.

Encapsidation of sgRNA3a. (A) BMV RNA was extracted from a purified virus preparation either by two cycles of PEG precipitation or by PEG precipitation and additional ultracentrifugation at 100,000 × g. The extracted RNA was separated in an agarose denaturing gel and stained with ethidium bromide. Lane 1, nt 1 to 1221 transcribed RNA3 size standard; lanes 2 and 3, virion RNA after two PEG precipitations at 2 and 10 days postinoculation; lanes 4 and 5, virion RNA after ultracentrifugation at 2 and 10 days postinfection. The encapsidated sgRNA3a and other BMV RNAs are indicated by arrows. (B) Northern blot analysis of BMV RNA extracted from a virus preparation 10 days postinoculation by using an RNA3-specific probe as for Fig. 1. Lanes 1 and 2, BMV RNA from a viral preparation after two PEG precipitations and after additional ultracentrifugation, respectively; lane 3, nt 1 to 1221 transcribed sgRNA3a size standard.

FIG. 3.

Copying in vitro of minus-strand [(-)strand] RNA3 templates with BMV replicase. (A) Diagram of RW-IDrev1 RNA template. The positive-sense RNA initiation sequence [(+)promoter] is represented by a solid bar, with flanking nucleotide positions corresponding to those of the wt RNA3 shown below. The sequences complementary to the 3a and CP ORFs are shown as open boxes, and the oligo(U)-containing subgenomic promoter as a shaded box. The sgRNA3a molecule (lower line) carries the 3′-oligo(A) tail that is represented by a gray box. The locations of the RNA linker and those of both DNA primers used for PCR amplification are shown as a black line and arrows, respectively. (B) Electrophoretic analysis of RNA products after in vitro copying of RW-IDrev1 RNA3. The RNA (1 μg) was copied in the RdRp assay reaction (see Materials and Methods) by using a BMV replicase preparation, and the radioactive products were separated by electrophoresis in a 4% polyacrylamide-12 M urea denaturing gel (lane 1). Besides a full-length copy of the input RW-IDrev1 RNA3 template, shorter products of the premature termination reaction at the oligo(U) tract (1,400 nt) and subgenomic sgRNA4 (800 nt) are visible. Lanes M1 and M2 show separate migrations of radioactive in vitro-transcribed RNAs used as size standards: the 800-nt RNA transcript the size of sgRNA4 and the 1,221-nt marker corresponding to the size of sgRNA3a. Repl., BMV replicase. (C) The effect of ATP on the copying of RW-IDrev1 RNA3. The copying reactions at increased ATP concentrations (indicated at the bottom) and analysis of the products were performed as described for panel B.

FIG. 4.

Effect of deletions in the oligo(A) tract on accumulation of sgRNA3a and sgRNA4 in vivo. (A) Schematic representation of the oligo(A) deletion constructs. The upper scheme shows the genomic RNA3 molecule and its sgp region sequence, while the lower part depicts deletion mutants. Δ26 lacks the entire oligo(A) sequence plus a short upstream region, and construct Δ17 carries only four A residues in the oligo(A) tract. (B) Northern blot analysis of accumulation of sgRNAs in total RNA extracts from Chenopodium quinoa. The RNA, extracted after 14 dpi as described in Materials and Methods, was separated in 1% denaturing agarose gel, transferred to a nylon membrane, and probed with an RNA3-specific probe (to detect sgRNA3a) or with a 3′-end probe (to detect sgRNA4). The inoculated RNA3 deletion constructs are indicated by the numbers on top, and the positions of individual RNAs are indicated on the right. (C) Northern blot analysis of accumulation of sgRNAs in the encapsidated BMV RNA from C. quinoa after 14 days psi, using two different probes, as specified. Std., in vitro transcribed 5′ 1,200 nt of RNA3.

FIG. 5.

Translation activity of sgRNA3a in vitro and in the presence of polysomes. (A) Translation in rabbit reticulocyte system. The RNAs were incubated in rabbit reticulocyte extract at two different concentrations of MgCl₂, as indicated, and the products were separated in 12% SDS-polyacrylamide gel. The template RNAs are BMV RNA, total encapsidated viral RNA extracted from purified BMV preparation; sgRNA3a, the fraction of sgRNA3a purified from the encapsidated BMV RNA by cutting a band off the agarose formaldehyde/formamide gel; trRNA3 and trRNA3a, the RNA3 and sgRNA3a preparations synthesized by transcription in vitro and purified from unincorporated NTPs (on RNeasy columns; QIAGEN) prior to the translation reaction. (B) Translation in the wheat germ system. The RNAs were incubated in the wheat germ system with [³⁵S]methionine, and the products were analyzed in polyacrylamide-SDS gel, as described for panel A. The synthetic RNA3 or sgRNA3a templates were either capped or uncapped (see Materials and Methods), as indicated (lanes 1 through 4), whereas standard BMV RNA (containing the same amount of the RNA3 component) was translated in lane 5. The migration of protein 3a is shown on the right. tr, transcribed. (C) Detection of BMV RNA3-related sequences in polysomes. The polysomal RNA was extracted from BMV-infected barley leaves as described in Materials and Methods and separated by ultracentrifugation in sucrose gradient. Individual fractions (numbered 1 to 8) were analyzed by Northern blotting in 1% denaturing agarose gel and with the RNA3-specific probe. The positions of RNA3 and sgRNA3a were confirmed by comigration with the corresponding size transcripts in the “Std.” lane on the right.

FIG. 6.

Model illustrating the synthesis of sgRNA3a in view of multiple functions of the intergenic region in minus-strand RNA3. The BMV RdRp enzyme complex (represented by gray ovals) migrates alongside the minus-strand RNA template and pauses (represented by curved arrows) at the secondary structure or, most notably, at the oligo(U) tract, leading to the formation of subgenomic sgRNA3a. Yet another molecule of the RdRp enzyme binds to the sgp and initiates the de novo synthesis of sgRNA4. Also, the rehybridization of the sgRNA3a oligo(A) tail to the RNA3 minus template can resume full-length copying, which primes the observed RNA3-RNA3 recombination (5, 69). The positive and minus RNA strands are represented by thick lines and both the oligo(U) tract in the minus-strand template and the oligo(A) 3′-termini are exposed. The stem-and-loop structures adopted by the positive and minus strands upstream of their oligo(U) and oligo(A) tracts (3) are shown. The region that binds to protein 1a via the B box of the stem-loop structure in positive strands (28, 57) is shown.